Endoderm Gene Regulatory Networks Research Articles

SOX transcription factors direct TCF-independent WNT/β-catenin responsive transcription to govern cell fate in human pluripotent stem cells.

WNT/β-catenin signaling controls gene expression across biological contexts from development and stem cell homeostasis to diseases including cancer. How β-catenin is recruited to distinct enhancers to activate context-specific transcription is unclear, given that most WNT/ß-catenin-responsive transcription is thought to be mediated by TCF/LEF transcription factors (TFs). With time-resolved multi-omic analyses, we show that SOX TFs can direct lineage-specific WNT-responsive transcription during the differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm and neuromesodermal progenitors. We demonstrate that SOX17 and SOX2 are required to recruit β-catenin to lineage-specific WNT-responsive enhancers, many of which are not occupied by TCFs. At TCF-independent enhancers, SOX TFs establish a permissive chromatin landscape and recruit a WNT-enhanceosome complex to activate SOX/ß-catenin-dependent transcription. Given that SOX TFs and the WNT pathway are critical for specification of most cell types, these results have broad mechanistic implications for the specificity of WNT responses across developmental and disease contexts.

Sox17 and β-catenin co-occupy Wnt-responsive enhancers to govern the endoderm gene regulatory network.

Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN in Xenopus gastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and β-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and β-catenin synergistically activate transcription apparently independent of Tcfs, whereas on other enhancers, Sox17 represses β-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/β-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and β-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts.

Natural cryptic variation in epigenetic modulation of an embryonic gene regulatory network

Gene regulatory networks (GRNs) that direct animal embryogenesis must respond to varying environmental and physiological conditions to ensure robust construction of organ systems. While GRNs are evolutionarily modified by natural genomic variation, the roles of epigenetic processes in shaping plasticity of GRN architecture are not well understood. The endoderm GRN in Caenorhabditis elegans is initiated by the maternally supplied SKN-1/Nrf2 bZIP transcription factor; however, the requirement for SKN-1 in endoderm specification varies widely among distinct C. elegans wild isotypes, owing to rapid developmental system drift driven by accumulation of cryptic genetic variants. We report here that heritable epigenetic factors that are stimulated by transient developmental diapause also underlie cryptic variation in the requirement for SKN-1 in endoderm development. This epigenetic memory is inherited from the maternal germline, apparently through a nuclear, rather than cytoplasmic, signal, resulting in a parent-of-origin effect (POE), in which the phenotype of the progeny resembles that of the maternal founder. The occurrence and persistence of POE varies between different parental pairs, perduring for at least 10 generations in one pair. This long-perduring POE requires piwi-interacting RNA (piRNA) function and the germline nuclear RNA interference (RNAi) pathway, as well as MET-2 and SET-32, which direct histone H3K9 trimethylation and drive heritable epigenetic modification. Such nongenetic cryptic variation may provide a resource of additional phenotypic diversity through which adaptation may facilitate evolutionary changes and shape developmental regulatory systems.

Evolution and Developmental System Drift in the Endoderm Gene Regulatory Network of Caenorhabditis and Other Nematodes.

Developmental gene regulatory networks (GRNs) underpin metazoan embryogenesis and have undergone substantial modification to generate the tremendous variety of animal forms present on Earth today. The nematode Caenorhabditis elegans has been a central model for advancing many important discoveries in fundamental mechanistic biology and, more recently, has provided a strong base from which to explore the evolutionary diversification of GRN architecture and developmental processes in other species. In this short review, we will focus on evolutionary diversification of the GRN for the most ancient of the embryonic germ layers, the endoderm. Early embryogenesis diverges considerably across the phylum Nematoda. Notably, while some species deploy regulative development, more derived species, such as C. elegans, exhibit largely mosaic modes of embryogenesis. Despite the relatively similar morphology of the nematode gut across species, widespread variation has been observed in the signaling inputs that initiate the endoderm GRN, an exemplar of developmental system drift (DSD). We will explore how genetic variation in the endoderm GRN helps to drive DSD at both inter- and intraspecies levels, thereby resulting in a robust developmental system. Comparative studies using divergent nematodes promise to unveil the genetic mechanisms controlling developmental plasticity and provide a paradigm for the principles governing evolutionary modification of an embryonic GRN.

A biphasic role of non-canonical Wnt16 signaling during early anterior-posterior patterning and morphogenesis of the sea urchin embryo.

