Zebrafish Endoderm Research Articles

Gata6 functions in zebrafish endoderm to regulate late differentiating arterial pole cardiogenesis.

Mutations in GATA6 are associated with congenital heart disease, most notably conotruncal structural defects. However, how GATA6 regulates cardiac morphology during embryogenesis is undefined. We used knockout and conditional mutant zebrafish alleles to investigate the spatiotemporal role of gata6 during cardiogenesis. Loss of gata6 specifically impacts atrioventricular valve formation and recruitment of epicardium, with a prominent loss of arterial pole cardiac cells, including those of the ventricle and outflow tract. However, there are no obvious defects in cardiac progenitor cell specification, proliferation or death. Conditional loss of gata6 starting at 24 h is sufficient to disrupt the addition of late differentiating cardiomyocytes at the arterial pole, with decreased expression levels of anterior secondary heart field (SHF) markers spry4 and mef2cb. Conditional loss of gata6 in the endoderm is sufficient to phenocopy the straight knockout, resulting in a significant loss of ventricular and outflow tract tissue. Exposure to a Dusp6 inhibitor largely rescues the loss of ventricular cells in gata6-/- larvae. Thus, gata6 functions in endoderm are mediated by FGF signaling to regulate the addition of anterior SHF progenitor derivatives during heart formation.

Investigating the molecular guts of endoderm formation using zebrafish.

The vertebrate endoderm makes major contributions to the respiratory and gastrointestinal tracts and all associated organs. Zebrafish and humans share a high degree of genetic homology and strikingly similar endodermal organ systems. Combined with a multitude of experimental advantages, zebrafish are an attractive model organism to study endoderm development and disease. Recent functional genomics studies have shed considerable light on the gene regulatory programs governing early zebrafish endoderm development, while advances in biological and technological approaches stand to further revolutionize our ability to investigate endoderm formation, function and disease. Here, we discuss the present understanding of endoderm specification in zebrafish compared to other vertebrates, how current and emerging methods will allow refined and enhanced analysis of endoderm formation, and how integration with human data will allow modeling of the link between non-coding sequence variants and human disease.

Variability of an Early Developmental Cell Population Underlies Stochastic Laterality Defects

SummaryEmbryonic development seemingly proceeds with almost perfect precision. However, it is largely unknown how much underlying microscopic variability is compatible with normal development. Here, we quantify embryo-to-embryo variability in vertebrate development by studying cell number variation in the zebrafish endoderm. We notice that the size of a sub-population of the endoderm, the dorsal forerunner cells (DFCs, which later form the left-right organizer), exhibits significantly more embryo-to-embryo variation than the rest of the endoderm. We find that, with incubation of the embryos at elevated temperature, the frequency of left-right laterality defects is increased drastically in embryos with a low number of DFCs. Furthermore, we observe that these fluctuations have a large stochastic component among fish of the same genetic background. Hence, a stochastic variation in early development leads to a remarkably strong macroscopic phenotype. These fluctuations appear to be associated with maternal effects in the specification of the DFCs.

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.

Nuclear movement regulated by non-Smad Nodal signaling via JNK is associated with Smad signaling during zebrafish endoderm specification.

Asymmetric nuclear positioning is observed during animal development, but its regulation and significance in cell differentiation remain poorly understood. Using zebrafish blastulae, we provide evidence that nuclear movement towards the yolk syncytial layer, which comprises extraembryonic tissue, occurs in the first cells fated to differentiate into the endoderm. Nodal signaling is essential for nuclear movement, whereas nuclear envelope proteins are involved in movement through microtubule formation. Positioning of the microtubule-organizing center, which is proposed to be crucial for nuclear movement, is regulated by Nodal signaling and nuclear envelope proteins. The non-Smad JNK signaling pathway, which is downstream of Nodal signaling, regulates nuclear movement independently of the Smad pathway, and this nuclear movement is associated with Smad signal transduction toward the nucleus. Our study provides insight into the function of nuclear movement in Smad signaling toward the nucleus, and could be applied to the control of TGFβ signaling.

A role for planar cell polarity during early endoderm morphogenesis.

ABSTRACTThe zebrafish endoderm begins to develop at gastrulation stages as a monolayer of cells. The behaviour of the endoderm during gastrulation stages is well understood. However, knowledge of the morphogenic movements of the endoderm during somitogenesis stages, as it forms a mesenchymal rod, is lacking. Here we characterise endodermal development during somitogenesis stages, and describe the morphogenic movements as the endoderm transitions from a monolayer of cells into a mesenchymal endodermal rod. We demonstrate that, unlike the overlying mesoderm, endodermal cells are not polarised during their migration to the midline at early somitogenesis stages. Specifically, we describe the stage at which endodermal cells begin to leave the monolayer, a process we have termed ‘midline aggregation’. The planar cell polarity (PCP) signalling pathway is known to regulate mesodermal and ectodermal cell convergence towards the dorsal midline. However, a role for PCP signalling in endoderm migration to the midline during somitogenesis stages has not been established. In this report, we investigate the role for PCP signalling in multiple phases of endoderm development during somitogenesis stages. Our data exclude involvement of PCP signalling in endodermal cells as they leave the monolayer.

Fscn1 is required for the trafficking of TGF-β family type I receptors during endoderm formation.

