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Hand2 Loss-of-Function in Hand1-Expressing Cells Reveals Distinct Roles in Epicardial and Coronary Vessel Development
Abstract
The basic helix-loop-helix (bHLH) transcription factors Hand1 and Hand2 are essential for embryonic development. Given their requirement for cardiogenesis, it is imperative to determine their impact on cardiovascular function.
To deduce the role of Hand2 within the epicardium.
We engineered a Hand1 allele expressing Cre recombinase. Cardiac Hand1 expression is largely limited to cells of the primary heart field, overlapping little with Hand2 expression. Hand1 is expressed within the septum transversum, and the Hand1 lineage marks the proepicardial organ and epicardium. To examine Hand factor functional overlap, we conditionally deleted Hand2 from Hand1-expressing cells. Hand2 mutants display defective epicardialization and fail to form coronary arteries, coincident with altered extracellular matrix deposition and Pdgfr expression.
These data demonstrate a hierarchal relationship whereby transient Hand1 septum transversum expression defines epicardial precursors that are subsequently dependent on Hand2 function.
... [10][11][12]. Lineage trace analysis reveals that Hand1-lineage marks cells within the anterior ST, PEO, epicardium and its secondary EMT derivatives; however, Hand1 expression is only detectable within the anterior ST [13]. Hand2 expression is observed within the PEO and epicardium [13]. ...
... Lineage trace analysis reveals that Hand1-lineage marks cells within the anterior ST, PEO, epicardium and its secondary EMT derivatives; however, Hand1 expression is only detectable within the anterior ST [13]. Hand2 expression is observed within the PEO and epicardium [13]. Deletion of Hand2 using WT1 ERT2Cre [14], which is expressed specifically in the epicardium during development, results in a similar phenotype as the deletion of Hand2 within the Hand1-lineage, malformed coronaries [13]. ...
... Hand2 expression is observed within the PEO and epicardium [13]. Deletion of Hand2 using WT1 ERT2Cre [14], which is expressed specifically in the epicardium during development, results in a similar phenotype as the deletion of Hand2 within the Hand1-lineage, malformed coronaries [13]. ...
We have previously identified a Hand1 transcriptional enhancer that drives expression within the septum transversum, the origin of the cells that contribute to the epicardium. This enhancer directly overlaps a common exon of a predicted family of long non-coding RNAs (lncRNA) that are specific to mice. To interrogate the necessity of this Hand1 enhancer, as well as the importance of these novel lncRNAs, we deleted the enhancer sequences, including the common exon shared by these lncRNAs, using genome editing. Resultant homozygous Hand1 enhancer mutants (Hand1
Δ
ST/ΔST) present with no observable phenotype. Assessment of lncRNA expression reveals that Hand1
Δ
ST/ΔST mutants effectively eliminate detectable lncRNA expression. Expression analysis within Hand1
Δ
ST/ΔST mutant hearts indicates higher levels of Hand1 than in controls. The generation of Hand1 compound heterozygous mutants with the Hand1LacZ null allele (Hand1
Δ
ST/LacZ) also did not reveal any observable phenotypes. Together these data indicate that deletion of this Hand1 enhancer and by consequence a family of murine-specific lncRNAs does not impact embryonic development in observable ways.
... Differentiation of inner ear sensory neurons, cerebellum, and the hippocampus [166] Survival and migration of neuroendocrine lung carcinomas; cell motility and tumor formation of neuroblastoma; in cooperation with Otx2, controls Group 3 medulloblastoma active enhancer landscape [167] NEUROD2 Formation of corpus callosum, essential for communication between the two cerebral hemispheres [168] Tumor suppressor and prognostic biomarker in Glioblastoma; copy number gains of NEUROD2 in male breast cancer (prognostic value) [169] HAND1 Proliferation, differentiation, and morphogenesis of embryonic ventricle cardiomyocytes [170,171] Downregulated in medulloblastoma; facilitates proliferation and metastasis in gastrointestinal stromal tumor; silenced in over 90% of human primary colorectal tumors. Methylation of HAND1 associated with poor survival in gastric cancer; involved in thyroid carcinogenesis [172] HAND2 Proliferation, differentiation, and morphogenesis of embryonic ventricle cardiomyocytes [170,171] Tumor suppressor in endometroid endometrial carcinoma. ...
... Differentiation of inner ear sensory neurons, cerebellum, and the hippocampus [166] Survival and migration of neuroendocrine lung carcinomas; cell motility and tumor formation of neuroblastoma; in cooperation with Otx2, controls Group 3 medulloblastoma active enhancer landscape [167] NEUROD2 Formation of corpus callosum, essential for communication between the two cerebral hemispheres [168] Tumor suppressor and prognostic biomarker in Glioblastoma; copy number gains of NEUROD2 in male breast cancer (prognostic value) [169] HAND1 Proliferation, differentiation, and morphogenesis of embryonic ventricle cardiomyocytes [170,171] Downregulated in medulloblastoma; facilitates proliferation and metastasis in gastrointestinal stromal tumor; silenced in over 90% of human primary colorectal tumors. Methylation of HAND1 associated with poor survival in gastric cancer; involved in thyroid carcinogenesis [172] HAND2 Proliferation, differentiation, and morphogenesis of embryonic ventricle cardiomyocytes [170,171] Tumor suppressor in endometroid endometrial carcinoma. HAND2 suppression upregulates Fgfs in endometriosis [173]. ...
... Besides their extensive role in neural development, bHLH TFs have also been well-studied in the development of other structures. Math1, Neurogenin 3, and NeuroD1 play a sequential role in the development of gastrointestinal entero-endocrine cells-specification, segregation to the secretory lineage and differentiation [284][285][286][287]. Hand1 and Hand2 play critical roles in the proliferation, differentiation, and the morphogenesis of embryonic ventricle cardiomyocytes [170,171,288]. Twist1 and Twist2 play a major role in bone formation or osteogenesis. ...
Development requires the careful orchestration of several biological events in order to create any structure and, eventually, to build an entire organism. On the other hand, the fate transformation of terminally differentiated cells is a consequence of erroneous development, and ultimately leads to cancer. In this review, we elaborate how development and cancer share several biological processes, including molecular controls. Transcription factors (TF) are at the helm of both these processes, among many others, and are evolutionarily conserved, ranging from yeast to humans. Here, we discuss four families of TFs that play a pivotal role and have been studied extensively in both embryonic development and cancer—high mobility group box (HMG), GATA, paired box (PAX) and basic helix-loop-helix (bHLH) in the context of their role in development, cancer, and their conservation across several species. Finally, we review TFs as possible therapeutic targets for cancer and reflect on the importance of natural resistance against cancer in certain organisms, yielding knowledge regarding TF function and cancer biology.
... Hand1 cardiac expression is first detectable in the mouse embryo at E8.5 in the posterior ventricle as well as a small domain of the developing OFT termed the myocardial cuff ( Fig. 1; Barnes et al. 2011). As heart looping proceeds, Hand1 expression is robust within the left ventricle (LV) between E9.5-E13.5, is detected in both cNCC and SHF-derived myocardial cuff of the OFT, and pericardium (Barbosa et al. 2007; Barnes et al. 2010). ...
... As heart looping proceeds, Hand1 expression is robust within the left ventricle (LV) between E9.5-E13.5, is detected in both cNCC and SHF-derived myocardial cuff of the OFT, and pericardium (Barbosa et al. 2007; Barnes et al. 2010). Hand1 expression is not detectable within endocardial or epicardial cells; however, the epicardium, and all its derivatives, is Hand1-lineage derived (Barnes et al. , 2011. ...
... Hand2 expression within cardiac and endocardial progenitors is detectable at E7.75 within the cardiac crescent ( Fig. 1; Barnes et al. 2011). Hand2 is robustly expressed within the SHF pharyngeal mesoderm that underlies and contributes to the growing heart tube (Barnes et al. 2011;Tsuchihashi et al. 2011). ...
Congenital heart defects account for 1% of infant mortality and 10% of in utero deaths. As the vertebrate embryo develops, multiple tissue types develop in tandem to morphologically pattern the functional heart. Underlying cardiac development is a network of transcription factors known to tightly control these morphological events. Members of the Twist family of basic helix–loop–helix transcription factors, Hand1 and Hand2, are essential to this process. The expression patterns and functional role of Hand factors in neural crest cells, endocardium, myocardium, and epicardium is indicative of their importance during cardiogenesis; however, to date, an extensive understanding of the transcriptional targets of Hand proteins and their overall mechanism of action remain unclear. In this review, we summarize the recent findings that further outline the crucial functions of Hand factors during heart development and in post‐natal heart function. Anat Rec, 302:101–107, 2019. © 2018 Wiley Periodicals, Inc.
... We used this Hand1 enhancer to make an LV-specific Cre driver. Lineage analysis using the Hand1 eGFPCre knock-in allele revealed that Hand1-lineage cells are restricted to the LV myocardium and to a ring of SHF-derived myocardium occupying the OFT, termed the myocardial cuff [7,10]. Using the 2.7kb Hand1 basal promoter to provide the eGFPCre with a transcriptional start site, we cloned the Hand1 LV-enhancer 5' (Fig 1A) and generated several F0 transgenic lines. ...
... As we have established that the Hand1-LV myocardial lineage is not required for cardiogenesis, we next investigated whether the expression of Hand1 and Hand2 within the embryonic LV is required for normal heart development. Although early embryonic expression analysis shows that the majority of Hand2 expression within the heart is restricted to the endocardium, epicardium, and SHF derived myocardium [10,18,19], at later embryonic stages, Hand2 mRNA becomes detectable within E11.5 LV myocardium in a pattern overlapping with Hand1 (S4C and S4D Fig). We subsequently generated compound heterozygous Hand1 LV -Cre;Hand1 fx/+ ; Hand2 fx/+ male mice and crossed them to Hand1 fx/fx ;Hand2 fx/fx females to generate Hand1 LV -Cre;Hand1 fx/fx ;Hand2 fx/fx offspring (Fig 3). ...
... The Indiana University Transgenic and Knock-Out Mouse Core generated the Hand1 LV -Cre transgenic mouse line on a C3HeB/FeJ background. Genotyping of the Hand1 tm2Eno , Hand2 tm1Cse , Gt(ROSA)26Sor tm1(DTA)Jpmb , and Gt(ROSA)26Sor tm1Sor alleles has previously been described [9,10,35,36]. These mice were maintained on a mixed C57Bl/6;129S background. ...
Coordinated cardiomyocyte growth, differentiation, and morphogenesis are essential for heart formation. We demonstrate that the bHLH transcription factors Hand1 and Hand2 play critical regulatory roles for left ventricle (LV) cardiomyocyte proliferation and morphogenesis. Using an LV-specific Cre allele (Hand1LV-Cre), we ablate Hand1-lineage cardiomyocytes, revealing that DTA-mediated cardiomyocyte death results in a hypoplastic LV by E10.5. Once Hand1-linage cells are removed from the LV, and Hand1 expression is switched off, embryonic hearts recover by E16.5. In contrast, conditional LV loss-of-function of both Hand1 and Hand2 results in aberrant trabeculation and thickened compact zone myocardium resulting from enhanced proliferation and a breakdown of compact zone/trabecular/ventricular septal identity. Surviving Hand1;Hand2 mutants display diminished cardiac function that is rescued by concurrent ablation of Hand-null cardiomyocytes. Collectively, we conclude that, within a mixed cardiomyocyte population, removal of defective myocardium and replacement with healthy endogenous cardiomyocytes may provide an effective strategy for cardiac repair.
... Curiously, zebrafish only retained hand2 in their genome, and hand2 mutants develop cardiac and branchial arch defects, hypoplastic pectoral fins and reduced mesothelium that results in herniation (Garavito-Aguilar et al., 2010;Hu et al., 2011;Perens et al., 2016;Prummel et al., 2022;Yelon and Stainier, 2005;Yelon et al., 2000). Compared to genes encoding other cardiac transcription factors, HAND1/2 seem to be less frequently mutated in congenital disease, possibly due to their partial redundancy and potential to compensate for one another upon perturbation (Barnes et al., 2011;Charité et al., 2000;McFadden et al., 2005). Notably, partial trisomy of distal chromosome 4q (4q+; no OMIM reference), which contains the HAND2 locus, is associated with cardiac, digit, kidney and craniofacial defects, indicating broader LPM perturbation during development (Battaglia et al., 2005;Lundin et al., 2002;Lurie, 2005). ...
... The mouse Tbx5 locus harbors several regulatory elements, including a dedicated forelimb enhancer (Cunningham et al., 2018;Minguillon et al., 2012) and multiple cardiac enhancers (Richter et al., 2020;Smemo et al., 2012); more regulatory elements (and their upstream control) await discovery (Bickley and Logan, 2014;Bruneau et al., 1999). Similarly, zebrafish hand2 and mouse Hand2 mutants have provided key insights into the developmental mechanisms in heart, limbs and additional LPM organs influenced by this deeply conserved transcription factor (Barnes et al., 2011;Firulli et al., 1998;George et al., 2022;Hashimoto et al., 2019;Keegan et al., 2005;Laurent et al., 2017;McFadden et al., 2005;Osterwalder et al., 2014;Perens et al., 2016;Prummel et al., 2022;Vincentz et al., 2021;Yamagishi et al., 2000;Yelon and Stainier, 2005). The upstream regulation of Hand2 in mouse and zebrafish, beyond the pharyngeal arches (Charité et al., 2001;Iklé et al., 2012), remains vastly uncharacterized, as regulatory elements for broad early LPM expression or for heart and limb/pectoral fin activity have not been identified to date. ...
Syndromic birth defects are rare diseases that can present with seemingly pleiotropic comorbidities. Prime examples are rare congenital heart and cardiovascular anomalies that can be accompanied by forelimb defects, kidney disorders and more. Whether such multi-organ defects share a developmental link remains a key question with relevance to the diagnosis, therapeutic intervention and long-term care of affected patients. The heart, endothelial and blood lineages develop together from the lateral plate mesoderm (LPM), which also harbors the progenitor cells for limb connective tissue, kidneys, mesothelia and smooth muscle. This developmental plasticity of the LPM, which founds on multi-lineage progenitor cells and shared transcription factor expression across different descendant lineages, has the potential to explain the seemingly disparate syndromic defects in rare congenital diseases. Combining patient genome-sequencing data with model organism studies has already provided a wealth of insights into complex LPM-associated birth defects, such as heart-hand syndromes. Here, we summarize developmental and known disease-causing mechanisms in early LPM patterning, address how defects in these processes drive multi-organ comorbidities, and outline how several cardiovascular and hematopoietic birth defects with complex comorbidities may be LPM-associated diseases. We also discuss strategies to integrate patient sequencing, data-aggregating resources and model organism studies to mechanistically decode congenital defects, including potentially LPM-associated orphan diseases. Eventually, linking complex congenital phenotypes to a common LPM origin provides a framework to discover developmental mechanisms and to anticipate comorbidities in congenital diseases affecting the cardiovascular system and beyond.
... The epithelial-to-mesenchymal transition (EMT) is a complex event highly regulated by different transcription factors, such as Snai1, Snai2, Twist1, or Hand2, that mediate the change from epithelial cells into a mesenchymal phenotype and are implicated in mesenchymal cell proliferation and migration [26][27][28][30][31][32]. In the same way, some TGFβ and FGF superfamily members promote epicardial EMT and EPDC motility [33,34]. ...
... EPDC-derived lineage determination is a precisely regulated process mediated by complex interactions among signaling pathways and transcription factors, which can act as transcriptional activators and/or repressors [40]. For instance, Pdgfra expression promoted by Hand2 is required for epicardium-derived cardiac fibroblast specification, which is one of the most numerous cell populations in the heart, while Pdgfrb activation induces EPDCs differentiation into vascular smooth muscle cells [32,[41][42][43][44][45]. Tcf21 regulates EPDC differentiation into smooth muscle and fibroblast lineages. ...