ABSTRACTA Wnt signaling network governs early anterior-posterior (AP) specification and patterning of the deuterostome sea urchin embryo. We have previously shown that non-canonical Fzl1/2/7 signaling antagonizes the progressive posterior-to-anterior downregulation of the anterior neuroectoderm (ANE) gene regulatory network (GRN) by canonical Wnt/β-catenin and non-canonical Wnt1/Wnt8-Fzl5/8-JNK signaling. This study focuses on the non-canonical function of the Wnt16 ligand during early AP specification and patterning. Maternally supplied wnt16 is expressed ubiquitously during cleavage and zygotic wnt16 expression is concentrated in the endoderm/mesoderm beginning at mid-blastula stage. Wnt16 antagonizes the ANE restriction mechanism and this activity depends on a functional Fzl1/2/7 receptor. Our results also show that zygotic wnt16 expression depends on both Fzl5/8 and Wnt/β-catenin signaling. Furthermore, Wnt16 is necessary for the activation and/or maintenance of key regulatory endoderm/mesoderm genes and is essential for gastrulation. Together, our data show that Wnt16 has two functions during early AP specification and patterning: (1) an initial role activating the Fzl1/2/7 pathway that antagonizes the ANE restriction mechanism; and (2) a subsequent function in activating key endoderm GRN factors and the morphogenetic movements of gastrulation.

Extensive intraspecies cryptic variation in an ancient embryonic gene regulatory network.

Innovations in metazoan development arise from evolutionary modification of gene regulatory networks (GRNs). We report widespread cryptic variation in the requirement for two key regulatory inputs, SKN-1/Nrf2 and MOM-2/Wnt, into the C. elegans endoderm GRN. While some natural isolates show a nearly absolute requirement for these two regulators, in others, most embryos differentiate endoderm in their absence. GWAS and analysis of recombinant inbred lines reveal multiple genetic regions underlying this broad phenotypic variation. We observe a reciprocal trend, in which genomic variants, or knockdown of endoderm regulatory genes, that result in a high SKN-1 requirement often show low MOM-2/Wnt requirement and vice-versa, suggesting that cryptic variation in the endoderm GRN may be tuned by opposing requirements for these two key regulatory inputs. These findings reveal that while the downstream components in the endoderm GRN are common across metazoan phylogeny, initiating regulatory inputs are remarkably plastic even within a single species.

In Vivo Regulation of the Zebrafish Endoderm Progenitor Niche by T-Box Transcription Factors

SummaryT-box transcription factors T/Brachyury homolog A (Ta) and Tbx16 are essential for correct mesoderm development in zebrafish. The downstream transcriptional networks guiding their functional activities are poorly understood. Additionally, important contributions elsewhere are likely masked due to redundancy. Here, we exploit functional genomic strategies to identify Ta and Tbx16 targets in early embryogenesis. Surprisingly, we discovered they not only activate mesodermal gene expression but also redundantly regulate key endodermal determinants, leading to substantial loss of endoderm in double mutants. To further explore the gene regulatory networks (GRNs) governing endoderm formation, we identified targets of Ta/Tbx16-regulated homeodomain transcription factor Mixl1, which is absolutely required in zebrafish for endoderm formation. Interestingly, we find many endodermal determinants coordinately regulated through common genomic occupancy by Mixl1, Eomesa, Smad2, Nanog, Mxtx2, and Pou5f3. Collectively, these findings augment the endoderm GRN and reveal a panel of target genes underlying the Ta, Tbx16, and Mixl1 mutant phenotypes.

Co-expression of Foxa.a, Foxd and Fgf9/16/20 defines a transient mesendoderm regulatory state in ascidian embryos.

In many bilaterian embryos, nuclear β-catenin (nβ-catenin) promotes mesendoderm over ectoderm lineages. Although this is likely to represent an evolutionary ancient developmental process, the regulatory architecture of nβ-catenin-induced mesendoderm remains elusive in the majority of animals. Here, we show that, in ascidian embryos, three nβ-catenin transcriptional targets, Foxa.a, Foxd and Fgf9/16/20, are each required for the correct initiation of both the mesoderm and endoderm gene regulatory networks. Conversely, these three factors are sufficient, in combination, to produce a mesendoderm ground state that can be further programmed into mesoderm or endoderm lineages. Importantly, we show that the combinatorial activity of these three factors is sufficient to reprogramme developing ectoderm cells to mesendoderm. We conclude that in ascidian embryos, the transient mesendoderm regulatory state is defined by co-expression of Foxa.a, Foxd and Fgf9/16/20.

