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Promoter analysis and histone methylation patterns of candidate genes. (A) Previous work has identified the gene regulatory network (GRN) involved in the DV patterning of the zebrafish embryo. While in the dorsal side canonical Wnt signaling activates directly or indirectly the genes involved in the induction of the organizer, ventrally Vox/Vent/Ved and Pou5f1 represses the expression of these genes. (B) We tested the upstream 4 kb genomic region of the candidate genes for a possible enrichment in the binding sites of key transcription factors. There was no significant difference between the validated genes and a random gene set. (As expected, significant difference in the number of TcfLef binding sites could be detected between the promoter region of the confirmed Wnt-target subset of our gene-set, and the randomized set of genes (p,0.05).) (C) Using a previously published dataset we were able to demonstrate that the histones associated to our candidate genes often show a ''bivalent status'', carrying activating (H3K4me3) and repressing (H3K27me3) methylation marks at the same time. This is in stark contrast with the histonemethylation patterns of randomly selected genes. doi:10.1371/journal.pone.0070053.g005
Source publication
Understanding the molecular interactions that lead to the establishment of the major body axes during embryogenesis is one of the main goals of developmental biology. Although the past two decades have revolutionized our knowledge about the genetic basis of these patterning processes, the list of genes involved in axis formation is unlikely to be c...
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... the expression of typical dorsal genes, on the ventral side maternal and then zygotic Pou5f1/Spg (the zebrafish homolog of mamma- lian Oct4), in combination with the homeobox repressors Vox, Vent and Ved inhibits the activation of the same dorsal genes ( Figure 5A). ...
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... with a random set of zebrafish genes, we could not detect a statistically significant difference in the number of Pou5f1 and TcfLef binding sites ( Figure 5B). When examining only the promoter regions of previously confirmed Wnt target genes in our gene-set, we did observe a significant enrichment in TcfLef sites, suggesting that our approach was sound. ...
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... seen in the data, yet this difference was not large enough to be statistically significant ( Figure 5B). ...
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... validated ''dorsal'' gene set show an increase in the activating H3 K4 trimethylation marks in the promoter region, compared with the random set of genes, and an overabundance of ''bivalent'' histone-marks at the onset of zygotic transcription ( Figure 5C), showing that dorsal genes are epigenetically marked prior to transcriptional activation. (Of note, according to the Lindeman et al. dataset [46] dharma, one of the earliest transcribed genes in the zebrafish genome [48], carries only H3K27 trimethylation during the time of its transcription. ...
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... given the importance of Wnts during early zebrafish development a tight regulation can be expected. The close relationship between the pathway and its regulators is also underscored by an apparent enrichment in TcfLef binding sites in the promoter regions of the respective genes ( Figure 5B). Furthermore, the ''bivalent'' epigenetic status observed at the majority of the Wnt-modulators ( Figure 6) also suggests a tight and efficient regulation of this regulatory module. ...
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... an unbiased full transcriptome sequencing approach we generated a comprehensive list of post-MBT dorsally induced genes in the zebrafish embryo ( Figure 1B). Our results suggest that the majority of these genes are epigenetically marked during the activation of zygotic transcription, often with ''bivalent'' H3 K4/ K27 trimethylation ( Figure 5C). Given that in Xenopus dorsal b- catenin has a role in establishing a ''poised'' chromatin state prior to MBT [54], we propose that ich embryos, due to insufficient b- catenin-2 levels [25], lack these chromatin modifications and consequently fail to induce their dorsal developmental programs. ...
Citations
... This surprising finding has kindled tremendous interest in uncovering the molecular composition of the Organizer; this began shortly after its discovery with analyses of oxygen consumption (reviewed in refs. [8][9][10] and in more recent times primarily through expression cloning and transcriptomic screens (11)(12)(13)(14)(15)(16). However, our understanding of the proteome and metabolome, the comprehensive suite of proteins and small non-peptide molecules that carry out molecular functions, including signaling, is still lacking in the Organizer for any animal. ...
Molecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been mostly limited to transcripts and a few proteins, the latter due to limitations in detection and sensitivity. The Spemann–Mangold Organizer (SMO) in the South African Clawed Frog ( X. laevis ), a popular model of development, has long been known to be the origin of signals that pattern the mesoderm and central nervous system. Molecular screens of the SMO have identified several genes responsible for the ability of the SMO to establish the body axis. Nonetheless, a comprehensive study of proteins and metabolites produced specifically in the SMO and their functional roles has been lacking. Here, we pioneer a deep discovery proteomic and targeted metabolomic screen of the SMO in comparison to the remainder of the embryo using high-resolution mass spectrometry (HRMS). Quantification of ~4,600 proteins and a panel of targeted metabolites documented differential expression for 460 proteins and multiple intermediates of energy metabolism in the SMO. Upregulation of oxidative phosphorylation and redox regulatory proteins gave rise to elevated oxidative stress and an accumulation of reactive oxygen species in the SMO. Imaging experiments corroborated these findings, discovering enrichment of hydrogen peroxide in the SMO. Chemical perturbation of the redox gradient perturbed mesoderm involution during early gastrulation. HRMS expands the bioanalytical toolbox of cell and developmental biology, providing previously unavailable information on molecular classes to challenge and refine our classical understanding of the Organizer and its function during early patterning of the embryo.
... Classical embryological experiments in many vertebrate species have demonstrated the inductive prowess of the Organizer, wherein transplantation of this tissue to the ventral side of an embryo resulted in the formation of a secondary body axis containing properly patterned mesoderm and induced neural ectoderm (3)(4)(5)(6). This surprising finding has kindled tremendous interest in uncovering the molecular composition of the Organizer, which so far has been studied primarily through expression cloning and transcriptomic screens (7)(8)(9)(10)(11). However, our understanding about the was not certified by peer review) is the author/funder. ...
Molecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been limited to transcripts and some proteins due to limitations in detection and sensitivity. The Spemann-Mangold Organizer (SMO) in the South African Clawed Frog (X. laevis), a popular model of development, has long been discovered to induce the patterning of the central nervous system. Molecular screens on the tissue have identified several genes, such as goosecoid, chordin, and noggin, with independent ability to establish a body axis. A comprehensive study of proteins and metabolites produced in the SMO and their functional roles has been lacking. Here, we pioneer a deep discovery proteomic and targeted metabolomic screen of the SMO in comparison to the rest of the embryo using liquid chromatography high-resolution mass spectrometry (HRMS). Quantification of ~4,600 proteins and a panel of metabolites documented differential expression for ~450 proteins and multiple intermediates of energy metabolism in the SMO. Upregulation of oxidative phosphorylation (OXPHOS) and redox regulatory proteins gave rise to elevated oxidative stress and an accumulation of reactive oxygen species in the Organizer. Imaging experiments corroborated these findings, discovering enrichment of hydrogen peroxide in the SMO tissue. Chemical perturbation of the redox gradient affected mesoderm involution during early tissue movements of gastrulation. HRMS expands the bioanalytical toolbox of cell and developmental biology, providing previously unavailable information on molecular classes to challenge and refine our classical understanding of the Organizer and its function during early patterning of the embryo.
... Comparative analyses between different cell types or different genotypes are informative about the differences in genes expressed or proteins present between the groups, which, in many cases, are the cause for their distinct phenotypes. For instance, a comparative RNA-sequencing analysis between untreated and rescued zebrafish ichabod mutants, which exhibit severe ventralization due to defects in the Wnt/β-catenin pathway, led to the identification of genes differentially upregulated in the rescued embryos, suggesting that their expression is positively regulated by Wnt/β-catenin signaling (41,80). Further analyses revealed that the transcripts of some of these genes localize specifically to the dorsal part of the embryo, further strengthening the idea that they may be required for dorsoventral patterning. ...