The epicardium is the outermost cell layer in the vertebrate heart that originates during development from mesothelial precursors located in the proepicardium and septum transversum. The epicardial layer plays a key role during cardiogenesis since a subset of epicardial-derived cells (EPDCs) undergo an epithelial–mesenchymal transition (EMT); migrate into the myocardium; and differentiate into distinct cell types, such as coronary vascular smooth muscle cells, cardiac fibroblasts, endothelial cells, and presumably a subpopulation of cardiomyocytes, thus contributing to complete heart formation. Furthermore, the epicardium is a source of paracrine factors that support cardiac growth at the last stages of cardiogenesis. Although several lineage trace studies have provided some evidence about epicardial cell fate determination, the molecular mechanisms underlying epicardial cell heterogeneity remain not fully understood. Interestingly, seminal works during the last decade have pointed out that the adult epicardium is reactivated after heart damage, re-expressing some embryonic genes and contributing to cardiac remodeling. Therefore, the epicardium has been proposed as a potential target in the treatment of cardiovascular disease. In this review, we summarize the previous knowledge regarding the regulation of epicardial cell contribution during development and the control of epicardial reactivation in cardiac repair after damage.
... 10 In turn, heart and neural crest derivatives expressed-2 (HAND2), islet-1, fibroblast growth factor-8, fibroblast growth factor-10, and paired-like homeodomain-2 are mostly expressed in the secondary heart field. [11][12][13][14] In mice, from embryonic day 7.75, Hand2 expression is detectable within the cardiac crescent and it becomes robustly expressed within the secondary heart field pharyngeal mesoderm that underlies and contributes to the growing heart tube. 12,15 During cardiac looping, myocardial expression of Hand2 is very low but very accentuated in the endocardium. ...
... [11][12][13][14] In mice, from embryonic day 7.75, Hand2 expression is detectable within the cardiac crescent and it becomes robustly expressed within the secondary heart field pharyngeal mesoderm that underlies and contributes to the growing heart tube. 12,15 During cardiac looping, myocardial expression of Hand2 is very low but very accentuated in the endocardium. 16,17 Loss-of-function studies in mice revealed the importance of these transcription factors during development of the heart. ...
Aims:
Research on the pathophysiology of right ventricular (RV) failure has, in spite of the associated high mortality and morbidity, lagged behind compared to the left ventricle (LV).Previous work from our lab revealed that the embryonic basic helix-loop-helix transcription factor heart and neural crest derivatives expressed-2 (Hand2) is re-expressed in the adult heart and activates a 'fetal gene program' contributing to pathological cardiac remodeling under conditions of LV pressure overload. As such, ablation of cardiac expression of Hand2 conferred protection to cardiac stress and abrogated the maladaptive effects that were observed upon increased expression levels. In this study, we aimed to understand the contribution of Hand2 to RV remodeling in response to pressure overload induced by pulmonary artery banding (PAB).
Methods and results:
In the present study, Hand2F/F and MCM- Hand2F/F mice were treated with tamoxifen (control and knockout, respectively) and subjected to six weeks of RV pressure overload induced by PAB. Echocardiographic- and MRI-derived hemodynamic parameters as well as molecular remodeling were assessed for all experimental groups and compared to sham-operated controls. Six weeks after PAB, levels of Hand2 expression increased in the control banded animals but, as expected, remained absent in the knockout hearts. Despite the dramatic differences in Hand2 expression, pressure overload resulted in impaired cardiac function independently of the genotype. In fact, Hand2 depletion seems to sensitize the RV to pressure overload as these mice develop more hypertrophy and more severe cardiac dysfunction. Higher expression levels of HAND2 were also observed in RV samples of human hearts from patients with pulmonary hypertension. In turn, the LV of RV-pressure overloaded hearts was also dramatically affected as reflected by changes in shape, decreased LV mass and impaired cardiac function. RNA sequencing revealed a distinct set of genes that are dysregulated in the pressure-overloaded RV, compared to the previously described pressure-overloaded LV.
Conclusions:
Cardiac-specific depletion of Hand2 is associated with severe cardiac dysfunction in conditions of RV pressure overload. While inhibiting Hand2 expression can prevent cardiac dysfunction in conditions of LV pressure overload, the same does not hold true for conditions of RV pressure overload. This study highlights the need to better understand the molecular mechanisms driving pathological remodeling of the RV in contrast to the LV, in order to better diagnose and treat patients with RV or LV failure.
Translational perspective:
RV failure associated with pulmonary hypertension reduces long-term survival rate to 55% within 3 years, suggesting that 3 years after diagnosis almost half of the patients will die. To revert these numbers an adequate RV-specific and, therefore, more efficient treatment is needed. Our work suggests that current therapies and potential mechanisms underlying LV failure may not be suitable for RV failure. While Hand2 deletion is favorable in LV response to stress, it is particularly detrimental in the RV under similar conditions, and thus, highlighting potential severe consequences of not differentiating therapeutic targets or treatment for RV or LV failure.
... Section in situ hybridizations were performed on 10-µm paraffin sections as described [20,21]. Antisense digoxygenin-labeled riboprobes were synthesized using T7, T3, or SP6 polymerases (Promega) and DIG-Labeling Mix (Roche) using the following plasmid templates: Dlx5, Dlx6, Hand2, Sox9 (provided by Benoit De Crombrugghe), and Ret (provided by Jean-Francois Brunet). ...
... In this study, we set out to interrogate how NCC gene regulatory networks that include HAND transcription factors facilitate NCC specialization into specific tissue fates. Hand1 and Hand2 mark both cranial and cardiac NCC populations [36,37], exhibit genetic interactions that, when disrupted, result in a phenotype [4,19,21,38], and set up tissue boundaries that are essential normal tissue morphogenesis within the post migration NCCs occupying the pharyngeal arches [6,8]. ...
Congenital heart defects (CHDs) occur with such a frequency that they constitute a significant cause of morbidity and mortality in both children and adults. A significant portion of CHDs can be attributed to aberrant development of the cardiac outflow tract (OFT), and of one of its cellular progenitors known as the cardiac neural crest cells (NCCs). The gene regulatory networks that identify cardiac NCCs as a distinct NCC population are not completely understood. Heart and neural crest derivatives (HAND) bHLH transcription factors play essential roles in NCC morphogenesis. The Hand1PA/OFT enhancer is dependent upon bone morphogenic protein (BMP) signaling in both cranial and cardiac NCCs. The Hand1PA/OFT enhancer is directly repressed by the endothelin-induced transcription factors DLX5 and DLX6 in cranial but not cardiac NCCs. This transcriptional distinction offers the unique opportunity to interrogate NCC specification, and to understand why, despite similarities, cranial NCC fate determination is so diverse. We generated a conditionally active transgene that can ectopically express DLX5 within the developing mouse embryo in a Cre-recombinase-dependent manner. Ectopic DLX5 expression represses cranial NCC Hand1PA/OFT-lacZ reporter expression more effectively than cardiac NCC reporter expression. Ectopic DLX5 expression induces broad domains of NCC cell death within the cranial pharyngeal arches, but minimal cell death in cardiac NCC populations. This study shows that transcription control of NCC gene regulatory programs is influenced by their initial specification at the dorsal neural tube.
... Previous cell lineage analysis using Hand1 eGFPCre shows that within the heart, Hand1-lineage is restricted to the compact zone, trabecular, and small domain within the IVS myocardium of the LV and within myocardial cuff of the RV with no detectable expression observed within the endocardium. 56,57 Although the Hand1-lineage directly contributes to the epicardium, Hand1 is not expressed within the proepicardial organ or epicardium. 56,57 These observations suggest that the observed CHDs observed in H1CKOs are cell autonomous to the myocardium and that cardiomyocyte deletion of Hand1 results in an increase of LV trabecular tissue and that VSD and valve phenotypes are consistent with similar published embryonic analysis. ...
... 56,57 Although the Hand1-lineage directly contributes to the epicardium, Hand1 is not expressed within the proepicardial organ or epicardium. 56,57 These observations suggest that the observed CHDs observed in H1CKOs are cell autonomous to the myocardium and that cardiomyocyte deletion of Hand1 results in an increase of LV trabecular tissue and that VSD and valve phenotypes are consistent with similar published embryonic analysis. 23 ...
Aims
To examine the role of the basic Helix-loop-Helix (bHLH) transcription factor HAND1 in embryonic and adult myocardium.
Methods and results
Hand1 is expressed within the cardiomyocytes of the left ventricle (LV) and myocardial cuff between embryonic days (E) 9.5–13.5. Hand gene dosage plays an important role in ventricular morphology and the contribution of Hand1 to congenital heart defects requires further interrogation. Conditional ablation of Hand1 was carried out using either Nkx2.5 knockin Cre (Nkx2.5Cre) or α-myosin heavy chain Cre (αMhc-Cre) driver. Interrogation of transcriptome data via ingenuity pathway analysis reveals several gene regulatory pathways disrupted including translation and cardiac hypertrophy-related pathways. Embryo and adult hearts were subjected to histological, functional, and molecular analyses. Myocardial deletion of Hand1 results in morphological defects that include cardiac conduction system defects, survivable interventricular septal defects, and abnormal LV papillary muscles (PMs). Resulting Hand1 conditional mutants are born at Mendelian frequencies; but the morphological alterations acquired during cardiac development result in, the mice developing diastolic heart failure.
Conclusion
Collectively, these data reveal that HAND1 contributes to the morphogenic patterning and maturation of cardiomyocytes during embryogenesis and although survivable, indicates a role for Hand1 within the developing conduction system and PM development.
... Specific inactivation in developing heart tissues has revealed essential Hand2 functions in the cardiac neural crest cells that contribute to cardiac cushions in the OFT, survival of SHF progenitors, heart chamber trabeculation, and epicardial cell differentiation (Barnes et al., 2011;Holler et al., 2010;Tsuchihashi et al., 2011;VanDusen et al., 2014a). Previous studies had also pointed to HAND2 functions in cardiac cushion formation, but the potential essential functions have not been identified (Holler et al., 2010;Liu et al., 2009;VanDusen et al., 2014a). ...
... We also provide evidence that HAND2 directly regulates Snai1 transcription in other embryonic tissues. The notion that the direct transcriptional regulation of Snai1 by HAND2 maybe of more general importance is supported by genetic analysis as Hand2 and Snai1 are both essential for the EMT of epicardial cells and morphogenesis of craniofacial structures such as the palate (Barnes et al., 2011;Murray et al., 2007;Tao et al., 2013;Xiong et al., 2009). In summary, our study identifies the HAND2 target GRN that controls the initiation of cardiac valve formation and provides evidence for its general role in regulating the expression of Snai1 during mouse embryogenesis. ...
The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost from Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development.
... Embryonically, HAND2 induces type 1 (embryonic) MET patterning of NCCs to branching aortic vessels and of mesodermal second heart field (SHF) cells to the right ventricle (RV), parts of the atria, interventricular septum, and outflow tract [25][26][27]. HAND2 murine knockout (KO) is lethal at ED9. yielding RV hypoplasia, and vascular malformations [25,28]. Molecularly, HAND2 is a crucial downstream regulator of the endocardial VEGF-Notch signaling pathway during cardiogenesis and coronary vasculogenesis [27]. ...
We previously developed several successful decellularization strategies that yielded porcine cardiac extracellular matrices (pcECMs) exhibiting tissue-specific bioactivity and bioinductive capacity when cultured with various pluripotent and multipotent stem cells. Here, we study the tissue-specific effects of the pcECM on seeded human mesenchymal stem cell (hMSC) phenotypes using reverse transcribed quantitative polymerase chain reaction (RT-qPCR) arrays for cardiovascular related gene expression. We further corroborated interesting findings at the protein level (flow cytometry and immunological stains) as well as bioinformatically using several mRNA sequencing and protein databases of normal and pathologic adult and embryonic (organogenesis stage) tissue expression. We discovered that upon the seeding of hMSCs on the pcECM, they displayed a partial mesenchymal-to-epithelial transition (MET) toward endothelial phenotypes (CD31+) and morphologies, which were preceded by an early spike (~Day 3 onward after seeding) in HAND2 expression at both the mRNA and protein levels compared to that in plate controls. The CRISPR-Cas9 knockout (KO) of HAND2 and its associated antisense long non-coding RNA (HAND2-AS1) regulatory region resulted in proliferation arrest, hypertrophy, and senescent-like morphology. Bioinformatic analyses revealed that HAND2 and HAND2-AS1 are highly correlated in expression and are expressed in many different tissue types albeit at distinct yet tightly regulated expression levels. Deviation (downregulation or upregulation) from these basal tissue expression levels is associated with a long list of pathologies. We thus suggest that HAND2 expression levels may possibly fine-tune hMSCs’ plasticity through affecting senescence and mesenchymal-to-epithelial transition states, through yet unknown mechanisms. Targeting this pathway may open up a promising new therapeutic approach for a wide range of diseases, including cancer, degenerative disorders, and aging. Nevertheless, further investigation is required to validate these findings and better understand the molecular players involved, potential inducers and inhibitors of this pathway, and eventually potential therapeutic applications.
... Analyses of potential HOXD10 interaction partners further revealed an association with other posterior HOX genes, especially HOXA11, HOXD9, and HOXD13 as well as genes important for extracellular matrix remodeling as potential fibroblast-specific features. For example, HAND2 overexpression was particularly shown to mediate fibroblast marker expression and regulate extracellular matrix organization and function by maintaining the balance between integrins and fibronectin (58,59). Likewise, EGR1, a known connective tissue gene regulating extracellular matrix deposition and organization, was found to be differentially expressed in fibroblasts and potentially linked to differentially expressed HOXD10 here (60). ...
Introduction
Mesenchymal stem cells (MSCs) are considered to be the most promising stem cell type for cell-based therapies in regenerative medicine. Based on their potential to home to diseased body sites following a therapeutically application, these cells could (i) differentiate then into organ-specific cell types to locally restore injured cells or, most prominently, (ii) foster tissue regeneration including immune modulations more indirectly by secretion of protective growth factors and cytokines. As tissue-resident stem cells of mesenchymal origin, these cells are morphologically and even molecularly- at least concerning the classical marker genes- indistinguishable from similar lineage cells, particularly fibroblasts.
Methods
Here we used microarray-based gene expression and global DNA methylation analyses as well as accompanying computational tools in order to specify differences between MSCs and fibroblasts, to further unravel potential identity genes and to highlight MSC signaling pathways with regard to their trophic and immunosuppressive action.
Results
We identified 1352 differentially expressed genes, of which in the MSCs there is a strong signature for e.g., KRAS signaling, known to play essential role in stemness maintenance, regulation of coagulation and complement being decisive for resolving inflammatory processes, as well as of wound healing particularly important for their regenerative capacity. Genes upregulated in fibroblasts addressed predominately transcription and biosynthetic processes and mapped morphological features of the tissue. Concerning the cellular identity, we specified the already known HOX code for MSCs, established a potential HOX code for fibroblasts, and linked certain HOX genes to functional cell-type-specific properties. Accompanied methylation profiles revealed numerous regions, especially in HOX genes, being differentially methylated, which might provide additional biomarker potential.
Discussion
Conclusively, transcriptomic together with epigenetic signatures can be successfully be used for the definition (cellular identity) of MSCs versus fibroblasts as well as for the determination of the superior functional properties of MSCs, such as their immunomodulatory potential.
... In fact, Scleraxis induces the expression of Col1a2 in adult cardiac fibroblasts [97]. Finally, Hand2 is necessary for normal development of the epicardium where it activates PDGFRα [98]. This receptor of PDGF is required for epicardial EMT and the differentiation of EPDCs [99]. ...