Brachyury and SMAD signalling collaboratively orchestrate distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells.

The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors. We show that BRA has distinct genome-wide binding landscapes in these two cell populations, and that BRA interacts and collaborates with SMAD1 or SMAD2/3 signalling to regulate the expression of its target genes in a cell-specific manner. Importantly, by manipulating the levels of BRA in cells exposed to different signalling environments, we demonstrate that BRA is essential for mesoderm but not for endoderm formation. Together, our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context dependent. Our study reinforces the importance of analysing the functions of a transcription factor in different cellular and signalling environments.

Elucidation of the endoderm gene regulatory network by integrating computational methods and perturbation analyses in Xenopus

Elucidation of the endoderm gene regulatory network by integrating computational methods and perturbation analyses in Xenopus

The endoderm gene regulatory network in sea urchin embryos up to mid-blastula stage

As the result of early specification processes, sea urchin embryos eventually form various mesodermal cell lineages and a gut consisting of fore-, mid- and hindgut. The progression of specification as well as the overall spatial organization of the organism is encoded in its gene regulatory networks (GRNs). We have analyzed the GRN driving endoderm specification up to the onset of gastrulation and present in this paper the mechanisms which determine this process up to mid-blastula stage. At this stage, the embryo consists of two separate lineages of endoderm precursor cells with distinct regulatory states. One of these lineages, the veg2 cell lineage, gives rise to endoderm and mesoderm cell types. The separation of these cell fates is initiated by the spatially confined activation of the mesoderm GRN superimposed on a generally activated endoderm GRN within veg2 descendants. Here we integrate the architecture of regulatory interactions with the spatial restriction of regulatory gene expression to model the logic control of endoderm development.

Functional analysis of the evolutionarily conserved cis-regulatory elements on the sox17 gene in zebrafish

The Sox17 is an important transcription factor for endodermal cells (Danio rerio). According to the predictions of the GRNs, based on perturbation experiments and literature search, the sox17 gene is engaged with two other regulatory genes, sox32 and pou5f1. Nodal signaling operated on several endoderm-specific transcription factors to determine the endoderm specification. In addition, endoderm specification requires the Fgf and Bmp signaling pathways to be repressed in the cells which will become endoderm. It is predicted that Nodal activates sox32 and works synergistically with Pou5f1 to activate sox17. Bmp represses the expression of sox17 on the ventral side and Fgf represses it on the dorsal side. The regulatory inputs of sox17 at the genomic sequence level are not known. Here, we have uncovered the relevant sox17 cis-regulatory elements, and examined the specific input predictions of the GRNs. We discovered three conserved modules, A, B, and C, with a synergistic effect among them. We revealed that the Pou5f1-binding element on the B module and the Sox32-binding element on the C module work synergistically. Furthermore, an evolutionarily non-conserved R module exhibits a repressive effect on both the ventral and dorsal side. We have directly demonstrated the structural and functional relationships of the genomic code at this key node of the endoderm GRNs in zebrafish development. This information provides new insight into the complexity of endoderm formation and serves as a valuable resource for the establishment of a complete endoderm gene regulatory network.

Global analysis of the transcriptional network controllingXenopusendoderm formation

A conserved molecular pathway has emerged controlling endoderm formation in Xenopus zebrafish and mice. Key genes in this pathway include Nodal ligands and transcription factors of the Mix-like paired homeodomain class, Gata4-6 zinc-finger factors and Sox17 HMG domain proteins. Although a linear epistatic pathway has been proposed, the precise hierarchical relationships between these factors and their downstream targets are largely unresolved. Here, we have used a combination of microarray analysis and loss-of-function experiments to examine the global regulatory network controlling Xenopus endoderm formation. We identified over 300 transcripts enriched in the gastrula endoderm, including most of the known endoderm regulators and over a hundred uncharacterized genes. Surprisingly only 10% of the endoderm transcriptome is regulated as predicted by the current linear model. We find that Nodal genes, Mixer and Sox17 have both shared and distinct sets of downstream targets, and that a number of unexpected autoregulatory loops exist between Sox17 and Gata4-6, between Sox17 and Bix1/Bix2/Bix4, and between Sox17 and Xnr4. Furthermore, we find that Mixer does not function primarily via Sox17 as previously proposed. These data provides new insight into the complexity of endoderm formation and will serve as valuable resource for establishing a complete endoderm gene regulatory network.