Multicellular organisms develop complex shapes from much simpler, single-celled zygotes through a process commonly called morphogenesis. Morphogenesis involves an interplay between several factors, ranging from the gene regulatory networks determining cell fate and differentiation to the mechanical processes underlying cell and tissue shape changes. Thus, the study of morphogenesis has historically been based on multidisciplinary approaches at the interface of biology with physics and mathematics. Recent technological advances have further improved our ability to study morphogenesis by bridging the gap between the genetic and biophysical factors through the development of new tools for visualizing, analyzing, and perturbing these factors and their biochemical intermediaries. Here, we review how a combination of genetic, microscopic, biophysical, and biochemical approaches has aided our attempts to understand morphogenesis and discuss potential approaches that may be beneficial to such an inquiry in the future.
Expected final online publication date for the Annual Review of Genetics, Volume 55 is November 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Further, Ism1 has been identified as a clefting and craniofacial patterning gene in humans (Lansdon et al., 2018) and is required for normal hematopoiesis in developing zebrafish (Berrun et al., 2018), apart from acting as an angiogenesis inhibitor and preventing tumor growth (Xiang et al., 2011). In other independent studies, Ism1 expression has been described in conjunction with transcriptome analysis of dorsal-ventral patterning genes and in a genomewide RNA tomography study of zebrafish embryos (Bennett et al., 2007;Fodor et al., 2013;Junker et al., 2014). ...
Extracellular signals play essential roles during embryonic patterning by providing positional information in a concentration-dependent manner, and many such signals, like Wnt, fibroblast growth factor (FGF), Hedgehog (Hh), and retinoic acid, act by being secreted into the extracellular space, thereby triggering receptor-mediated responses in other cells. Isthmin1 (ism1) is a secreted protein whose gene expression pattern coincides with that of early dorsal determinants, nodal ligand genes like sqt and cyc, and with fgf8 during various phases of zebrafish development. Ism1 functions in early embryonic patterning and development are poorly understood; however, it has recently been shown to interact with nodal pathway genes to control organ asymmetry in chicken. Here, we show that misexpression of ism1 deletion constructs disrupts embryonic patterning in zebrafish and exhibits genetic interactions with both Fgf and nodal signaling. Unlike Fgf and nodal pathway mutants, CRISPR/Cas9-engineered ism1 mutants did not show obvious developmental defects. Further, in vivo single molecule fluorescence correlation spectroscopy (FCCS) showed that Ism1 diffuses freely in the extra-cellular space, with a diffusion coefficient similar to that of Fgf8a; however, our measurements do not support direct molecular interactions between Ism1 and either nodal ligands or Fgf8a in the developing zebrafish embryo. Together, data from gain- and loss-of-function experiments suggest that zebrafish Ism1 plays a complex role in regulating extracellular signals during early embryonic development.
... Whole transcriptome sequencing was performed as described previously [43]. Briefly, RNA quality and quantity measurements were performed on Bioanalyzer 2100 (Agilent Technologies Santa Clara, California, USA) and Qubit (Thermo Fisher Scientific, Waltham, MA USA; Q12210). ...
Microorganisms or LPS (lipopolysaccharide), an outer membrane component of Gram‐negative bacteria, can induce a systemic inflammatory response that leads to sepsis, multiple organ dysfunction, and mortality. Here, we investigated the role of cyclophilin D (CypD)‐dependent mitochondrial permeability transition (mPT) in the immunosuppressive phase of LPS‐induced endotoxic shock. The liver plays an important role in immunity and organ dysfunction; therefore, we used liver RNA sequencing (RNAseq) data, Ingenuity® Pathway Analysis (IPA ®) to investigate the complex role of mPT formation in inflammatory reprogramming and disease progression. LPS induced significant changes in the expression of 2844 genes, affecting 179 pathways related to mitochondrial dysfunction, defective oxidative phosphorylation, nitric oxide (NO) and reactive oxygen species (ROS) accumulation, nuclear factor, erythroid 2 like 2 (Nrf2), Toll‐like receptors (TLRs), and tumor necrosis factor α receptor (TNFR)‐mediated processes in wild‐type mice. The disruption of CypD reduced LPS‐induced alterations in gene expression and pathways involving TNFRs and TLRs, in addition to improving survival and attenuating oxidative liver damage and the related NO‐ and ROS‐producing pathways. CypD deficiency diminished the suppressive effect of LPS on mitochondrial function, nuclear‐ and mitochondrial‐encoded genes, and mitochondrial DNA (mtDNA) quantity, which could be critical in improving survival. Our data propose that CypD‐dependent mPT is an amplifier in inflammatory reprogramming and promotes disease progression. The mortality in human sepsis and shock is associated with mitochondrial dysfunction. Prevention of mPT by CypD disruption reduces inflammatory reprogramming, mitochondrial dysfunction, and lethality; therefore, CypD can be a novel drug target in endotoxic shock and related inflammatory diseases.