The embryonic epicardium originates from the proepicardium, an extracardiac primordium constituted by a cluster of mesothelial cells. In early embryos, the embryonic epicardium is characterized by a squamous cell epithelium resting on the myocardium surface. Subsequently, it invades the subepicardial space and thereafter the embryonic myocardium by means of an epithelial–mesenchymal transition. Within the myocardium, epicardial-derived cells present multilineage potential, later differentiating into smooth muscle cells and contributing both to coronary vasculature and cardiac fibroblasts in the mature heart. Over the last decades, we have progressively increased our understanding of those cellular and molecular mechanisms driving proepicardial/embryonic epicardium formation. This study provides a state-of-the-art review of the transcriptional and emerging post-transcriptional mechanisms involved in the formation and differentiation of the embryonic epicardium.
... Embryonically, HAND2 induces type 1 (embryonic) MET patterning of NCCs to branching aortic vessels and of mesodermal second heart field (SHF) cells to the right ventricle (RV), parts of the atria, interventricular septum, and outflow tract [25][26][27] . HAND2 murine knockout (KO) is lethal at E9.5-10.5, yielding RV hypoplasia, single chambered hearts, and vascular malformations 25,28 . Molecularly, HAND2 is a crucial downstream regulator of the endocardial VEGF-Notch signaling pathway during cardiogenesis and coronary vasculogenesis 27 . ...
We have previously developed several successful decellularization strategies yielding porcine cardiac extracellular matrices (pcECMs), which exhibit tissue-specific bioactivity and bioinductive capacity when cultured with various pluri- and multipotent stem cells. Here, we studied the tissue-specific effects of the pcECM on seeded human mesenchymal stem cells (hMSCs) phenotype using reverse transcribed quantitative polymerase chain reaction (RT-qPCR) arrays for cardio-vascular related genes. We further corroborated interesting findings at the protein level (flow cytometry and immunological stains) as well as bioinformatically using several mRNA sequencing and protein databases of normal and pathologic adult tissue expression, as well as during human embryonic organogenesis. We discovered that upon seeding of human mesenchymal stem cells (hMSCs) on the pcECM they displayed partial MET toward endothelial phenotypes (CD31+) and morphologies, which were preceded by an early spike (~day 3 onward after seeding) in HAND2 expression at both the mRNA and protein levels compared to plate controls. CRISPR-Cas9 knockout (KO) of HAND2 and its associated antisense long non-coding RNA (HAND2-AS1) regulatory region resulted in proliferation arrest, hypertrophy, and senescent-like morphology. Bioinformatic analyses revealed that HAND2 and HAND2-AS1 are highly correlated in expression, are expressed in many different tissue types albeit at distinct yet tightly regulated expression levels. Deviation (down or up regulation) from these basal tissue expression levels are associated with a long list of pathologies. We thus suggest that HAND2 expression levels may finetune cell plasticity possibly affecting senescence and mesenchymal-to-epithelial transition states, through yet unknown mechanisms. Targeting this pathway may represent a promising new therapeutic approach for a wide range of diseases, including cancer, degenerative disorders, and aging. Nevertheless, further investigations are required to better understand the molecular players involved, potential inducers and inhibitors of this pathway, and eventually potential therapeutic applications.
... To investigate whether SDSCs have properties similar to pluripotent stem cells, we transplanted these SDSCs colonies under the kidney capsules of immune-deficient mice, and solid tumors formed after 10 weeks of transplantation (Fig. 3a). Subsequently, PCR analysis revealed that three germ layers markers, including the ectodermal marker genes NOTCH1 and GFAP [50,51], mesodermal marker genes KDR and HAND1 [52,53], and endodermal marker genes AFP and FOXA2 [50,54], were identified in ectopic developed SDSC-derived solid tumors (Fig. 3b-d). Importantly, we confirmed by H&E staining that tumors contained tissues derived from three germ layers, including ectodermderived neuroepithelium, mesoderm-derived muscle and adipose, and endoderm-derived original kidney (Fig. 3e). ...
According to estimations, approximately about 15% of couples worldwide suffer from infertility, in which individuals with azoospermia or oocyte abnormalities cannot be treated with assisted reproductive technology. The skin-derived stem cells (SDSCs) differentiation into primordial germ cell-like cells (PGCLCs) is one of the major breakthroughs in the field of stem cells intervention for infertility treatment in recent years. However, the cellular origin of SDSCs and their dynamic changes in transcription profile during differentiation into PGCLCs in vitro remain largely undissected. Here, the results of single-cell RNA sequencing indicated that porcine SDSCs are mainly derived from multipotent dermal fibroblast progenitors (MDFPs), which are regulated by growth factors (EGF/bFGF). Importantly, porcine SDSCs exhibit pluripotency for differentiating into three germ layers and can effectively differentiate into PGCLCs through complex transcriptional regulation involving histone modification. Moreover, this study also highlights that porcine SDSC-derived PGCLCs specification exhibit conservation with the human primordial germ cells lineage and that its proliferation is mediated by the MAPK signaling pathway. Our findings provide substantial novel insights into the field of regenerative medicine in which stem cells differentiate into germ cells in vitro, as well as potential therapeutic effects in individuals with azoospermia and/or defective oocytes.
... Although such genes are influenced by HAND2 function, they are likely not transcriptionally regulated by HAND2 directly; nevertheless, their altered expression fits with HAND2 function in previous studies. In epicardial Hand2 deletion, although Fn1 expression is unaltered when comparing control with mutants, FN1 organization is altered within H2CKO epicardial cells (Barnes et al., 2011). During jaw morphogenesis, Hand2 has been established as lying downstream of EDN1 signaling and plays an important negative feedback role once activated, by repressing Dlx5 and Dlx6 expression within the ventral-most portion of the mandible mesoderm (Barron et al., 2011;Charité et al., 2001;Clouthier et al., 2000;Vincentz et al., 2016). ...
The transcription factor HAND2 plays critical roles during cardiogenesis. Hand2 endocardial deletion (H2CKO) results in tricuspid atresia or double inlet left ventricle with accompanying intraventricular septum defects, hypo-trabeculated ventricles, and an increased density of coronary lumens. To understand the regulatory mechanisms of these phenotypes, single cell transcriptome analysis of E11.5 H2CKO hearts was performed revealing a number of disrupted endocardial regulatory pathways. Utilizing HAND2 DNA occupancy data, we identify several HAND2-dependent enhancers, including two endothelial enhancers for the shear-stress master regulator, KLF2. A 1.8kb enhancer located 50kb upstream of the Klf2 TSS imparts specific endothelial/endocardial expression within the vasculature and endocardium. This enhancer is HAND2-dependent for ventricular endocardium expression but HAND2-independent for Klf2 vascular and valve expression. Deletion of this Klf2 enhancer results in reduced Klf2 expression within ventricular endocardium. These data reveal that HAND2 functions within endocardial gene regulatory networks including shear-stress response.
... Although such genes are influenced by HAND2 function, they are likely not transcriptionally regulated by HAND2 directly; nevertheless, their altered expression fits with HAND2 function in previous studies. In epicardial Hand2 deletion, although Fn1 expression is unaltered when comparing control with mutants, FN1 organization is altered within H2CKO epicardial cells (Barnes et al., 2011). During jaw morphogenesis, Hand2 has been established as lying downstream of EDN1 signaling and plays an important negative feedback role once activated, by repressing Dlx5 and Dlx6 expression within the ventral-most portion of the mandible mesoderm (Barron et al., 2011;Charité et al., 2001;Clouthier et al., 2000;Vincentz et al., 2016). ...
The transcription factor HAND2 plays critical roles during cardiogenesis. Hand2 endocardial deletion (H2CKO) results in tricuspid atresia or double inlet left ventricle with accompanying intraventricular septum defects, hypo-trabeculated ventricles, and an increased density of coronary lumens. To understand the regulatory mechanisms of these phenotypes, single cell transcriptome analysis of E11.5 H2CKO hearts was performed revealing a number of disrupted endocardial regulatory pathways. Utilizing HAND2 DNA occupancy data, we identify several HAND2-dependent enhancers, including two endothelial enhancers for the sheer-stress master regulator, KLF2. A 1.8kb enhancer located 50kb upstream of the Klf2 transcriptional start site imparts specific endothelial/endocardial expression within the vasculature and endocardium. This enhancer is HAND2-dependent for ventricular endocardium expression but HAND2-independent for Klf2 vascular and valve expression. Deletion of this Klf2 enhancer reveals reduced Klf2 expression within ventricular endocardium. These data reveal that HAND2 functions within endocardial gene regulatory networks including sheer stress response.
... Consistent with these findings, we observe TCF21 to be an early to mid-regulator of the cardiac fibroblast trajectory. We also observe molecular signatures of activity for MEF2C (Desjardins and Naya 2016) in ventricular cardiomyocytes, GATA4 in atrial cardiomyocytes (Misra et al. 2012) and HAND2 in vascular smooth muscle cells (Barnes et al. 2011). Apart from revealing the precise timing of activity at cREs for these known master regulators, we also nominate putative novel regulatory TFs. ...
Congenital heart defects, the most common birth disorders, are the clinical manifestation of anomalies in fetal heart development - a complex process involving dynamic spatiotemporal coordination among various precursor cell lineages. This complexity underlies the incomplete understanding of the genetic architecture of congenital heart diseases (CHDs). To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We identified similarities and differences of regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts. We interpreted deep learning models that predict cell-type resolved, base-resolution chromatin accessibility profiles from DNA sequence to decipher underlying TF motif lexicons and infer the regulatory impact of non-coding variants. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in CHD cases versus controls. We used CRISPR-based perturbations to validate an enhancer harboring a nominated regulatory CHD mutation, linking it to effects on the expression of a known CHD gene JARID2 . Together, this work defines the cell-type resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements as a component of the genetic etiology of CHD.
... The other three clusters were less relevant and were thus excluded from the subsequent analyses. One of these clusters was a Gata4-expressing population, which likely involved cardiac (Hand2 + ) and liver (Oit3 + ) VECs, 13,17,38 presumably due to accidental incorporation during the dissection of embryos, as the heart and visceral bud should be prospectively excluded before sampling (Supplementary information, Fig. S3d-g). The features of the five major clusters corresponded closely with the clusters identified by the well-based scRNA-seq, among which Vwa1 + VEC population was transcriptomically similar to EP7, with both expressing a set of arterial genes along with several specific genes, such as Vwa1 and Nid2 ( Fig. 1e; Supplementary information, Fig. S3e, h). ...
Arteriogenesis rather than unspecialized capillary expansion is critical for restoring effective circulation to compromised tissues in patients. Deciphering the origin and specification of arterial endothelial cells during embryonic development will shed light on the understanding of adult arteriogenesis. However, during early embryonic angiogenesis, the process of endothelial diversification and molecular events underlying arteriovenous fate settling remain largely unresolved in mammals. Here, we constructed the single-cell transcriptomic landscape of vascular endothelial cells (VECs) during the time window for the occurrence of key vasculogenic and angiogenic events in both mouse and human embryos. We uncovered two distinct arterial VEC types, the major artery VECs and arterial plexus VECs, and unexpectedly divergent arteriovenous characteristics among VECs that are located in morphologically undistinguishable vascular plexus intra-embryonically. Using computational prediction and further lineage tracing of venous-featured VECs with a newly developed Nr2f2 CrexER mouse model and a dual recombinase-mediated intersectional genetic approach, we revealed early and widespread arterialization from the capillaries with considerable venous characteristics. Altogether, our findings provide unprecedented and comprehensive details of endothelial heterogeneity and lineage relationships at early angiogenesis stages, and establish a new model regarding the arteriogenesis behaviors of early intra-embryonic vasculatures.
... Our finding that expression of the transcription factor Hand2 demarcates the merging mesothelial progenitors within the post-gastrulation LPM provides new means to study the mesothelium in development and disease. Hand gene function centrally contributes to cardiac, limb, and pharyngeal arch development across several models (Barnes et al., 2011;Firulli et al., 1998;Han and Olson, 2005;Shin et al., 2009;Yelon et al., 2000). Our results indicate that Hand genes also act as conserved regulators of mesothelial fates. ...
The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration, and their dysregulation can result in congenital anomalies of the viscera, ventral wall defects, and mesothelioma tumors. Nonetheless, the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here, we combine genetic lineage tracing, in toto live imaging, and single-cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm (LPM). Our single-cell analysis uncovers a post-gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos, we chart the origin of mesothelial progenitors to the lateral-most, hand2 -expressing LPM and confirm evolutionary conservation in mouse. Our time-lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation, documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally, hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium, we find de novo expression of LPM-associated transcription factors, and in particular of Hand2, indicating the re-initiation of a developmental transcriptional program in mesothelioma. Taken together, our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2, contributing to our understanding of mesothelial pathologies and mesothelioma.
... A cell-autonomous role in formation and modification of the surrounding ECM landscape, including fibronectin fibrils, drives autocrine regulation of epicardial EMT (epiEMT) and, moreover, is postulated to signal myocardial growth and compaction, and to provide a foundation for coronary sprouting from the SV. 49 The importance of these features will be described in the next section. ...
Research into potential targets for cardiac repair encompasses recognition of tissue‐resident cells with intrinsic regenerative properties. The adult vertebrate heart is covered by mesothelium, named the epicardium, which becomes active in response to injury and contributes to repair, albeit suboptimally. Motivation to manipulate the epicardium for treatment of myocardial infarction is deeply rooted in its central role in cardiac formation and vasculogenesis during development. Moreover, the epicardium is vital to cardiac muscle regeneration in lower vertebrate and neonatal mammalian‐injured hearts. In this review, we discuss our current understanding of the biology of the mammalian epicardium in development and injury. Considering present challenges in the field, we further contemplate prospects for reinstating full embryonic potential in the adult epicardium to facilitate cardiac regeneration.
... This method consistently yields cardiac organoids comprised of approximately 59% cardiomyocytes, 16% epicardial cells, 14% endocardial cells, 12% cardiac fibroblasts, and 1.6% endothelial cells and shows robust beating throughout the entire structure within a week from differentiation initiation and up to at least 8 weeks in culture (longer culture times were not attempted). The organization and specification of these cell types may be related to HAND transcription factor expression, as HAND1 and HAND2 lineage-derived cells contribute to the developing myocardium, epicardium, endocardium, and vasculature 37,[64][65][66] . The fact that both FHF and SHF HAND markers are present suggests that they could play a role in the development of the structural and cell type complexity seen in our hHOs. ...
... These results suggest that Hand2 and Gata6 may transcriptionally reprogram MEFs toward an epicardial-like state. Consistent with these findings, mouse genetic studies have previously shown that Gata6 and Hand2 function in epicardial development and maintenance (Barnes et al., 2011;Ko-lander et al., 2014). ...
Ectopic expression of transcription factors (TFs) can reprogram cell state. However, because of the large combinatorial space of possible TF cocktails, it remains difficult to identify TFs that reprogram specific cell types. Here, we develop Reprogram-Seq to experimentally screen thousands of TF cocktails for reprogramming performance. Reprogram-Seq leverages organ-specific cell-atlas data with single-cell perturbation and computational analysis to predict, evaluate, and optimize TF combinations that reprogram a cell type of interest. Focusing on the cardiac system, we perform Reprogram-Seq on MEFs using an undirected library of 48 cardiac factors and, separately, a directed library of 10 epicardial-related TFs. We identify a combination of three TFs, which efficiently reprogram MEFs to epicardial-like cells that are transcriptionally, molecularly, morphologically, and functionally similar to primary epicardial cells. Reprogram-Seq holds promise to accelerate the generation of specific cell types for regenerative medicine. : Direct reprogramming of a cellular state holds promise for regenerative medicine. Duan et al. present Reprogram-Seq to identify, evaluate, and optimize transcription factor cocktails that drive direct reprogramming of a cell state. They apply Reprogram-Seq to generate epicardial-like cells and show how the approach can be leveraged for rational cellular reprogramming. Keywords: cellular reprogramming, single-cell RNA-Seq, single-cell perturbation, transcription factor, cardiac
... We detected three subpopulations of the endocardial/endothelial lineage: hematoendothelial precursors, specified endothelial/endocardial cells and endocardial cells undergoing an endothelial-to-mesenchymal transition typical of valve development (Fig. 1d). We also identified non-cardiomyocyte cell lineages that contribute to cardiac morphogenesis, including epicardial cells and septum transversum cells, from which epicardial cells are derived (Fig. 1b, d) 13 . Intriguingly, this analysis revealed a heterogeneous population (labelled as E7.75 myocardium), comprising cells largely captured at E7.75 that expressed the Nodal co-receptor Tdgf1 14 Table 2). ...
