[Show abstract][Hide abstract] ABSTRACT: Fragile X syndrome (FXS) is a neurodevelopmental disorder whose biochemical manifestations involve dysregulation of mGluR5-dependent pathways, which are widely modeled using cultured neurons. In vitro phenotypes in cultured neurons using standard morphological, functional, and chemical approaches have demonstrated considerable variability. Here, we study transcriptomes obtained in situ in the intact brain tissues of a murine model of FXS to see how they reflect the in vitro state.
We used genome-wide mRNA expression profiling as a robust characterization tool for studying differentially expressed pathways in fragile X mental retardation 1 (Fmr1) knockout (KO) and wild-type (WT) murine primary neuronal cultures and in embryonic hippocampal and cortical murine tissue. To study the developmental trajectory and to relate mouse model data to human data, we used an expression map of human development to plot murine differentially expressed genes in KO/WT cultures and brain.
We found that transcriptomes from cell cultures showed a stronger signature of Fmr1KO than whole tissue transcriptomes. We observed an over-representation of immunological signaling pathways in embryonic Fmr1KO cortical and hippocampal tissues and over-represented mGluR5-downstream signaling pathways in Fmr1KO cortical and hippocampal primary cultures. Genes whose expression was up-regulated in Fmr1KO murine cultures tended to peak early in human development, whereas differentially expressed genes in embryonic cortical and hippocampal tissues clustered with genes expressed later in human development.
The transcriptional profile in brain tissues primarily centered on immunological mechanisms, whereas the profiles from cell cultures showed defects in neuronal activity. We speculate that the isolation and culturing of neurons caused a shift in neurological transcriptome towards a “juvenile” or “de-differentiated” state. Moreover, cultured neurons lack the close coupling with glia that might be responsible for the immunological phenotype in the intact brain. Our results suggest that cultured cells may recapitulate an early phase of the disease, which is also less obscured with a consequent “immunological” phenotype and in vivo compensatory mechanisms observed in the embryonic brain. Together, these results suggest that the transcriptome of cultured primary neuronal cells, in comparison to whole brain tissue, more robustly demonstrated the difference between Fmr1KO and WT mice and might reveal a molecular phenotype, which is typically hidden by compensatory mechanisms present in vivo. Moreover, cultures might be useful for investigating the perturbed pathways in early human brain development and genes previously implicated in autism.
[Show abstract][Hide abstract] ABSTRACT: Rationale:
The fetal origins of disease hypothesis suggests that variations in the course of prenatal lung development may affect lifelong pulmonary function growth, decline and pathobiology. Many studies support the existence of differences in the developing lung trajectory in males and females, and sex specific differences in the prevalence of chronic lung diseases such as asthma and bronchopulmonary dysplasia (BPD).
To investigate the early developing fetal lung for transcriptomic correlates of post conception age (maturity) and sex, and their associations with chronic lung diseases.
We analyzed whole lung transcriptome profiles of 61 females and 78 males at 54-127 days post conception (dpc) from non-smoking mothers using unsupervised principal component analysis and supervised linear regression models.
We identified dominant transcriptomic correlates for post conception age and sex with corresponding gene sets that were enriched for developing lung structural and functional ontologies. We observed that the transcriptomic sex difference was not a uniform global time shift/lag, rather lungs of males appear to be more mature that females before 96 dpc, and females appear to be more mature than males after 96 dpc. The age correlate gene set was consistently enriched for asthma and BPD genes, but the sex correlate gene sets were not.
Despite sex differences in the developing fetal lung transcriptome, post conception age appears to be more dominant than sex in the effect of early fetal lung developments on disease risk during this early pseudoglandular phase of development.
No preview · Article · Nov 2015 · American Journal of Respiratory Cell and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Objective:
To test several key glucocorticoid genes that are enhanced in lung development for associations with respiratory distress syndrome (RDS) after antenatal corticosteroid use.
A prospective cohort of women received betamethasone to accelerate fetal lung maturity for threatened preterm delivery. DNA was obtained from mothers and newborns. Neonatal RDS was the primary outcome. Genotyping for single-nucleotide polymorphisms (SNPs) in 68 glucocorticoid genes found to be differentially expressed during lung development was performed. Multivariable analysis tested for associations of SNPs in the candidate genes with RDS.
Genotypic results for 867 SNPs in 96 mothers and 73 babies were included. Thirty-nine (53.4%) babies developed RDS. Maternal SNPs in the centromeric protein E (CENPE), GLRX, CD9, and AURKA genes provided evidence of association with RDS (P < .01). In newborns, SNPs in COL4A3, BHLHE40, and SRGN provided evidence of association with RDS (P < .01).