... The zygotic expression of both ligands initiates in the organizer and margin of the embryo (Fig. 4) (Langdon & Mullins, 2011;Tuazon & Mullins, 2015). These same domains also express the FGF antagonists sprouty2, sprouty4, and sef1, as well as the Wnt antagonist dkk1b (Fodor et al., 2013;Zinski et al., 2018). The interaction between the ligands and their antagonists establishes a high Wnt/Fgf signaling region along the margin, with levels decreasing toward the animal pole, translating the initial animal-vegetal polarity of the oocyte into the AP axis of the embryo (Green et al., 2015;Tuazon & Mullins, 2015). ...
Axis specification of the zebrafish embryo begins during oogenesis and relies on proper formation of well-defined cytoplasmic domains within the oocyte. Upon fertilization, maternally-regulated cytoplasmic flow and repositioning of dorsal determinants establish the coordinate system that will build the structure and developmental body plan of the embryo. Failure of specific genes that regulate the embryonic coordinate system leads to catastrophic loss of body structures. Here, we review the genetic principles of axis formation and discuss how maternal factors orchestrate axis patterning during zebrafish early embryogenesis. We focus on the molecular identity and functional contribution of genes controlling critical aspects of oogenesis, egg activation, blastula, and gastrula stages. We examine how polarized cytoplasmic domains form in the oocyte, which set off downstream events such as animal-vegetal polarity and germ line development. After gametes interact and form the zygote, cytoplasmic segregation drives the animal-directed reorganization of maternal determinants through calcium- and cell cycle-dependent signals. We also summarize how maternal genes control dorsoventral, anterior-posterior, mesendodermal, and left-right cell fate specification and how signaling pathways pattern these axes and tissues during early development to instruct the three-dimensional body plan. Advances in reverse genetics and phenotyping approaches in the zebrafish model are revealing positional patterning signatures at the single-cell level, thus enhancing our understanding of genotype-phenotype interactions in axis formation. Our emphasis is on the genetic interrogation of novel and specific maternal regulatory mechanisms of axis specification in the zebrafish.
... The lack of mesodermal involution and likely convergent extension in amphioxus gastrulation indicates that the developmental program for mesodermal involution in vertebrates is absent in amphioxus. Disruption of cadherin-mediated cell-cell adhesion is essential during mesodermal involution and convergent extension in vertebrates, and fibronectin leucine-rich-repeat transmembrane 3 (Flrt3) and a small GTPase (Rnd1) control C-cadherin degradation [26,34,52,53]. A BLAST search revealed a homologue of rnd1 in amphioxus, but not of flrt3 (Additional file 1: Figure S5A and B). ...
Introduction:
The vertebrate head is characterized by unsegmented head mesoderm the evolutionary origin of which remains enigmatic. The head mesoderm is derived from the rostral part of the dorsal mesoderm, which is regionalized anteroposteriorly during gastrulation. The basal chordate amphioxus resembles vertebrates due to the presence of somites, but it lacks unsegmented head mesoderm. Gastrulation in amphioxus occurs by simple invagination with little mesodermal involution, whereas in vertebrates gastrulation is organized by massive cell movements, such as involution, convergence and extension, and cell migration.