Organogenesis involves integration of myriad cell types with reciprocal interactions, each progressing through successive stages of lineage specification and differentiation. Establishment of unique gene networks within each cell dictates fate determination, and mutations of transcription factors that drive such networks can result in birth defects. Congenital heart defects are the most common malformations and are caused by disruption of discrete subsets of progenitors, however, determining the transcriptional changes in individual cells that lead to organ-level defects in the heart, or other organs, has not been tractable. Here, we employed single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular sub-populations can have catastrophic consequences. A network-based computational method for single-cell RNA-sequencing that predicts lineage specifying transcription factors identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite failure of right ventricular formation in Hand2-null mice. Temporal single-cell transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium differentiated but failed to migrate into the anterior pole of the developing heart. Dysregulation of retinoic acid signaling, responsible for anterior-posterior patterning, was associated with posteriorization of anterior cardiac progenitors in Hand2-null mutant hearts and ectopic atrial gene expression in outflow tract and right ventricle precursors. This work reveals transcriptional determinants in individual cells that specify cardiac progenitor cell fate and differentiation and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework to investigate congenital heart defects.
... As we observe a thin walled myocardium in the Nkx2.5 Cre ; Hand1 A126FS/þ mutants, we also looked at endocardial and epicardial gene expression which can influence myocardial growth [47][48][49] and production of cardiac myofibroblasts and coronary vasculature. [50][51][52][53] Expression analysis of the endocardial markers Hand2, Flt1, Tie2, Nrg1, Nrp1, VegfR2, and NfactC1 and epicardial markers WT1 and Tcf21 by QPCR from E10.5 RNA control and mutant hearts revealed no observable changes in expression (see Supplementary material online, Figure S1). ...
Aims:
To test if a human Hand1 frame shift mutation identified in human samples is causative of hypoplastic left heart syndrome (HLHS).
Methods and results:
HLHS is a poorly understood single ventricle congenital heart defect that affects two to three infants in every 10 000 live births. The aetiologies of HLHS are largely unknown. The basic helix-loop-helix transcription factor HAND1 is required for normal heart development. Interrogation of HAND1 sequence from fixed HLHS tissues identified a somatic frame-shift mutation at Alanine 126 (NP_004812.1 p.Ala126Profs13X defined as Hand1A126fs). Hand1A126fs creates a truncated HAND1 protein that predictively functions as dominant negative. To determine if this mutation is causative of HLHS, we engineered a conditional Hand1A126fs mouse allele. Activation of this allele with Nkx2.5Cre results in E14.5 lethality accompanied by cardiac outflow tract and intraventricular septum abnormalities. Using αMHC-Cre or Mef2CAHF-Cre to activate Hand1A126fs results in reduced phenotype and limited viability. Left ventricles of Hand1A126FS mutant mice are not hypoplastic.
Conclusions:
Somatically acquired Hand1A126FS mutation is not causative of HLHS. Hand1A126FS mutation does exhibit embryonic lethal cardiac defects that reflect a dominant negative function supporting the critical role of Hand1 in cardiogenesis.
... H1 dimer mutants display noncell-autonomous craniofacial phenotypes outside of the distal cap (6). The H1 Cre knock-in allele provides a tool with which to interrogate cellautonomous roles of Hand factors within the MD1 (7,8). To examine the development of the H1 lineage relative to the developing incisors, we performed X-Gal staining of R26R lacZ/+ ;H1 Cre/+ embryos at E15.5. ...
Cranial neural crest cells (crNCCs) migrate from the neural tube to the pharyngeal arches (PAs) of the developing embryo and, subsequently , differentiate into bone and connective tissue to form the mandible. Within the PAs, crNCCs respond to local signaling cues to partition into the proximo-distally oriented subdomains that convey positional information to these developing tissues. Here, we show that the distal-most of these subdomains, the distal cap, is marked by expression of the transcription factor Hand1 (H1) and gives rise to the ectomesenchymal derivatives of the lower incisors. We uncover a H1 enhancer sufficient to drive reporter gene expression within the crNCCs of the distal cap. We show that bone morphogenic protein (BMP) signaling and the transcription factor HAND2 (H2) syner-gistically regulate H1 distal cap expression. Furthermore, the homeodomain proteins distal-less homeobox 5 (DLX5) and DLX6 reciprocally inhibit BMP/H2-mediated H1 enhancer regulation. These findings provide insights into how multiple signaling pathways direct transcriptional outcomes that pattern the developing jaw. Bmp | DLX | HAND1 | cranial neural crest cells | development A fter migrating to specific locations within the developing embryo, neural crest cells (NCCs), a multipotent cell population originating from the dorsal lip of the neural tube, respond to local morphogenetic signaling cues to pattern and differentiate (1). Following migration to the pharyngeal arches (PAs), cranial NCCs (crNCCs) respond to endothelin 1 (EDN1) and bone mor-phogenic protein (BMP) signaling from surrounding pharyngeal epithelia to subdivide the PA ectomesenchyme into discrete proximo-distal domains (2). In the mandibular arch (MD1), these nested PA subdomains, characterized by the expression of DLX homeobox and/or HAND basic helix–loop–helix (bHLH) transcription factors, are integral to the development of specific jaw structures, including bone, tongue mesenchyme, and heterogeneous teeth (3). HAND factors regulate mandibular incisor development (4), whereas DLX proteins influence maxillary molar development (2). The mechanisms by which DLX-and HAND-dependent transcriptional programs establish proximo-distal PA subdomains are poorly understood. Indeed, the dearth of identified cis-regulatory elements active in postmigratory NCCs has hampered the elucidation of the gene regulatory networks that establish regional identity within the developing mandible. BMP and EDN1 signaling initially overlaps within the distal murine MD1, but by embryonic day (E)10.5, these signaling pathways become spatially segregated. The distal-most tip of the PAs, known as the distal cap, is transiently devoid of active EDN1 sig-naling. BMP signaling is ostensibly restricted to this distal cap by the localized expression of Bmp antagonists (3). Here, we provide evidence that DLX5 and DLX6 act as transcriptional repressors of BMP signaling specifically within the cranial PAs. We show that, within the crNCCs, the BMP-dependent transcription factors Smads, H2, and GATA2/3 provide positive transcriptional inputs that serve to counteract the activity of Dlx proteins, thereby relieving EDN1-meditated repression. Together, these findings integrate the communication between BMP and EDN1 signaling that establishes the distal cap of the mandible. Results The H1 Cre Lineage Gives Rise to the Lower Incisors, in a HAND Factor-Dependent Manner. H1 expression is confined to the distal cap ecto-mesenchyme (4, 5). H1 dimer mutants display noncell-autonomous craniofacial phenotypes outside of the distal cap (6). The H1 Cre knock-in allele provides a tool with which to interrogate cell-autonomous roles of Hand factors within the MD1 (7, 8). To examine the development of the H1 lineage relative to the developing incisors, we performed X-Gal staining of R26R lacZ/+ ;H1 Cre/+ embryos at E15.5. Consistent with H1 lacZ expression, the H1 lineage is restricted to the midline of the tongue and Meckel's cartilage within the developing jaw; however, the dental papilla (dp) of the man-dibular incisors, which exhibits undetectable H1 lacZ expression (4) (Fig. S1A) is derived from H1 Cre-marked cells (Fig. S1B). These findings suggest that Hand factors function cell-autonomously during early mandibular incisor development. Significance Within the developing mandible, proper specification and positioning of bone, tongue, and teeth are controlled by secreted morphogens. Among these morphogenetic cues, endothelin 1 (EDN1) and bone morphogenic proteins (BMPs) divide the nascent mandible into subdomains along a proximo-distal axis. The transcriptional mechanisms by which mandibular progenitor cells interpret morphogenetic signals to establish these subdomains are poorly understood. Here, we characterize a Hand1 enhancer that drives gene expression specifically within the distal-most of these subdomains, the distal cap. Our findings show that Bmp-dependent transcription factors provide positive transcriptional inputs that serve to counteract the repressive activity of EDN1-dependent transcription factors within the distal cap, thus integrating the communication between BMP and EDN1 signaling that patterns the mandible.
... H1 dimer mutants display noncell-autonomous craniofacial phenotypes outside of the distal cap (6). The H1 Cre knock-in allele provides a tool with which to interrogate cellautonomous roles of Hand factors within the MD1 (7,8). To examine the development of the H1 lineage relative to the developing incisors, we performed X-Gal staining of R26R lacZ/+ ;H1 Cre/+ embryos at E15.5. ...
Significance
Within the developing mandible, proper specification and positioning of bone, tongue, and teeth are controlled by secreted morphogens. Among these morphogenetic cues, endothelin 1 (EDN1) and bone morphogenic proteins (BMPs) divide the nascent mandible into subdomains along a proximo-distal axis. The transcriptional mechanisms by which mandibular progenitor cells interpret morphogenetic signals to establish these subdomains are poorly understood. Here, we characterize a Hand1 enhancer that drives gene expression specifically within the distal-most of these subdomains, the distal cap. Our findings show that Bmp-dependent transcription factors provide positive transcriptional inputs that serve to counteract the repressive activity of EDN1-dependent transcription factors within the distal cap, thus integrating the communication between BMP and EDN1 signaling that patterns the mandible.
... The STM expresses Foxf1, Gata4, Lhx2, Hand1, Hlx, Mab21l2, Tbx18, Zfpm2, and Wt1. 28,[36][37][38][53][54][55][56][57] Because the STM is the source of epicardium in the heart, of the diaphragm, and of MCs in the liver, deletion of the Mab21l2, Tbx18, and Zfpm2 gene resulted in abnormal development of the heart, diaphragm, and liver, respectively. [55][56][57] Gata4-null mouse embryos showed heart defects due to underdevelopment of the STM. ...
Mesothelial cells (MCs) cover the surface of visceral organs and the parietal walls of cavities, and they synthesize lubricating fluids to create a slippery surface that facilitates movement between organs without friction. Recent studies have indicated that MCs play active roles in liver development, fibrosis, and regeneration. During liver development, the mesoderm produces MCs that form a single epithelial layer of the mesothelium. MCs exhibit an intermediate phenotype between epithelial cells and mesenchymal cells. Lineage tracing studies have indicated that during liver development, MCs act as mesenchymal progenitor cells that produce hepatic stellate cells, fibroblasts around blood vessels, and smooth muscle cells. Upon liver injury, MCs migrate inward from the liver surface and produce hepatic stellate cells or myofibroblast depending on the etiology, suggesting that MCs are the source of myofibroblasts in capsular fibrosis. Similar to the activation of hepatic stellate cells, transforming growth factor β induces the conversion of MCs into myofibroblasts. Further elucidation of the biological and molecular changes involved in MC activation and fibrogenesis will contribute to the development of novel approaches for the prevention and therapy of liver fibrosis.
... This impairment was subsequently rescued with the over-expression of Sox9, providing evidence that Sox9 is also important for epicardial EMT [58]. Similarly, HAND2 induces the expression of Pdgfrα, and loss of Hand2 results in a loss of epicardial integrity and impairment of epicardial EMT [59]. ...
The epicardium is an epithelial monolayer that plays a central role in heart development and the myocardial response to injury. Recent developments in our understanding of epicardial cell biology have revealed this layer to be a dynamic participant in fundamental processes underlying the development of the embryonic ventricles, the coronary vasculature, and the cardiac valves. Likewise, recent data have identified the epicardium as an important contributor to reparative and regenerative processes in the injured myocardium. These essential functions of the epicardium rely on both non-cell autonomous and cell-autonomous mechanisms, with the latter featuring the process of epicardial Epithelial-to-Mesenchymal Transition (EMT). This review will focus on the induction and regulation of epicardial EMT, as it pertains to both cardiogenesis and the response of the myocardium to injury.
... Finally, mechanical tension can also disrupt β-catenin localization in the adherens junctions to prime the cell for further stimulation or cause calcium influx which activates calcineurin/NFAT signaling. known as Capsulin or POD1), HAND2, and Myocardin-Related Transcription Factors (MRTFs) which influence EMT and epicardial-derived progenitor cell (EPDC) differentiation [51][52][53][54][55][56][57]. This gene signature is silenced after birth, but is reactivated by disease or injury, potentially mobilizing EPDCs and leading to the generation of nascent fibroblasts. ...
Cardiac fibroblasts help maintain the normal architecture of the healthy heart and are responsible for scar formation and the healing response to pathological insults. Various genetic, biomechanical, or humoral factors stimulate fibroblasts to become contractile smooth muscle-like cells called myofibroblasts that secrete large amounts of extracellular matrix. Unfortunately, unchecked myofibroblast activation in heart disease leads to pathological fibrosis, which is a major risk factor for the development of cardiac arrhythmias and heart failure. A better understanding of the molecular mechanisms that control fibroblast plasticity and myofibroblast activation is essential to develop novel strategies to specifically target pathological cardiac fibrosis without disrupting the adaptive healing response. This review highlights the major transcriptional mediators of fibroblast origin and function in development and disease. The contribution of the fetal epicardial gene program will be discussed in the context of fibroblast origin in development and following injury, primarily focusing on Tcf21 and C/EBP. We will also highlight the major transcriptional regulatory axes that control fibroblast plasticity in the adult heart, including transforming growth factor β (TGFβ)/Smad signaling, the Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF) axis, and Calcineurin/transient receptor potential channel (TRP)/nuclear factor of activated T-Cell (NFAT) signaling. Finally, we will discuss recent strategies to divert the fibroblast transcriptional program in an effort to promote cardiomyocyte regeneration. This article is a part of a Special Issue entitled "Fibrosis and Myocardial Remodeling".
Background
The bHLH transcription factor HAND2 plays important roles in the development of the embryonic heart, face, limbs, and sympathetic and enteric nervous systems. To define how and when HAND2 regulates these developmental systems, requires understanding the transcriptional regulation of Hand2.
Results
Remarkably, Hand2 is flanked by an extensive upstream gene desert containing a potentially diverse enhancer landscape. Here, we screened the regulatory interval 200 kb proximal to Hand2 for putative enhancers using evolutionary conservation and histone marks in Hand2‐expressing tissues. H3K27ac signatures across embryonic tissues pointed to only two putative enhancer regions showing deep sequence conservation. Assessment of the transcriptional enhancer potential of these elements using transgenic reporter lines uncovered distinct in vivo enhancer activities in embryonic stomach and limb mesenchyme, respectively. Activity of the identified stomach enhancer was restricted to the developing antrum and showed expression within the smooth muscle and enteric neurons. Surprisingly, the activity pattern of the limb enhancer did not overlap Hand2 mRNA but consistently yielded a defined subectodermal anterior expression pattern within multiple transgenic lines.
Conclusions
Together, these results start to uncover the diverse regulatory potential inherent to the Hand2 upstream regulatory interval.