Single-nucleotide polymorphisms in several glucocorticoid responsive genes suggest association with neonatal RDS after antenatal corticosteroid use.
No preview · Article · Oct 2015 · Reproductive sciences (Thousand Oaks, Calif.)
[Show abstract][Hide abstract] ABSTRACT: From coffee beans flowing in a chute to cells remodelling in a living tissue, a wide variety of close-packed collective systems-both inert and living-have the potential to jam. The collective can sometimes flow like a fluid or jam and rigidify like a solid. The unjammed-to-jammed transition remains poorly understood, however, and structural properties characterizing these phases remain unknown. Using primary human bronchial epithelial cells, we show that the jamming transition in asthma is linked to cell shape, thus establishing in that system a structural criterion for cell jamming. Surprisingly, the collapse of critical scaling predicts a counter-intuitive relationship between jamming, cell shape and cell-cell adhesive stresses that is borne out by direct experimental observations. Cell shape thus provides a rigorous structural signature for classification and investigation of bronchial epithelial layer jamming in asthma, and potentially in any process in disease or development in which epithelial dynamics play a prominent role.
[Show abstract][Hide abstract] ABSTRACT: In utero smoke exposure has been shown to have detrimental effects on lung function and to be associated with persistent wheezing and asthma in children. One potential mechanism of IUS effects could be alterations in DNA methylation, which may have life-long implications. The goal of this study was to examine the association between DNA methylation and nicotine exposure in fetal lung and placental tissue in early development; nicotine exposure in this analysis represents a likely surrogate for in-utero smoke. We performed an epigenome-wide analysis of DNA methylation in fetal lung tissue (n = 85, 41 smoke exposed (48%), 44 controls) and the corresponding placental tissue samples (n = 80, 39 smoke exposed (49%), 41 controls) using the Illumina HumanMethylation450 BeadChip array. Differential methylation analyses were conducted to evaluate the variation associated with nicotine exposure. The most significant CpG sites in the fetal lung analysis mapped to the PKP3 (P = 2.94×10(-03)), ANKRD33B (P = 3.12×10(-03)), CNTD2 (P = 4.9×10(-03)) and DPP10 (P = 5.43×10(-03)) genes. In the placental methylome, the most significant CpG sites mapped to the GTF2H2C and GTF2H2D genes (P = 2.87×10(-06) - 3.48×10(-05)). One hundred and one unique CpG sites with P-values < 0.05 were concordant between lung and placental tissue analyses. Gene Set Enrichment Analysis demonstrated enrichment of specific disorders, such as asthma and immune disorders. Our findings demonstrate an association between in utero nicotine exposure and variable DNA methylation in fetal lung and placental tissues, suggesting a role for DNA methylation variation in the fetal origins of chronic diseases.
Preview · Article · Nov 2014 · Epigenetics: official journal of the DNA Methylation Society
[Show abstract][Hide abstract] ABSTRACT: Rationale: Antenatal corticosteroids enhance lung maturation. However, the importance of glucocorticoid genes on early lung development, asthma susceptibility and treatment response remains unknown. We investigated whether glucocorticoid genes are important during lung development, and their role in asthma susceptibility and treatment response. Methods: Genes that were differentially expressed by corticosteroids in two of three genomic datasets: lymphoblastoid cell lines of participants in the Childhood Asthma Management Program, a glucocorticoid ChIP-seq experiment, or a murine model, were identified (GCGS). Using gene expression profiles from 38 human and C57BL/J6 murine fetal lungs to represent the developing lung, we found that the top 5% of genes contributing to the principal components (PCs) most highly associated with post-conception age or that were identified by linear models of post-conception age within the developing lungs and tested for enrichment with glucocorticoid genes. This developmental glucocorticoid gene set was then tested for enrichment between asthmatic subjects and controls, and before and after treatment with inhaled corticosteroids in asthmatic subjects. Results: 232 genes were included in the GCGS. Analysis of gene expression demonstrated that glucocorticoid genes were enriched in lung development (p=7.02 x 10-26). Furthermore, the developmental GCGS was enriched for genes that are differentially expressed between asthmatics and controls (p=4.26 x 10-3) and were enriched after treatment of asthmatic subjects with inhaled corticosteroids (p<2.72 x 10-4). Conclusions: Glucocorticoid genes are over-represented among genes implicated in fetal lung development. These genes influence asthma susceptibility and treatment response suggesting their involvement in the early ontogeny of asthma.