Results:
To identify key developmental events in the evolution of the vertebrate head mesoderm, we compared anterior/posterior (A/P) patterning mechanisms of the dorsal mesoderm in amphioxus and vertebrates. The dorsal mesodermal genes gsc, bra, and delta are expressed in similar patterns in early embryos of both animals, but later in development, these expression domains become anteroposteriorly segregated only in vertebrates. Suppression of mesodermal involution in vertebrate embryos by inhibition of convergence and extension recapitulates amphioxus-like dorsal mesoderm formation.
Conclusions:
Reorganization of ancient mesoderm was likely involved in the evolution of the vertebrate head.
... Whole transcriptome sequencing was performed as described previously [18]. Briefly, total RNA samples from three biological replicates were pooled in equimolar concentrations and processed using the SOLiD total RNA-Seq Kit (Life Technologies), according to the manufacturer's instructions. ...
To better understand the molecular events underlying vulvovaginal candidiasis, we established an in vitro system. Immortalized vaginal epithelial cells were infected with live, yeast form C. albicans and C. albicans cultured in the same medium without vaginal epithelial cells were used as control. In both cases a yeast to hyphae transition was robustly induced. Whole transcriptome sequencing was used to identify specific gene expression changes in C. albicans. Numerous genes leading to a yeast to hyphae transition and hyphae specific genes were upregulated in the control hyphae and the hyphae in response to vaginal epithelial cells. Strikingly, the GlcNAc pathway was exclusively triggered by vaginal epithelial cells. Functional analysis in our in vitro system revealed that the GlcNAc biosynthesis is involved in the adherence to, and the ability to kill, vaginal epithelial cells in vitro, thus indicating the key role for this pathway in the virulence of C. albicans upon vulvovaginal candidiasis.
... Nodal signaling induces and patterns mesendoderm and establishes left-right asymmetry (Conlon et al., 1994;Shen, 2007;Grande and Patel, 2009;Schier, 2009;Duboc et al., 2010;Shiratori and Hamada, 2014). The Nodal signaling pathway regulates dozens of genes, ranging from transcription factors to cytoskeletal components, in order to pattern embryonic tissues (Bennett et al., 2007;Liu et al., 2011;Fodor et al., 2013). In embryonic stem cells, Nodal signaling is required for self-renewal as well as specification of endoderm and mesoderm (James et al., 2005;Vallier et al., 2005;Schier, 2009;Oshimori and Fuchs, 2012;Chen et al., 2013). ...
Morphogen gradients expose cells to different signal concentrations and induce target genes with different ranges of expression. To determine how the Nodal morphogen gradient induces distinct gene expression patterns during zebrafish embryogenesis, we measured the activation dynamics of the signal transducer Smad2 and the expression kinetics of long-and short-range target genes. We found that threshold models based on ligand concentration are insufficient to predict the response of target genes. Instead, morphogen interpretation is shaped by the kinetics of target gene induction: the higher the rate of transcription and the earlier the onset of induction, the greater the spatial range of expression. Thus, the timing and magnitude of target gene expression can be used to modulate the range of expression and diversify the response to morphogen gradients.
... Seurat inferred that these 'apoptotic-like' cells were scattered throughout the developing embryo, although they originated more frequently toward the animal and ventral poles (Fig. 5g, purple). Notably, these cells were not an artifact of the isolation process: they were identified in 10 separate embryos, in each experimental batch, and previous in situ analysis for foxo3b, aplnrb, and isg15 revealed their individual scattered expression 38,39 . ...
Spatial localization is a key determinant of cellular fate and behavior, but methods for spatially resolved, transcriptome-wide gene expression profiling across complex tissues are lacking. RNA staining methods assay only a small number of transcripts, whereas single-cell RNA-seq, which measures global gene expression, separates cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos and generated a transcriptome-wide map of spatial patterning. We confirmed Seurat's accuracy using several experimental approaches, then used the strategy to identify a set of archetypal expression patterns and spatial markers. Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems.