Pleiotropic genetic factors (e.g., DNA polymorphisms) may be involved in the initiation of neuroblastoma (NB) and coronary artery disease (CAD) given their common origin from defects in neural crest development.To discover novel NB susceptibility genes, we conducted a three-stage survey including a meta-analysis of NB and CAD genome-wide association data, prioritization of NB causal variants, and validation in an independent case-controls cohort.The lead SNP, rs13337397 at the 16q23.1 locus, associated with both diseases in the meta-analysis and with NB in the validation study. All the SNPs in linkage disequilibrium with rs13337397 were annotated using the H3K27ac epigenetic marker of neural crest cells (NCC) and NB cell lines. Indeed, we identified the functional SNP rs13337017, mapping within an enhancer of NCCs and NB cell lines and showing long-range interactions with CFDP1 by Hi-C analysis. Luciferase assays indicated that the risk allele of rs13337017 increased CFDP1 expression in NB cell lines. Of note, CFDP1 high expression associated with unfavorable prognostic markers in an analysis including 498 NB transcriptomes. Moreover, depletion of CFDP1 markedly decreased viability and migration and increased apoptotic rates in NB cell lines. Finally, transcriptome and qPCR analyses revealed that the depletion of CFDP1 may affect noradrenergic neuron differentiation by downregulating master regulators of sympathetic noradrenergic identity, including PHOX2B, HAND2 and GATA3.Our data strongly suggest that CFDP1 acts as oncogene in NB. Additionally, we provide evidence that genetic predisposition to NB can be mediated by the alteration of noradrenergic lineage-specific gene expression.
We investigated possible cardioprotective mechanisms of l-arginine coated nanoparticles (L-ACN) combined with swimming exercise (SE) in aging male rats considering heart and neural crest derivatives-expressed protein 2 (HAND2) and t-box transcription factor 5 (TBX5). Thirty-five male Wistar rats were randomly assigned into five groups: young, old, old + L-ACN, old + SE, and old + L-ACN + SE (n = 7 in each). l-arginine coated with chitosan nanoparticles was given to L-ACN groups via gavage at 500 mg/kg/day. SE groups performed a swimming exercise program 5 days per week for 6 weeks. The exercise program started with 20 min, gradually increasing to 60 min after four sessions, which was then constant until the completion of the training period. After the protocol completion, the rats were sacrificed, and the heart was fixed and frozen to carry out histological, immunohistochemistry (IHC), and gene expression analyses. The expression of HAND2 protein, HAND2 mRNA, and TBX5 mRNA of the heart tissue was significantly higher in the young group than in all older groups (P < 0.05). The old + L-ACN, old + SE, and old + L-ACN + SE groups showed a significant increase in these factors compared to the old group (P < 0.05). Nano-l-arginine supplement, along with swimming exercises, seems to have cardioprotective potential and improve cardiac function in old age by strengthening cardiomyocyte signaling, especially HAND2 and TBX5. However, more research is required, particularly on human samples.
The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies. The mesothelium supports homeostasis and regeneration, yet its development origins remain unclear. Here, the authors uncovered the earliest mesothelium progenitor cells in zebrafish, linking Hand2 gene function to mesothelium formation and its re-activation to mesothelioma tumors.
Congenital heart defects constitute the most common human birth defect, however understanding of how these disorders originate is limited by our ability to model the human heart accurately in vitro. Here we report a method to generate developmentally relevant human heart organoids by self-assembly using human pluripotent stem cells. Our procedure is fully defined, efficient, reproducible, and compatible with high-content approaches. Organoids are generated through a three-step Wnt signaling modulation strategy using chemical inhibitors and growth factors. Heart organoids are comparable to age-matched human fetal cardiac tissues at the transcriptomic, structural, and cellular level. They develop sophisticated internal chambers with well-organized multi-lineage cardiac cell types, recapitulate heart field formation and atrioventricular specification, develop a complex vasculature, and exhibit robust functional activity. We also show that our organoid platform can recreate complex metabolic disorders associated with congenital heart defects, as demonstrated by an in vitro model of pregestational diabetes-induced congenital heart defects.
Congenital heart defects (CHDs) affecting the cardiac outflow tract (OFT) constitute a significant cause of morbidity and mortality. The OFT develops from migratory cell populations which include the cardiac neural crest cells (cNCCs) and secondary heart field (SHF) derived myocardium and endocardium. The related transcription factors HAND1 and HAND2 have been implicated in human CHDs involving the OFT. Although Hand1 is expressed within the OFT, Hand1 NCC-specific conditional knockout mice (H1CKOs) are viable. Here we show that these H1CKOs present a low penetrance of OFT phenotypes, whereas SHF-specific Hand1 ablation does not reveal any cardiac phenotypes. Further, HAND1 and HAND2 appear functionally redundant within the cNCCs, as a reduction/ablation of Hand2 on an NCC-specific H1CKO background causes pronounced OFT defects. Double conditional Hand1 and Hand2 NCC knockouts exhibit persistent truncus arteriosus (PTA) with 100% penetrance. NCC lineage-tracing and Sema3c in situ mRNA expression reveal that Sema3c-expressing cells are mis-localized, resulting in a malformed septal bridge within the OFTs of H1CKO;H2CKO embryos. Interestingly, Hand1 and Hand2 also genetically interact within the SHF, as SHF H1CKOs on a heterozygous Hand2 background exhibit Ventricular Septal Defects (VSDs) with incomplete penetrance. Previously, we identified a BMP, HAND2, and GATA-dependent Hand1 OFT enhancer sufficient to drive reporter gene expression within the nascent OFT and aorta. Using these transcription inputs as a probe, we identify a novel Hand2 OFT enhancer, suggesting that a conserved BMP-GATA dependent mechanism transcriptionally regulates both HAND factors. These findings support the hypothesis that HAND factors interpret BMP signaling within the cNCCs to cooperatively coordinate OFT morphogenesis.
Congenital heart defects (CHD) constitute the most common birth defect in humans, affecting approximately 1% of all live births. Our ability to understand how these disorders originate is hindered by our limited ability to model the complexity of the human heart in vitro. There is a pressing need to develop more faithful organ-like platforms recapitulating complex in vivo phenotypes to study human development and disease in vitro. Here we report a novel method to generate human heart organoids by self-assembly using pluripotent stem cells. Our method is fully defined, highly efficient, scalable, shows high reproducibility and is compatible with screening and high-throughput approaches. Human heart organoids (hHOs) are generated using a two-step canonical Wnt signaling modulation strategy using a combination of chemical inhibitors and growth factors in completely defined culture conditions. hHOs faithfully recapitulate human cardiac development and are similar to age-matched fetal cardiac tissues at the transcriptomic, structural and cellular level. hHOs develop sophisticated internal chambers with well-organized multi-lineage cell-type regional identities reminiscent of the heart fields and the atrial and ventricular chambers, as well as the epicardium, endocardium, and coronary vasculature, and exhibit functional activity. We also show that hHOs can recreate complex metabolic disorders associated with CHD by establishing the first in vitro human model of diabetes during pregnancy (DDP) to study embryonic CHD. morphological and metabolically effects of increased glucose and insulin, showing the capability of modeling the effects of diabetes during pregnancy (DDP). Our heart organoid model constitutes a powerful novel tool for translational studies in human cardiac development and disease.
In Brief A comprehensive atlas of mouse liver emergence is described at single-cell resolution starting at endoderm progenitor specification, including data detailing divergence of vascular and sinusoidal endothelia, hepatoblast specification, and the emergence of a distinct, migratory hepatomesenchymal cell type. Single-cell analysis of endoderm and early liver cell-cell interactions E7.5 E8.75 E9.5 E10.5 endothelium mesenchyme hepatoblasts 45,334 cells in vivo validation trajectory analyses emergence of HSECs and mesenchyme hepatomesenchymal hybrid cell type sinusoidal signaling niche mesenchymal hepatic SUMMARY The cellular complexity and scale of the early liver have constrained analyses examining its emergence during organogenesis. To circumvent these issues, we analyzed 45,334 single-cell transcriptomes from embryonic day (E)7.5, when endoderm progenitors are specified, to E10.5 liver, when liver parenchymal and non-paren-chymal cell lineages emerge. Our data detail divergence of vascular and sinusoidal endothelia, including a distinct transcriptional profile for sinusoidal endothelial specification by E8.75. We characterize two distinct mesothelial cell types as well as early hepatic stellate cells and reveal distinct spatiotemporal distributions for these populations. We capture transcriptional profiles for hepatoblast specification and migration, including the emergence of a hepatomesenchymal cell type and evidence for hepatoblast collective cell migration. Further, we identify cell-cell interactions during the organization of the primitive sinusoid. This study provides a comprehensive atlas of liver lineage establishment from the endoderm and mesoderm through to the organization of the primitive sinusoid at single-cell resolution.
The embryonic epicardium, originating from the proepicardial organ (PEO), provides a source of multipotent progenitors for cardiac lineages, including pericytes, fibroblasts, and vascular smooth muscle cells. Maximizing the regenerative capacity of the adult epicardium depends on recapitulating embryonic cell fates. The potential of the epicardium to contribute coronary endothelium is unclear, due to conflicting Cre-based lineage trace data. Controversy also surrounds when epicardial cell fate becomes restricted. Here, we systematically investigate expression of five widely used epicardial markers, Wt1, Tcf21, Tbx18, Sema3d, and Scx, over the course of development. We show overlap of markers in all PEO and epicardial cells until E13.5, and find no evidence for discrete proepicardial sub-compartments that might contribute coronary endothelium via the epicardial layer. Our findings clarify a number of prevailing discrepancies and support the notion that epicardium-derived cell fate, to form fibroblasts or mural cells, is specified after epithelial-mesenchymal transition, not pre-determined within the PEO.
Cardiac fibroblasts and fibrosis contribute to the pathogenesis of heart failure, a prevalent cause of mortality. Therefore, a majority of the existing information regarding cardiac fibroblasts is focused on their function and behavior after heart injury. Less is understood about the signaling and transcriptional networks required for the development and homeostatic roles of these cells. This review is devoted to describing our current understanding of cardiac fibroblast development. I detail cardiac fibroblast formation during embryogenesis including the discovery of a second embryonic origin for cardiac fibroblasts. Additional information is provided regarding the roles of the genes essential for cardiac fibroblast development. It should be noted that many questions remain regarding the cell-fate specification of these fibroblast progenitors, and it is hoped that this review will provide a basis for future studies regarding this topic.
Ectopic expression of transcription factors (TFs) can reprogram cell state. However, due to the large combinatorial space of possible TF cocktails, it remains difficult to identify TFs that reprogram specific cell types. Here, we develop Reprogram-Seq to experimentally screen thousands of TF cocktails for reprogramming performance. Reprogram-Seq leverages organ-specific cell atlas data with single-cell perturbation and computational analysis to predict, evaluate, and optimize TF combinations that reprogram a cell type of interest. Focusing on the cardiac system, we perform Reprogram-Seq on MEFs using an undirected library of 48 cardiac factors and separately on a directed library of 10 epicardial-related TFs. We identify a novel combination of 3 TFs which efficiently reprogram MEFs to epicardial-like cells that are transcriptionally, molecularly, morphologically, and functionally similar to primary epicardial cells. Reprogram-Seq holds promise to accelerate the generation of specific cell types for regenerative medicine.
Document S1. Supplemental Experimental Procedures, Figures S1–S7, and Tables S1, S2, and S8
The mammalian post-implantation embryo has been extensively investigated at the tissue level. However, to unravel the molecular basis for the cell-fate plasticity and determination, it is essential to study the characteristics of individual cells. Especially, the individual definitive endoderm (DE) cells have not been characterized in vivo. Here, we report gene expression patterns in single cells freshly isolated from mouse embryos on days 5.5 and 6.5. Initial transcriptome data from 124 single cells yielded signature genes for the epiblast, visceral endoderm, and extra-embryonic ectoderm and revealed a unique distribution pattern of fibroblast growth factor (Fgf) ligands and receptors. Further analysis indicated that early-stage epiblast cells do not segregate into lineages of the major germ layers. Instead, some cells began to diverge from epiblast cells, displaying molecular features of the pre-mesendoderm by expressing higher levels of mesendoderm markers and lower levels of Sox3 transcripts. Analysis of single-cell high-throughput quantitative RT-PCR data from 441 cells identified a late stage of the day 6.5 embryo in which mesoderm and DE cells emerge, with many of them coexpressing Oct4 and Gata6. Analysis of single-cell RNA-seq data from 112 cells of the late-stage day 6.5 embryos revealed differentially expressed signaling genes and networks of transcription factors that might underlie the segregation of the mesoderm and DE lineages. Moreover, we discovered a subpopulation of mesoderm cells that possess molecular features of the extraembryonic mesoderm. This study provides fundamental insight into the molecular basis for lineage segregation in post-implantation mouse embryos.
In vertebrate embryonic development, the heart is the first organ to shape and start functioning. In higher developed vertebrates, it is organized by four chambers (two atria, two ventricles) that have distinct differentiation during embryonic development and either derives from the first heart field, the second heart field, or the cardiac neural crest. Interestingly, at least cardiomyocytes lose their ability to proliferate early after birth in higher developed vertebrates. Key factors triggering cardiac differentiation have been identified, such as Nkx2-5, GATA4, Tbx, Mef, Islet-1, and others. Although they are not strictly specific for cardiogenesis, they play a prominent role in cardiac differentiation. Recent advantages in the field have identified specific types of miRNA to orchester cardiac differentiation. The regulation of cardiac differentiation will be addressed in more detail in this chapter.
The coronary vascular system is a sophisticated, highly patterned anatomical entity, and therefore a wide range of congenital malformations of the coronary vasculature has been described. Despite the clinical interest of congenital coronary artery anomalies (CCA), very few attempts have been made to relate specific embryonic developmental mechanisms to the congenital anomalies of these blood vessels. This is so because developmental data about the morphogenesis of the coronary vascular system is derived from complex studies carried out in animals (mostly transgenic mice) and may not be noted by the clinicians who take the care of these patients. We will try to offer embryological explanations for a variety of CCA based on the analysis of multiple animal models for the study of cardiac embryogenesis, and suggest to the reader developmental mechanistic explanations for the pathogenesis of these anomalies.
Atrial septal defects (ASDs) are a common human congenital heart disease (CHD) that can be induced by genetic abnormalities.
Our previous studies have demonstrated a genetic interaction between Tbx5 and Osr1 in the second heart field (SHF) for atrial septation. We hypothesized that Osr1 and Tbx5 share a common signaling networking and downstream targets for atrial septation. To identify this molecular networks, we
acquired the RNA-Seq transcriptome data from the posterior SHF of wild-type, Tbx5+/−, Osr1+/−, Osr1−/− and Tbx5+/−/Osr1+/− mutant embryos. Gene set analysis was used to identify the Kyoto Encyclopedia of Genes and Genomes pathways that were affected
by the doses of Tbx5 and Osr1. A gene network module involving Tbx5 and Osr1 was identified using a non-parametric distance metric, distance correlation. A subset of 10 core genes and gene–gene interactions
in the network module were validated by gene expression alterations in posterior second heart field (pSHF) of Tbx5 and Osr1 transgenic mouse embryos, a time-course gene expression change during P19CL6 cell differentiation. Pcsk6 was one of the network module genes that were linked to Tbx5. We validated the direct regulation of Tbx5 on Pcsk6 using immunohistochemical staining of pSHF, ChIP-quantitative polymerase chain reaction and luciferase reporter assay. Importantly,
we identified Pcsk6 as a novel gene associated with ASD via a human genotyping study of an ASD family. In summary, our study implicated a gene
network involving Tbx5, Osr1 and Pcsk6 interaction in SHF for atrial septation, providing a molecular framework for understanding the role of Tbx5 in CHD ontogeny.