No preview · Article · Sep 2014 · American Journal of Respiratory Cell and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Lung function tracks from the earliest age that it can be reliably measured. Genome wide association studies suggest that most variants identified for common complex traits are regulatory in function and active during fetal development. Fetal programming of gene expression during development is critical to the formation of a normal lung. An understanding of how fetal developmental genes related to diseases of the lungs and airways is a critical area for research. This review article considers the developmental origins hypothesis, the stages of normal lung development and a variety of environmental exposures that might influence the developmental process: in utero cigarette smoke exposure, vitamin D and folate. We conclude with some information on developmental genes and asthma.
[Show abstract][Hide abstract] ABSTRACT: Poor maternal vitamin D intake is a risk factor for subsequent childhood asthma, suggesting that in utero changes related to vitamin D responsive genes might play a crucial role in later disease susceptibility. We hypothesized that vitamin D pathway genes are developmentally active in the fetal lung and that these developmental genes would be associated with asthma susceptibility and regulation in asthma.
Vitamin D pathway genes were derived from PubMed and Gene Ontology surveys. Principal component analysis was used to identify characteristic lung development genes.
Vitamin D regulated genes were markedly over-represented in normal human (odds ratio OR 2.15, 95% confidence interval CI: 1.69-2.74) and mouse (OR 2.68, 95% CI: 2.12-3.39) developing lung transcriptomes. 38 vitamin D pathway genes were in both developing lung transcriptomes with >63% of genes more highly expressed in the later than earlier stages of development. In immortalized B-cells derived from 95 asthmatics and their unaffected siblings, 12 of the 38 (31.6%) vitamin D pathway lung development genes were significantly differentially expressed (OR 3.00, 95% CI: 1.43-6.21), whereas 11 (29%) genes were significantly differentially expressed in 43 control versus vitamin D treated immortalized B-cells from Childhood Asthma Management Program subjects (OR 2.62, 95% CI: 1.22-5.50). 4 genes, LAMP3, PIP5K1B, SCARB2 and TXNIP were identified in both groups; each displays significant biologic plausibility for a role in asthma.
Our findings demonstrate a significant association between early lung development and asthma--related phenotypes for vitamin D pathway genes, supporting a genomic mechanistic basis for the epidemiologic observations relating maternal vitamin D intake and childhood asthma susceptibility.
Full-text · Article · Nov 2013 · BMC Medical Genomics
[Show abstract][Hide abstract] ABSTRACT: Surgical resection of pulmonary tissue exerts a pro-regenerative stretch stimulus in the remaining lung units. Whether this regeneration process reenacts part or whole of lung morphogenesis developmental program remains unclear. To address this question, we analyzed the stretch-induced regenerating lung transcriptome in mice after left pneumonectomy (PNX) in its developmental context. We created a C57BL/6 mice lung regeneration transcriptome time course at 3, 7, 14, 28 and 56 days post-PNX, profiling the cardiac and medial lobes and whole right lung. Prominent expression at days 3 and 7 of genes related to cell proliferation (Ccnb1, Bub1 and Cdk1), extracellular matrices (Col1a1, Eln and Tnc) and proteases (Serpinb2 and Mmp9) indicated regenerative processes that tapered off after 56 days. We projected the post-PNX transcriptomic time course into the transcriptomic principal component space of the C57BL/6 mouse developing lung time series from embryonic day 9.5 to postnatal day 56. All post-PNX samples were localized around late postnatal stage of developing lungs. Shortly after PNX, the temporal trajectory of regenerating lobes and right lung reversed course relative to the developing lungs in a process reminiscent of de-differentiation. This reversal was limited to the later postnatal stage of lung development. The post-PNX temporal trajectory then moves forward in lung development time close to its pre-PNX state after days 28 to 56 in a process resembling re-development. A plausible interpretation is that remaining pulmonary tissue reverts to a more primitive stage of development with higher potential for growth to generate tissue in proportion to the loss.