The placenta and cardiovascular system are the first organ systems to form during mammalian embryogenesis. We show here that a single gene is critical for development of both. The Hand1 gene, previously called Hxt, eHAND and Thing1, encodes a basic helix-loop-helix (bHLH) transcription factor that starts to be expressed during pre-implantation development. After implantation, Hand1 expression is restricted to placental trophoblast cells and later to embryonic cardiac and neural crest cells. We generated Hand1-null mutant mice by gene targetting. Homozygous mutant embryos arrested by embryonic day (E) 7.5 of gestation with defects in trophoblast giant cell differentiation. This early mortality could be rescued by aggregation of mutant embryos with wild-type tetraploid embryos, which contribute wild-type cells to the trophoblast, but not the embryo. By E10.5, however, the Hand1-null fetuses derived from tetraploid chimaeras died due to cardiac failure. Their heart tubes showed abnormal looping and ventricular myocardial differentiation. Therefore, Hand1 is essential for differentiation of both trophoblast and cardiomyocytes, which are embryologically distinct cell lineages.
The basic helix-loop-helix (bHLH) transcription factors, Hand1 and Hand2 (refs 1,2), also called eHand/Hxt/Thing1 and dHand/Hed/Thing2 (refs 3,4), respectively, are expressed in the heart and certain neural-crest derivatives during embryogenesis. In addition, Hand1 is expressed in extraembryonic membranes, whereas Hand2 is expressed in the deciduum. Previous studies have demonstrated that Hand2 is required for formation of the right ventricle of the heart and the aortic arch arteries. We have generated a germline mutation in the mouse Hand1 gene by replacing the first coding exon with a beta-galactosidase reporter gene. Embryos homozygous for the Hand1 null allele died between embryonic days 8.5 and 9.5 and exhibited yolk sac abnormalities due to a deficiency in extraembryonic mesoderm. Heart development was also perturbed and did not progress beyond the cardiac-looping stage. Our results demonstrate important roles for Hand1 in extraembryonic mesodermal and heart development.
Heart formation requires the fusion of bilateral cardiomyocyte populations as they move towards the embryonic midline. The bHLH transcription factor Hand2 is essential for cardiac fusion; however, the effector genes that execute this function of Hand2 are unknown. Here, we provide in zebrafish the first evidence for a downstream component of the Hand2 pathway that mediates cardiac morphogenesis. Although hand2 is expressed in cardiomyocytes, mosaic analysis demonstrates that it plays a non-autonomous role in regulating cardiomyocyte movement. Gene expression profiles reveal heightened expression of fibronectin 1 (fn1) in hand2 mutant embryos. Reciprocally, overexpression of hand2 leads to decreased Fibronectin levels. Furthermore, reduction of fn1 function enables rescue of cardiac fusion in hand2 mutants: bilateral cardiomyocyte populations merge and exhibit improved tissue architecture, albeit without major changes in apicobasal polarity. Together, our data provide a novel example of a tissue creating a favorable environment for its morphogenesis: the Hand2 pathway establishes an appropriate environment for cardiac fusion through negative modulation of Fn1 levels.
Coronary artery disease is the leading cause of death worldwide. Determining the coronary artery developmental program could aid understanding of the disease and lead to new treatments, but many aspects of the process, including their developmental origin, remain obscure. Here we show, using histological and clonal analysis in mice and cardiac organ culture, that coronary vessels arise from angiogenic sprouts of the sinus venosus-the vein that returns blood to the embryonic heart. Sprouting venous endothelial cells dedifferentiate as they migrate over and invade the myocardium. Invading cells differentiate into arteries and capillaries; cells on the surface redifferentiate into veins. These results show that some differentiated venous cells retain developmental plasticity, and indicate that position-specific cardiac signals trigger their dedifferentiation and conversion into coronary arteries, capillaries and veins. Understanding this new reprogramming process and identifying the endogenous signals should suggest more natural ways of engineering coronary bypass grafts and revascularizing the heart.
Cardiac fibroblasts are the most populous nonmyocyte cell type within the mature heart and are required for extracellular matrix synthesis and deposition, generation of the cardiac skeleton, and to electrically insulate the atria from the ventricles. Significantly, cardiac fibroblasts have also been shown to play an important role in cardiomyocyte growth and expansion of the ventricular chambers during heart development. Although there are currently no cardiac fibroblast-restricted molecular markers, it is generally envisaged that the majority of the cardiac fibroblasts are derived from the proepicardium via epithelial-to-mesenchymal transformation. However, still relatively little is known about when and where the cardiac fibroblasts cells are generated, the lineage of each cell, and how cardiac fibroblasts move to reside in their final position throughout all four cardiac chambers. In this review, we summarize the present understanding regarding the function of Periostin, a useful marker of the noncardiomyocyte lineages, and its role during cardiac morphogenesis. Characterization of the cardiac fibroblast lineage and identification of the signals that maintain, expand and regulate their differentiation will be required to improve our understanding of cardiac function in both normal and pathophysiological states.
Connexin 43 knockout (Cx43 KO) mice exhibit conotruncal malformations and coronary artery defects. We observed epicardial blisters in the Cx43 KO hearts that suggest defects in epicardial epithelial-mesenchymal transformation (EMT), a process that generates coronary vascular progenitors. Analysis using a three-dimensional collagen gel invasion assay showed that Cx43 KO epicardial cells are less invasive and that, unlike wild-type epicardial cells, they fail to organize into thin vessel-like projections. Examination of Cx43 KO hearts using Wt1 as an epicardial marker revealed a disorganized pattern of epicardial cell infiltration. Time-lapse imaging and motion analysis using epicardial explants showed a defect in directional cell migration. This was associated with changes in the actin/tubulin cytoskeleton. A defect in cell polarity was indicated by a failure of the microtubule-organizing center to align with the direction of cell migration. Forced expression of Cx43 constructs in epicardial explants showed the Cx43 tubulin-binding domain is required for Cx43 modulation of cell polarity and cell motility. Pecam staining revealed early defects in remodeling of the primitive coronary vascular plexuses in the Cx43 KO heart. Together, these findings suggest an early defect in coronary vascular development arising from a global perturbation of the cytoarchitecture of the cell. Consistent with this, we found aberrant myocardialization of the outflow tract, a process also known to be EMT dependent. Together, these findings suggest cardiac defects in the Cx43 KO mice arise from the disruption of cell polarity, a process that may be dependent on Cx43-tubulin interactions.
Dysfunction of lymphatic valves underlies human lymphedema, yet the process of valve morphogenesis is poorly understood. Here, we show that during embryogenesis, lymphatic valve leaflet formation is initiated by upregulation of integrin-alpha9 expression and deposition of its ligand fibronectin-EIIIA (FN-EIIIA) in the extracellular matrix. Endothelial cell-specific deletion of Itga9 (encoding integrin-alpha9) in mouse embryos results in the development of rudimentary valve leaflets characterized by disorganized FN matrix, short cusps, and retrograde lymphatic flow. Similar morphological and functional defects are observed in mice lacking the EIIIA domain of FN. Mechanistically, we demonstrate that in primary human lymphatic endothelial cells, the integrin-alpha9-EIIIA interaction directly regulates FN fibril assembly, which is essential for the formation of the extracellular matrix core of valve leaflets. Our findings reveal an important role for integrin-alpha9 signaling during lymphatic valve morphogenesis and implicate it as a candidate gene for primary lymphedema caused by valve defects.
Members of the Twist-family of bHLH proteins play a pivotal role in a number of essential developmental programs. Twist-family bHLH proteins function by dimerizing with other bHLH members and binding to cis- regulatory elements, called E-boxes. While Twist-family members may simply exhibit a preference in terms of high-affinity binding partners, a complex, multilevel cascade of regulation creates a dynamic role for these bHLH proteins. We summarize in this review information on each Twist-family member concerning expression pattern, function, regulation, downstream targets, and interactions with other bHLH proteins. Additionally, we focus on the phospho-regulatory mechanisms that tightly control posttranslational modification of Twist-family member bHLH proteins.
The development of the embryonic heart is dependent upon the generation and incorporation of different mesenchymal subpopulations that derive from intra- and extra-cardiac sources, including the endocardium, epicardium, neural crest, and second heart field. Each of these populations plays a crucial role in cardiovascular development, in particular in the formation of the valvuloseptal apparatus. Notwithstanding shared mechanisms by which these cells are generated, their fate and function differ profoundly by their originating source. While most of our early insights into the origin and fate of the cardiac mesenchyme has come from experimental studies in avian model systems, recent advances in transgenic mouse technology has enhanced our ability to study these cell populations in the mammalian heart. In this article, we will review the current understanding of the role of cardiac mesenchyme in cardiac morphogenesis and discuss several new paradigms based on recent studies in the mouse.
alpha 4 integrins are cell surface receptors that mediate cell-extracellular matrix (ECM) and cell-cell adhesions by interacting with fibronectin (FN) and vascular cell adhesion molecule 1 (VCAM-1), respectively. We have generated a null mutation in the gene for the alpha 4 integrin subunit. Homozygous null embryos express no alpha 4 integrins and show two unexpected defects, both of which lead to embryonic lethality. The first defect is failure of fusion of the allantois with the chorion during placentation. The second is in the development of the epicardium and coronary vessels leading to cardiac hemorrhage. Both processes clearly involve alpha 4 integrin interactions that were previously unsuspected. alpha 4 integrin and VCAM-1 are expressed at the sites of these interactions. These results raise the possibility of abortifacients targeting alpha 4 integrins, and raise serious questions about potential side effects of drugs currently being designed to block alpha 4 integrin functions in inflammation.
Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation, and aspects of cell growth control. Programmed cell death (apoptosis) is crucial for maintaining appropriate cell number and tissue organization. It was therefore of interest to determine whether cell-matrix interactions affect apoptosis. The present report demonstrates that apoptosis was induced by disruption of the interactions between normal epithelial cells and extracellular matrix. We have termed this phenomenon "anoikis." Overexpression of bcl-2 protected cells against anoikis. Cellular sensitivity to anoikis was apparently regulated: (a) anoikis did not occur in normal fibroblasts; (b) it was abrogated in epithelial cells by transformation with v-Ha-ras, v-src, or treatment with phorbol ester; (c) sensitivity to anoikis was conferred upon HT1080 cells or v-Ha-ras-transformed MDCK cells by reverse-transformation with adenovirus E1a; (d) anoikis in MDCK cells was alleviated by the motility factor, scatter factor. The results suggest that the circumvention of anoikis accompanies the acquisition of anchorage independence or cell motility.
Cell growth control in non-transformed cells depends, in part, on adhesive interactions with the extracellular matrix. Following injury, excess or altered fibronectin deposition into the extracellular matrix may contribute to the pathogenesis of fibrosis and atherosclerosis by triggering changes in specific cell functions associated with wound repair, including cell proliferation and migration. To assess the role of fibronectin polymerization on cell growth, we isolated mouse embryonic cells that lack endogenous fibronectin (fibronectin-null cells) and established them in culture under serum-free conditions. These fibronectin-null cells do not produce any detectable fibronectin, but are capable of assembling a fibronectin matrix when cultured in the presence of exogenously added fibronectin. Our data indicate that adhesion-dependent growth in fibronectin-null cells is dramatically increased (>2-5x) by culturing cells in the presence of fibronectin. This fibronectin-induced cell growth was blocked by inhibiting fibronectin matrix assembly. Arg-Gly-Asp peptides or fragments of fibronectin that contain the Arg-Gly-Asp cell binding site promoted clustering of the (&agr ;)5beta1 integrin in focal adhesions, but did not enhance cell growth. These data indicate that the polymerization of fibronectin into the extracellular matrix positively regulates cell growth, and that occupancy and clustering of fibronectin-binding integrins alone are not sufficient to trigger increased cell growth.
Mouse strains expressing the site-specific recombinase Cre (or Flp) facilitate conditional ablation of gene function when one or several exons of the gene of interest are flanked by loxP (or FRT) sites1. Cre expression achieved by classic transgenesis or targeting to an appropriate locus might be tissue specific, temporally restricted or inducible2, 3. In such experimental outlines, it is necessary to monitor Cre activity at desired time points as well as to verify that Cre was not active previously during development. Other investigators have generated transgenic4, 5 or knock-in6 lines in which lacZ expression is conditional on the removal of an intervening segment. However, such lines are most useful if lacZ can be expressed in all cell types and hence is driven off a constitutively active promoter in the mouse.
A subpopulation of neural crest termed the cardiac neural crest is required in avian embryos to initiate reorganization of the outflow tract of the developing cardiovascular system. In mammalian embryos, it has not been previously experimentally possible to study the long-term fate of this population, although there is strong inference that a similar population exists and is perturbed in a number of genetic and teratogenic contexts. We have employed a two-component genetic system based on Cre/lox recombination to label indelibly the entire mouse neural crest population at the time of its formation, and to detect it at any time thereafter. Labeled cells are detected throughout gestation and in postnatal stages in major tissues that are known or predicted to be derived from neural crest. Labeling is highly specific and highly efficient. In the region of the heart, neural-crest-derived cells surround the pharyngeal arch arteries from the time of their formation and undergo an altered distribution coincident with the reorganization of these vessels. Labeled cells populate the aorticopulmonary septum and conotruncal cushions prior to and during overt septation of the outflow tract, and surround the thymus and thyroid as these organs form. Neural-crest-derived mesenchymal cells are abundantly distributed in midgestation (E9.5-12.5), and adult derivatives of the third, fourth and sixth pharyngeal arch arteries retain a substantial contribution of labeled cells. However, the population of neural-crest-derived cells that infiltrates the conotruncus and which surrounds the noncardiac pharyngeal organs is either overgrown or selectively eliminated as development proceeds, resulting for these tissues in a modest to marginal contribution in late fetal and postnatal life.
Alternative splicing of the fibronectin gene transcript gives rise to forms that include the EIIIA (or ED-A) segment. EIIIA-containing fibronectins are prominently expressed during embryogenesis and wound healing and appear to mediate changes in cell adhesion and gene expression. Nonetheless, integrins that bind the EIIIA segment have not been identified. We previously mapped the epitope for two function-blocking monoclonal antibodies to the C-C' loop region of the EIIIA segment (Liao, Y.-F., Wieder, K. G., Classen, J. M., and Van De Water, L. (1999) J. Biol. Chem. 274, 17876-17884). The sequence of this epitope ((39)PEDGIHELFP(48)) resembles the sequence within tenascin-C to which the integrin alpha(9)beta(1) binds. We now report that either integrin alpha(9)beta(1) or alpha(4)beta(1) can mediate cell adhesion to the EIIIA segment. Moreover, this interaction is blocked both by epitope-mapped EIIIA antibodies as well as by the respective anti-integrins. Deletion mutants of the EIIIA segment that include the C-C' loop and flanking sequence bind cells expressing either alpha(9)beta(1) or alpha(4)beta(1). Adhesion of alpha(4)beta(1)-containing MOLT-3 cells to the EIIIA segment stimulates phosphorylation of p44/42 MAP kinase. Our observation that two integrins bind the EIIIA segment establishes a novel mechanism by which cell adhesion to fibronectin is regulated by alternative splicing.
The epicardium of the mammalian heart arises from progenitor cells outside the developing heart. The epicardial progenitor (EPP) cells migrate onto the heart through a cyst-mediated mechanism in which the progenitors are released from the tissue of origin as cysts; the cysts float in the fluid of the pericardial cavity and attach to the naked myocardial surface of the heart, and cells in the cysts then migrate out to form an epithelial sheet. In this paper, we show that the gene encoding the alpha4 subunit of alpha4beta1 integrin (alpha4beta1) is essential for this migratory process. We have generated a knockin mutation in mice replacing the alpha4 integrin gene with the lacZ reporter gene, placing lacZ under the control of the alpha4 integrin promoter. We show that in homozygous mutant embryos, the migration of EPP progenitor cells is impaired due to inefficient budding of the cysts and a failure of the cells in the cysts to migrate on the heart. This study provides direct genetic evidence for essential roles for alpha4beta1 integrin-mediated cell adhesion in the migration of progenitor cells to form the epicardium, in addition to a previous finding that alpha4beta1 is essential for maintaining the epicardium (Yang, J.T., H. Rayburn, and R.O. Hynes. 1995. Development. 121:549-560).
Cardiac and cephalic neural crest cells (NCCs) are essential components of the craniofacial and aortic arch mesenchyme. Genetic disruption of the platelet-derived growth factor receptor alpha (PDGFRalpha) results in defects in multiple tissues in the mouse, including neural crest derivatives contributing to the frontonasal process and the aortic arch. Using chimeric analysis, we show that loss of the receptor in NCCs renders them inefficient at contributing to the cranial mesenchyme. Conditional gene ablation in NCCs results in neonatal lethality because of aortic arch defects and a severely cleft palate. The conotruncal defects are first observed at E11.5 and are consistent with aberrant NCC development in the third, fourth and sixth branchial arches, while the bone malformations present in the frontonasal process and skull coincide with defects of NCCs from the first to third branchial arches. Changes in cell proliferation, migration, or survival were not observed in PDGFRalpha NCC conditional embryos, suggesting that the PDGFRalpha may play a role in a later stage of NCC development. Our results demonstrate that the PDGFRalpha plays an essential, cell-autonomous role in the development of cardiac and cephalic NCCs and provides a model for the study of aberrant NCC development.
Autosomal dominant mutations in the gene encoding the basic helix-loop-helix transcription factor Twist1 are associated with limb and craniofacial defects in humans with Saethre-Chotzen syndrome. The molecular mechanism underlying these phenotypes is poorly understood. We show that ectopic expression of the related basic helix-loop-helix factor Hand2 phenocopies Twist1 loss of function in the limb and that the two factors have a gene dosage-dependent antagonistic interaction. Dimerization partner choice by Twist1 and Hand2 can be modulated by protein kinase A- and protein phosphatase 2A-regulated phosphorylation of conserved helix I residues. Notably, multiple Twist1 mutations associated with Saethre-Chotzen syndrome alter protein kinase A-mediated phosphorylation of Twist1, suggesting that misregulation of Twist1 dimerization through either stoichiometric or post-translational mechanisms underlies phenotypes of individuals with Saethre-Chotzen syndrome.
Cardiogenesis is an exquisitely sensitive process. Any perturbation in the cells that contribute to the building of the heart leads to cardiac malformations, which frequently result in the death of the embryo. Previously, the myocardium was thought to be derived from a single source of cells. However, the recent identification of a second source of myocardial cells that make an important contribution to the cardiac chambers has modified the classical view of heart formation. It also has an important influence on the interpretation of mutant phenotypes in the mouse, with consequences for the classification and prognosis of human congenital heart defects.
Deepak Srivastava et al. Nature Genet. 16, 154-160 (1997).Due to a printing error, the contrast between colour shades in Fig. 1 was suboptimal, obscuring the difference in ventricular expression of dHAND (Fig. Ic). Fig. 1 Expression of dHAND and eHAND mRNA in staged mouse embryos by whole mount in situ hybridization.
Cardiogenesis is an exquisitely sensitive process. Any perturbation in the cells that contribute to the building of the heart leads to cardiac malformations, which frequently result in the death of the embryo. Previously, the myocardium was thought to be derived from a single source of cells. However, the recent identification of a second source of myocardial cells that make an important contribution to the cardiac chambers has modified the classical view of heart formation. It also has an important influence on the interpretation of mutant phenotypes in the mouse, with consequences for the classification and prognosis of human congenital heart defects.
Uncovering the origins of myocardial cells is important for understanding and treating heart diseases. Cai et al. suggest that Tbx18-expressing epicardium provides a substantial contribution to myocytes in the ventricular septum and the atrial and ventricular walls. Here we show that the T-box transcription factor gene 18 (Tbx18) itself is expressed in the myocardium, showing that their genetic lineage tracing system does not allow conclusions of an epicardial origin of cardiomyocytes in vivo to be drawn.
Alternative splicing of the fibronectin gene transcript gives rise to forms that include the EIIIA (or ED-A) segment. EIIIA-containing
fibronectins are prominently expressed during embryogenesis and wound healing and appear to mediate changes in cell adhesion
and gene expression. Nonetheless, integrins that bind the EIIIA segment have not been identified. We previously mapped the
epitope for two function-blocking monoclonal antibodies to the C-C′ loop region of the EIIIA segment (Liao, Y.-F., Wieder,
K. G., Classen, J. M., and Van De Water, L. (1999) J. Biol. Chem. 274, 17876–17884). The sequence of this epitope (39PEDGIHELFP48) resembles the sequence within tenascin-C to which the integrin α9β1binds. We now report that either integrin α9β1 or α4β1can mediate cell adhesion to the EIIIA segment. Moreover, this interaction is blocked both by epitope-mapped EIIIA antibodies
as well as by the respective anti-integrins. Deletion mutants of the EIIIA segment that include the C-C′ loop and flanking
sequence bind cells expressing either α9β1 or α4β1. Adhesion of α4β1-containing MOLT-3 cells to the EIIIA segment stimulates phosphorylation of p44/42 MAP kinase. Our observation that two integrins
bind the EIIIA segment establishes a novel mechanism by which cell adhesion to fibronectin is regulated by alternative splicing.
The basic Helix-Loop-Helix (bHLH) transcription factors Hand1 and Hand2 play critical roles in the development of multiple organ systems during embryogenesis. The dynamic expression patterns of these two factors within developing tissues obfuscate their respective unique and redundant organogenic functions. To define cell lineages potentially dependent upon Hand gene expression, we generated a mutant allele in which the coding region of Hand1 is replaced by Cre recombinase. Subsequent Cre-mediated activation of β-galactosidase or eYFP reporter alleles enabled lineage trace analyses that clearly define the fate of Hand1-expressing cells. Hand1-driven Cre marks specific lineages within the extra embryonic tissues, placenta, sympathetic nervous system, limbs, jaw, and several cell types within the cardiovascular system. Comparisons between Hand1 expression and Hand1-lineage greatly refine our understanding of its dynamic spatial-temporal expression domains and raise the possibility of novel Hand1 functions in structures not thought to be Hand1-dependent.
The reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) raises the possibility that a somatic cell could be reprogrammed to an alternative differentiated fate without first becoming a stem/progenitor cell. A large pool of fibroblasts exists in the postnatal heart, yet no single “master regulator” of direct cardiac reprogramming has been identified. Here, we report that a combination of three developmental transcription factors (i.e., Gata4, Mef2c, and Tbx5) rapidly and efficiently reprogrammed postnatal cardiac or dermal fibroblasts directly into differentiated cardiomyocyte-like cells. Induced cardiomyocytes expressed cardiac-specific markers, had a global gene expression profile similar to cardiomyocytes, and contracted spontaneously. Fibroblasts transplanted into mouse hearts one day after transduction of the three factors also differentiated into cardiomyocyte-like cells. We believe these findings demonstrate that functional cardiomyocytes can be directly reprogrammed from differentiated somatic cells by defined factors. Reprogramming of endogenous or explanted fibroblasts might provide a source of cardiomyocytes for regenerative approaches.
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During heart morphogenesis, epicardial cells undergo an epithelial-to-mesenchymal transition (EMT) and migrate into the subepicardium. The cellular signals controlling this process are poorly understood. Here, we show that epicardial cells exhibit two distinct mitotic spindle orientations, directed either parallel or perpendicular to the basement membrane. Cells undergoing perpendicular cell division subsequently enter the myocardium. We found that loss of beta-catenin led to a disruption of adherens junctions and a randomization of mitotic spindle orientation. Loss of adherens junctions also disrupted Numb localization within epicardial cells, and disruption of Numb and Numblike expression in the epicardium led to randomized mitotic spindle orientations. Taken together, these data suggest that directed mitotic spindle orientation contributes to epicardial EMT and implicate a junctional complex of beta-catenin and Numb in the regulation of spindle orientation.
Extracellular matrix (ECM) remodeling is critical for organogenesis, yet its molecular regulation is poorly understood. In zebrafish, asymmetric migration of the epithelial lateral plate mesoderm (LPM) displaces the gut leftward, allowing correct placement of the liver and pancreas. To observe LPM migration at cellular resolution, we transgenically expressed EGFP under the control of the regulatory sequences of the bHLH transcription factor gene hand2. We found that laminin is distributed along the LPM/gut boundary during gut looping, and that it appears to become diminished by the migrating hand2-expressing cells. Laminin diminishment is necessary for LPM migration and is dependent on matrix metalloproteinase (MMP) activity. Loss of Hand2 function causes reduced MMP activity and prolonged laminin deposition at the LPM/gut boundary, leading to failed asymmetric LPM migration and gut looping. Our study reveals an unexpected role for Hand2, a key regulator of cell specification and differentiation, in modulating ECM remodeling during organogenesis.
The establishment of the coronary circulation is critical for the development of the embryonic heart. Over the last several years, there has been tremendous progress in elucidating the pathways that control coronary development. Interestingly, many of the pathways that regulate the development of the coronary vasculature are distinct from those governing vasculogenesis in the rest of the embryo. It is becoming increasingly clear that coronary development depends on a complex communication between the epicardium, the subepicardial mesenchyme, and the myocardium mediated in part by secreted growth factors. This communication coordinates the growth of the myocardium with the formation of the coronary vasculature. This review summarizes our present understanding of the role of these growth factors in the regulation of coronary development. Continued progress in this field holds the potential to lead to novel therapeutics for the treatment of patients with coronary artery disease.
The basic helix-loop-helix DNA binding protein Hand2 has critical functions in cardiac development both in neural crest-derived and mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest has allowed us to genetically dissect Hand2-dependent defects specifically in outflow tract and cardiac cushion independent of Hand2 functions in mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest results in misalignment of the aortic arch arteries and outflow tract, contributing to development of double outlet right ventricle (DORV) and ventricular septal defects (VSD). These neural crest-derived developmental anomalies are associated with altered expression of Hand2-target genes we have identified by gene profiling. A number of Hand2 direct target genes have been identified using ChIP and ChIP-on-chip analyses. We have identified and validated a number of genes related to cell migration, proliferation/cell cycle and intracellular signaling whose expression is affected by Hand2 deletion in the neural crest and which are associated with development of VSD and DORV. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting expression of target genes associated with a number of functional interactions in neural crest-derived cells required for proper patterning of the outflow tract, generation of the appropriate number of neural crest-derived cells for elongation of the conotruncus and cardiac cushion organization. Our genetic model has made it possible to investigate the molecular genetics of neural crest contributions to outflow tract morphogenesis and cell differentiation.
The permanent cellular constituents of the heart include cardiac fibroblasts, myocytes, endothelial cells, and vascular smooth muscle cells. Previous studies have demonstrated that there are undulating changes in cardiac cell populations during embryonic development, through neonatal development and into the adult. Transient cell populations include lymphocytes, mast cells, and macrophages, which can interact with these permanent cell types to affect cardiac function. It has also been observed that there are marked differences in the makeup of the cardiac cell populations depending on the species, which may be important when examining myocardial remodeling. Current dogma states that the fibroblast makes up the largest cell population of the heart; however, this appears to vary for different species, especially mice. Cardiac fibroblasts play a critical role in maintaining normal cardiac function, as well as in cardiac remodeling during pathological conditions such as myocardial infarct and hypertension. These cells have numerous functions, including synthesis and deposition of extracellular matrix, cell-cell communication with myocytes, cell-cell signaling with other fibroblasts, as well as with endothelial cells. These contacts affect the electrophysiological properties, secretion of growth factors and cytokines, as well as potentiating blood vessel formation. Although a plethora of information is known about several of these processes, relatively little is understood about fibroblasts and their role in angiogenesis during development or cardiac remodeling. In this review, we provide insight into the various properties of cardiac fibroblasts that helps illustrate their importance in maintaining proper cardiac function, as well as their critical role in the remodeling heart.
Growth and expansion of ventricular chambers is essential during heart development and is achieved by proliferation of cardiac progenitors. Adult cardiomyocytes, by contrast, achieve growth through hypertrophy rather than hyperplasia. Although epicardial-derived signals may contribute to the proliferative process in myocytes, the factors and cell types responsible for development of the ventricular myocardial thickness are unclear. Using a coculture system, we found that embryonic cardiac fibroblasts induced proliferation of cardiomyocytes, in contrast to adult cardiac fibroblasts that promoted myocyte hypertrophy. We identified fibronectin, collagen, and heparin-binding EGF-like growth factor as embryonic cardiac fibroblast-specific signals that collaboratively promoted cardiomyocyte proliferation in a paracrine fashion. Myocardial beta1-integrin was required for this proliferative response, and ventricular cardiomyocyte-specific deletion of beta1-integrin in mice resulted in reduced myocardial proliferation and impaired ventricular compaction. These findings reveal a previously unrecognized paracrine function of embryonic cardiac fibroblasts in regulating cardiomyocyte proliferation.
The cardiac neural crest (cNC) lineage plays key roles in heart development by directly contributing to heart structures and regulating development of other heart lineages. The basic helix-loop-helix factor Hand2 regulates development of cardiovascular structures and NC-derived tissues including those that contribute to face and peripheral nervous system. Although Hand2 is expressed in cNC, its role has not been examined because of an early embryonic lethality when Hand2 is deleted in the NC lineage. We find that the lethality is attributable to loss of norepinephrine synthesis that can be overcome by activating adrenergic receptors. In rescued embryos, loss of Hand2 in the NC lineage leads to the misalignment of the outflow tract and aortic arch arteries. Defects include pulmonary stenosis, interrupted aortic artery, retroesophageal right subclavian artery, and ventricular septum defect, which resemble congenital heart defects attributed to defects in the NC. Hand2 functions in part by regulating signaling from the cNC to other cardiac lineages but not by regulating migration or survival of the cNC. Loss of Hand2 in NC also uncovered a novel role for the cNC in regulating proliferation and differentiation of the second heart field-derived myocardium that persists late into development. These results show that the cNC functions as a major signaling center for heart development and Hand2 plays a pivotal role in regulating both cell-autonomous and -nonautonomous functions of the cNC.
The epicardium plays an essential role in coronary artery formation and myocardial development, but signals controlling the development and differentiation of this tissue are not well understood. To investigate the role of platelet-derived growth factor receptor (PDGFR)beta in development of epicardial-derived vascular smooth muscle cells (VSMCs), we examined PDGFRbeta(-/-) and PDGFRbeta epicardial mutant hearts. We found that PDGFRbeta(-/-) hearts failed to form dominant coronary vessels on the ventral heart surface, had a thinned myocardium, and completely lacked coronary VSMCs (cVSMCs). This constellation of defects was consistent with a primary defect in the epicardium. To verify that these defects were specific to epicardial derivatives, we generated mice with an epicardial deletion of PDGFRbeta that resulted in reduced cVSMCs distal to the aorta. The regional absence of cVSMCs suggested that cVSMCs could arise from 2 sources, epicardial and nonepicardial, and that both were dependent on PDGFRbeta. In the absence of PDGFRbeta signaling, epicardial cells adopted an irregular actin cytoskeleton, leading to aberrant migration of epicardial cells into the myocardium in vivo. In addition, PDGF receptor stimulation promoted epicardial cell migration, and PDGFRbeta-driven phosphoinositide 3'-kinase signaling was critical for this process. Our data demonstrate that PDGFRbeta is required for the formation of 2 distinct cVSMC populations and that loss of PDGFRbeta-PI3K signaling disrupts epicardial cell migration.