Full-text · Article · Aug 2013 · AJP Lung Cellular and Molecular Physiology
[Show abstract][Hide abstract] ABSTRACT: In patients with Duchenne muscular dystrophy (DMD), the absence of a functional dystrophin protein results in sarcolemmal instability, abnormal calcium signaling, cardiomyopathy, and skeletal muscle degeneration. Using the dystrophin-deficient sapje zebrafish model, we have identified microRNAs (miRNAs) that, in comparison to our previous findings in human DMD muscle biopsies, are uniquely dysregulated in dystrophic muscle across vertebrate species. MiR-199a-5p is dysregulated in dystrophin-deficient zebrafish, mdx(5cv) mice, and human muscle biopsies. MiR-199a-5p mature miRNA sequences are transcribed from stem loop precursor miRNAs that are found within the introns of the dynamin-2 and dynamin-3 loci. The miR-199a-2 stem loop precursor transcript that gives rise to the miR-199a-5p mature transcript was found to be elevated in human dystrophic muscle. The levels of expression of miR-199a-5p are regulated in a serum response factor (SRF)-dependent manner along with myocardin-related transcription factors. Inhibition of SRF-signaling reduces miR-199a-5p transcript levels during myogenic differentiation. Manipulation of miR-199a-5p expression in human primary myoblasts and myotubes resulted in dramatic changes in cellular size, proliferation, and differentiation. MiR-199a-5p targets several myogenic cell proliferation and differentiation regulatory factors within the WNT signaling pathway, including FZD4, JAG1, and WNT2. Overexpression of miR-199a-5p in the muscles of transgenic zebrafish resulted in abnormal myofiber disruption and sarcolemmal membrane detachment, pericardial edema, and lethality. Together, these studies identify miR-199a-5p as a potential regulator of myogenesis through suppression of WNT-signaling factors that act to balance myogenic cell proliferation and differentiation.Cell Death and Differentiation advance online publication, 14 June 2013; doi:10.1038/cdd.2013.62.
Full-text · Article · Jun 2013 · Cell death and differentiation
[Show abstract][Hide abstract] ABSTRACT: Murine macrophages cultured on a more rigid (76.8 kPa) substrate exhibit relatively longer spontaneous dynamic extensions (see arrows) compared to less rigid substrate suggesting larger sampling area for phagocytosis.
[Show abstract][Hide abstract] ABSTRACT: Macrophages serve to maintain organ homeostasis in response to challenges from injury, inflammation, malignancy, particulate exposure, or infection. Until now, receptor ligation has been understood as being the central mechanism that regulates macrophage function. Using macrophages of different origins and species, we report that macrophage elasticity is a major determinant of innate macrophage function. Macrophage elasticity is modulated not only by classical biologic activators such as LPS and IFN-γ, but to an equal extent by substrate rigidity and substrate stretch. Macrophage elasticity is dependent upon actin polymerization and small rhoGTPase activation, but functional effects of elasticity are not predicted by examination of gene expression profiles alone. Taken together, these data demonstrate an unanticipated role for cell elasticity as a common pathway by which mechanical and biologic factors determine macrophage function.
[Show abstract][Hide abstract] ABSTRACT: Murine macrophages cultured on a more rigid (76.8 kPa) substrate. Movie demonstrates capture of a two beads via membrane projection and subsequent phagocytosis.
[Show abstract][Hide abstract] ABSTRACT: Rationale. The "fetal origins hypothesis" argued that physiological changes consequent to in utero exposures ultimately contribute to disease susceptibility in later life. The dramatic increase in asthma prevalence is attributed to early exposures acting on pre-existing asthma-susceptible genotypes. We showed previously that distinct transcriptome signatures distinguish the developmental respiratory phenotype of atopic (Brown Norway, BN) and normo-responsive (Lewis) rats. Objective. To determine whether maternal allergen exposure would influence asthma pathogenesis by reprogramming primary patterns of developmental lung gene expression. Methods. Post-natal offspring of dams sensitized to ovalbumin before mating and challenged during pregnancy were assessed for lung function, inflammatory biomarkers, and respiratory gene expression. Results. While maternal ovalbumin exposure resulted in characteristic features of an allergic response (BAL neutrophils, IgE, methacholine-induced lung resistance) in offspring of both strains, substantial strain-specific differences were observed in respiratory gene expression. Of 799 probes representing the top 5% of transcriptomic variation, only 112 (14%) were affected in both strains. Strain-specific gene signatures also exhibited marked differences in enrichment for gene ontologies; immune regulation and cell proliferation being prominent in the BN strain, cell cycle and microtubule assembly gene sets in the Lewis strain. Multiple ovalbumin-specific probes in both strains were also differentially expressed in lymphoblastoid cell lines from human asthmatic vs. non-asthmatic sibling pairs. Conclusion. Our data point to the existence of distinct, genetically programmed responses to maternal exposures in developing lung. These different response patterns, if recapitulated in human fetal development, can contribute to long-term pulmonary health including inter-individual susceptibility to asthma.