We employed the yeast two-hybrid technique to screen a mouse embryo cDNA library for novel tissue-specific Class B basic helix-loop-helix (bHLH) transcription factors, which heterodimerize with the ubiquitously expressed Class A bHLH protein E12. From this screen, we cloned a novel bHLH protein, which we named eHAND. Its low sequence identity with other bHLH family members and unique expression pattern during development suggest that eHAND defines a new subclass of Class B bHLH proteins. eHAND was expressed at high levels in trophoblast cells and extraembryonic membranes throughout development. The first site of eHAND expression in embryos was the heart, where it was expressed at high levels between 8.5 and 10.5 days post coitum (d.p.c.), after which transcript levels declined abruptly. By 13.5 d.p.c., eHAND expression in the heart was localized to regions of valve formation. Expression in other regions of the embryo was confined to tissues with a substantial neural crest component. eHAND was expressed in the first branchial arch and its derivatives, in the sympathoadrenal lineage, and in the enteric systems. The expression pattern of eHAND during development is distinct from that of other bHLH genes and suggests that it has a role in formation of extraembryonic tissues, heart, and neural crest derivatives.
Skeletal muscle development is controlled by a family of muscle-specific basic helix-loop-helix (bHLH) transcription factors.
Two bHLH genes, dHAND and eHAND, have now been isolated that are expressed in the bilateral heart primordia and subsequently throughout the primitive tubular
heart and its derivatives during chick and mouse embryogenesis. Incubation of stage 8 chick embryos with dHAND and eHAND antisense oligonucleotides revealed that either oligonucleotide alone had no effect on embryonic development, whereas together
they arrested development at the looping heart tube stage. Thus, dHAND and eHAND may play redundant roles in the regulation of the morphogenetic events of vertebrate heart development.
dHAND and eHAND are related basic helix-loop-helix (bHLH) transcription factors that are expressed in mesodermal and neural crest-derived structures of the developing heart. In contrast to their homogeneous expression during avian cardiogenesis, during mouse heart development we show that dHAND and eHAND are expressed in a complementary fashion and are restricted to segments of the heart tube fated to form the right and left ventricles, respectively. dHAND and eHAND represent the earliest cardiac chamber-specific transcription factors yet identified. Targeted gene deletion of dHAND in mouse embryos resulted in embryonic lethality at embryonic day 10.5 from heart failure. Our description of the cardiac phenotype of dHAND mutant embryos is the first demonstration of a single gene controlling the formation of the mesodermally derived right ventricle and the neural crest-derived aortic arches and reveals a novel cardiogenic subprogramme for right ventricular development.
The underlying mechanisms of lethal cardiovascular defects associated with the fibronectin-null (FN.null) mutation in mouse embryos were investigated by lineage analysis of myocardial, endocardial, and endothelial cells. A wide variation in phenotype was observed on two genetic backgrounds. In the less severe class (C57/BL6 background), FN.null embryos display a defective heart. Myocardial cells express the specific marker MF-20 and are correctly localized in the anterior trunk region, but myocardial organization is disrupted, resulting in a bulbous heart tube. Endocardial cells express the specific marker platelet-endothelial cell adhesion molecule-1 (PECAM-1) and are localized within the myocardium, but the endocardium appears collapsed. Endothelial cells of two vascular beds are specified, but the aortae are distended and lack contact with the surrounding mesenchyme, while no vessels form in the yolk sac. Defects in the more severe class suggest that FNs are essential earlier in development on the 129/Sv background. Myocardial and endocardial cells are specified, but morphogenesis of the myocardium and endocardium does not occur. Aortic endothelial cells are specified and localized normally, but remain scattered. Yolk sac endothelial cells resemble those of the less severe class. We conclude that FNs are essential for organization of heart and blood vessels, but are dispensable for cellular specification in the appropriate regions within the embryo.
dHAND and eHAND are basic helix-loop-helix transcription factors that play critical roles in cardiac development. The HAND genes have a complementary left-right cardiac asymmetry of expression with dHAND predominantly on the right side and eHAND on the left side of the looped heart tube. Here we show that although eHAND is asymmetrically expressed along the anterior-posterior and dorsal-ventral embryonic axes, it is symmetrically expressed along the left-right axis at early stages of embryonic and cardiac development. After cardiac looping, dHAND and eHAND are expressed in the right (pulmonary) and left (systemic) ventricles, respectively. The left-right (LR) sidedness of dHAND and eHAND expression is demonstrated to be anatomically reversed in situs inversus (inv/inv) mouse embryos; however, dHAND expression persists in the pulmonary ventricle and eHAND in the systemic ventricle regardless of anatomic position, indicating chamber specificity of expression. Previously we showed that dHAND-null mice fail to form a right-sided pulmonary ventricle. Here mice homozygous for the dHAND and inv mutations are demonstrated to have only a right-sided ventricle which is morphologically a left (systemic) ventricle. These data suggest that the HAND genes are involved in development of segments of the heart tube which give rise to specific chambers of the heart during cardiogenesis, rather than controlling the direction of cardiac looping by interpreting the cascade of LR embryonic signals.
The PML gene of acute promyelocytic leukaemia (APL) encodes a cell growth and tumour suppressor, however, the mechanisms by which PML suppresses tumorigenesis are poorly understood. We show here that Pml is required for Fas- and caspase-dependent DNA-damage-induced apoptosis. We also found that Pml is essential for induction of programmed cell death by Fas, tumour necrosis factor alpha (TNF), ceramide and type I and II interferons (IFNs). As a result, Pml-/- mice and cells are protected from the lethal effects of ionizing radiation and anti-Fas antibody. Pml is required for caspase 1 and caspase 3 activation upon exposure to these stimuli. The PML-RAR alpha fusion protein of APL renders haemopoietic progenitor cells resistant to Fas-, TNF- and IFN-induced apoptosis with a lack of caspase 3 activation, thus acting as a Pml dominant-negative product. These results demonstrate that Pml is a mediator of multiple apoptotic signals, and implicate inhibition of apoptosis in the pathogenesis of APL.
The Wilms' Tumour gene WT1 has important functions during development. Knock-out mice were shown to have defects in the urogenital system and to die at embryonic day E13.5, probably due to heart failure. Using a lacZ reporter gene inserted into a YAC construct, we demonstrate that WT1 is expressed in the early proepicardium, the epicardium and the subepicardial mesenchymal cells (SEMC). Lack of WT1 leads to severe defects in the epicardial layer and a concomitant absence of SEMCs, which explains the pericardial bleeding and subsequent embryonic death observed in Wt1 null embryos. We further show that a human-derived WT1 YAC construct is able to completely rescue heart defects, but only partially rescues defects in the urogenital system. Analysis of the observed hypoplastic kidneys demonstrate a continuous requirement for WT1 during nephrogenesis, in particular, in the formation of mature glomeruli. Finally, we show that the development of adrenal glands is also severely affected in partially rescued embryos. These data demonstrate a variety of new functions for WT1 and suggest a general requirement for this protein in the formation of organs derived from the intermediate mesoderm.
The HAND subclass of basic Helix-loop-helix factors is comprised of two members HAND1 and HAND2. HAND genes are present within the genomes of organisms ranging from flies to man. Experiments employing chick embryology, tissue culture, and gene targeting in mice show that HAND function is critical for the specification and/or differentiation of extraembryonic structures that include the yolk sac, placenta, and the cells of the trophoblast lineages. HAND factors also play key roles in cardiac, gut, sympathetic neuronal development and in the proper development of tissues populated by HAND-expressing neural crest cells, including regions of the developing vasculature, the limbs, the jaw, and teeth. Surprisingly, nearly 10 years after their initial identification and characterization, little is understood about the nature of the downstream target genes which HAND1 and HAND2 regulate, whether the nature of their transcriptional regulation is positive or negative, or if they modulate genetic programs common to these diverse tissue types or if they drive unique subsets of genes that contribute to tissue identity. At the core of these questions is by which mechanisms do HAND factors modulate biological activity? Do they behave like classical class B bHLH factors or is their function more complex requiring a rethinking of the dogma? What follows is a review of what is currently known about HAND factors and a reflection on why elucidating their role in the biological programs within which they participate has been so difficult.
Development of the coronary vascular system is an interesting model in developmental biology with major implications for the clinical setting. Although coronary vessel development is a form of vasculogenesis followed by angiogenesis, this system uses several unique developmental processes not observed in the formation of other blood vessels. This review summarizes the literature that describes the development of the coronary system, highlighting the unique aspects of coronary vessel development. It should be noted that many of the basic mechanisms that govern vasculogenesis in other systems have not been analyzed in coronary vessel development. In addition, we present recent advances in the field that uncover the basic mechanisms regulating the generation of these blood vessels and identify areas in need of additional studies.
The basic helix-loop-helix (bHLH) transcription factor HAND1 (also called eHAND) is expressed in numerous tissues during development including the heart, limbs, neural crest derivatives and extra-embryonic membranes. To investigate the role of Hand1 during development, we generated a Hand1 knockout mouse. Hand1-null mice survived to the nine somite stage at which time they succumbed to numerous developmental defects. One striking defect in Hand1-null embryos was the accumulation of hematopoietic cells between the yolk sac and the amnion because of defects in the yolk sac vasculature. In Hand1-null yolk sacs, vasculogenesis occurs but vascular refinement was arrested. Analysis of angiogenic genes in extra-embryonic membranes showed that most are expressed at normal levels in Hand1-null embryos but several, including Vegf, Ang1 and ephrin B2, and gene components of the Notch pathway are upregulated. In the absence of Hand1 the expression of the bHLH factor Hand2 is also enhanced. Although HAND1 and HAND2 share many structural features, and Hand2 is required for vasculature development in yolk sacs, enhanced expression of Hand2 is insufficient to compensate for the loss of Hand1. The most striking aspect of the vascular defect in Hand1 mutant yolk sacs is the abnormal distribution of smooth muscle cells. During normal angiogenesis, vascular smooth muscle precursors are recruited to the peri-endothelial tissue before differentiation, however, in Hand1 null yolk sacs, smooth muscle cells are not recruited but differentiate in clusters distributed throughout the mesoderm. These data indicate that Hand1 is required for angiogenesis and vascular smooth muscle recruitment in the yolk sac.
The role of GATA4 during the earliest stages of cardiogenesis has not been defined because Gata4 knockout embryos suffer an early developmental arrest caused by deficiencies in extraembryonic visceral endoderm function. We have used tetraploid embryo complementation to rescue these defects and generated clonal embryonic day 9.5 Gata4 –/– embryos directly from embryonic stem cells. GATA4-null embryos display heart defects characterized by disrupted looping morphogenesis, septation, and a hypoplastic ventricular myocardium. We find that myocardial gene expression is relatively normal in GATA4-null hearts including expression of GATA6. Moreover, GATA4 expression in the endocardium is dispensable for trabeculae formation. Remarkably, the proepicardium is absent in GATA4-null embryos, blocking formation of the epicardium. Therefore, we propose that the observed myocardial defects may be a secondary consequence of loss of the proepicardium. These findings definitively demonstrate a requirement for GATA4 during early cardiac development and identify an essential factor for generation of the proepicardium.
• heart development
• septum transversum mesenchyme
• proepicardial organ
The basic helix-loop-helix transcription factors Hand1 and Hand2 display dynamic and spatially restricted expression patterns in the developing heart. Mice that lack Hand2 die at embryonic day 10.5 from right ventricular hypoplasia and vascular defects, whereas mice that lack Hand1 die at embryonic day 8.5 from placental and extra-embryonic abnormalities that preclude analysis of its potential role in later stages of heart development. To determine the cardiac functions of Hand1, we generated mice harboring a conditional Hand1-null allele and excised the gene by cardiac-specific expression of Cre recombinase. Embryos homozygous for the cardiac Hand1 gene deletion displayed defects in the left ventricle and endocardial cushions, and exhibited dysregulated ventricular gene expression. However, these embryos survived until the perinatal period when they died from a spectrum of cardiac abnormalities. Creation of Hand1/2 double mutant mice revealed gene dose-sensitive functions of Hand transcription factors in the control of cardiac morphogenesis and ventricular gene expression. These findings demonstrate that Hand factors play pivotal and partially redundant roles in cardiac morphogenesis, cardiomyocyte differentiation and cardiac-specific transcription.
Myocardial differentiation is initiated by the activation of terminal-differentiation gene expression within a subset of cells in the anterior lateral plate mesoderm. We have previously shown that shortly after this activation, myocardial cells undergo epithelial maturation [1], suggesting that myocardial differentiation encompasses both molecular and cellular changes. To address the question of how the molecular programs driving myocardial gene expression and the formation of the myocardial epithelium are integrated, we analyzed the role of two essential myocardial terminal-differentiation factors, Hand2 and Gata5, in myocardial epithelia formation. hand2 and gata5 mutants exhibit a much-reduced number of myocardial cells and defects in myocardial gene expression [2,3]. We find that the few myocardial precursors that are present in hand2 mutants do not polarize. In contrast, embryos with reduced Gata5 function exhibit polarized myocardial epithelia despite a similar reduction in myocardial precursor number, indicating that proper cell number is not required for epithelial formation. Taken thogether, these results indicate that Hand2 is uniquely required for myocardial polarization, a previously unappreciated role for this critical transcription factor. Furthermore, these results demonstrate that two independent processes, the polarizaton of myocardial precursors and the allocation of proper cell number, contribute to myocardial development.
Paraquat (PQ)-induced pulmonary toxicity is characterized by initial development of pulmonary edema, infiltration of inflammatory cells, and damage to the alveolar epithelium, which may progress to severe fibrosis. However, the exact role of PQ in the progression of the pathogenesis has not been clearly established. To understand the mechanism of PQ in pulmonary toxicity, we developed an animal model of PQ-induced lung injury by intranasal instillation of PQ solution using C57Black/6J mice. Twenty microliters of PQ solution (0.01, 0.01, and 0.04 mg/mouse) was applied through the nares, and the same amount of vehicle was applied in control mice. The pathological progression of lung pathology in our mouse model was very similar to that of patients suffering from PQ poisoning. The lungs of some animals exposed to PQ showed acute fulmination, resulting in death from 5 days post-exposure, but others showed a more protracted injury, resulting in typical pulmonary fibrosis at 3 weeks. Using this PQ-poisoned mouse model, we examined the gene expression at the initial destructive phase (within 5 days) that fibrosis has not completely developed. We prepared RNAs after 6h, 24h, and 5 days and examined the changes of the expression levels for 45 selected genes. The genes showing >2-fold increase at 6h or a time-dependent decrease during this experimental period may be the early markers for the destructive phase. These genes are Mt1, Mt2, Hmox1, Gcl, GR, IL-6, IL-13, Txn1, Fas, FasL, Lpin2, Mmp1a, Mmp12, Sfp-B, Sfp-D, CAT, EC-SOD, GST, and Pltp. On the other hand, the genes involved in the development of fibrosis, such as procollagen, Fn1, Eln, SMA, and Mmp9, Timp1 were significantly increased on day 5, not at 6h nor at 24h, after PQ treatment (the late marker). The genes showing a significant increase (Mmp3 and Mmp8) or decrease (VEGFA) at 24h and 5 days and not at 6h may be also the late markers. These changes in gene expression, which are equalled to functional activities of proteins, will be the targets for future studies focused on the development on PQ-induced pulmonary damage.
The heart is the first organ to form and function during vertebrate development and is absolutely essential for life. The left ventricle is derived from the classical primary or first heart field (FHF), while the right ventricle and outflow tract are derived from a distinct second heart field (SHF). The recent discovery of the SHF has raised several fundamental and important questions about how the two heart fields are integrated into a single organ and whether unique molecular programs control the development of the two heart fields. This review briefly highlights the contributions of the SHF to the developing and mature heart and then focuses primarily on our current understanding of the transcriptional pathways that function in the development of the SHF and its derivatives.