Microtubules function in TGF-β signalling by facilitating the cytoplasmic trafficking of internalized receptors and the nucleocytoplasmic shuttling of Smads. However, nothing is known about whether actin filaments are required for these processes. Here we report that zebrafish actin-bundling protein fscn1a is highly expressed in mesendodermal precursors and its expression is directly regulated by the TGF-β superfamily member Nodal. Knockdown or knockout of fscn1a leads to a reduction of Nodal signal transduction and endoderm formation in zebrafish embryos. Fscn1 specifically interacts with TGF-β family type I receptors, and its depletion disrupts the association between receptors and actin filaments and sequesters the internalized receptors into clathrin-coated vesicles. Therefore, Fscn1 acts as a molecular linker between TGF-β family type I receptors and the actin filaments to promote the trafficking of internalized receptors from clathrin-coated vesicles to early endosomes during zebrafish endoderm formation.

Multiple Ribosomal Proteins Are Expressed at High Levels in Developing Zebrafish Endoderm and Are Required for Normal Exocrine Pancreas Development

Ribosomal protein L (rpl) genes are essential for assembly of the 60S subunit of the eukaryotic ribosome and may also carry out additional extra-ribosomal functions. We have identified a common expression pattern for rpl genes in developing zebrafish larvae. After initially widespread expression in early embryos, the expression of multiple rpl genes becomes increasingly restricted to the endoderm. With respect to the pancreas, rpl genes are highly expressed in ptf1a-expressing pancreatic progenitors at 48 hpf, suggesting possible functional roles in pancreatic morphogenesis and/or differentiation. Utilizing two available mutant lines, rpl23a(hi2582) and rpl6(hi3655b), we found that ptf1a-expressing pancreatic progenitors fail to properly expand in embryos homozygous for either of these genes. In addition to these durable homozygous phenotypes, we also demonstrated recoverable delays in ptf1a-expressing pancreatic progenitor expansion in rpl23a(hi2582) and rpl6(hi3655b) heterozygotes. Disruptions in ribosome assembly are generally understood to initiate a p53-dependent cellular stress response. However, concomitant p53 knockdown was unable to rescue normal pancreatic progenitor expansion in either rpl23a(hi2582) or rpl6(hi3655b) mutant embryos, suggesting required and p53-independent roles for rpl23a and rpl6 in pancreas development.

Sfrp5 Modulates Both Wnt and BMP Signaling and Regulates Gastrointestinal Organogensis in the Zebrafish, Danio rerio

Sfrp5 belongs to the family of secreted frizzled related proteins (Sfrp), secreted inhibitors of Wingless-MMTV Integration Site (Wnt) signaling, which play an important role in cancer and development. We selected sfrp5 because of its compelling expression profile in the developing endoderm in zebrafish, Danio rerio. In this study, overexpression of sfrp5 in embryos results in defects in both convergent extension (CE) by inhibition of non-canonical Wnt signaling and defects in dorsoventral patterning by inhibition of Tolloid-mediated proteolysis of the BMP inhibitor Chordin. From 25 hours post fertilization (hpf) to 3 days post fertilization (dpf), both overexpression and knockdown of Sfrp5 decrease the size of the endoderm, significantly reducing liver cell number. At 3 dpf, insulin-positive endodermal cells fail to coalesce into a single pancreatic islet. We show that Sfrp5 inhibits both canonical and non-canonical Wnt signaling during embryonic and endodermal development, resulting in endodermal abnormalities.

Control of Cell Identity in Pancreas Development and Regeneration

The endocrine and exocrine cells in the adult pancreas are not static, but can change their differentiation state in response to injury or stress. This concept of cells in flux means that there may be ways to generate certain types of cells (such as insulin-producing β-cells) and prevent formation of others (such as transformed neoplastic cells). We review different aspects of cell identity in the pancreas, discussing how cells achieve their identity during embryonic development and maturation, and how this identity remains plastic, even in the adult pancreas.

An evolutionarily conserved kernel of gata5, gata6, otx2 and prdm1a operates in the formation of endoderm in zebrafish

An evolutionarily conserved subcircuit (kernel) dedicated to a specific developmental function is found at the top of the gene regulatory networks (GRNs) hierarchy. Here we comprehensively demonstrate that a pan-deuterostome endoderm specification kernel exists in zebrafish. We analyzed interactions among gata5, gata6, otx2 and prdm1a using specific morpholino knockdowns and measured the gene expression profiles by quantitative real-time RT-PCR and in situ hybridization. The mRNA rescue experiment validated the specificity of the morpholino knockdown. We found that the interactions among gata5, gata6, otx2 and prdm1a determine the initial specification of the zebrafish endoderm. Although otx2 can activate both gata5 and gata6, and the prdm1a/krox homologue also activates some endoderm transcription factors, a feedback loop from Gata to otx2 and prdm1a is missing. Furthermore, we found the positive regulation between gata5 and gata6 to further lock-on the mesendoderm specification by the Gata family. Chromatin immunoprecipitation was used to further validate the recruitment of Otx2 to the gata5 and gata6 loci . Functional assays revealed that module B of gata6 and the basal promoter of gata5 drive the gene at the mesendoderm, and mutational analysis demonstrated that Otx2 and Gata5/6 contribute to reporter gene activation. This is the first direct evidence for evolutionarily conserved endoderm specification across echinoderms and vertebrates.

Long-range gene regulation links genomic type 2 diabetes and obesity risk regions to HHEX , SOX4 , and IRX3

Genome-wide association studies identified noncoding SNPs associated with type 2 diabetes and obesity in linkage disequilibrium (LD) blocks encompassing HHEX-IDE and introns of CDKAL1 and FTO [Sladek R, et al. (2007) Nature 445:881-885; Steinthorsdottir V, et al. (2007) Nat. Genet 39:770-775; Frayling TM, et al. (2007) Science 316:889-894]. We show that these LD blocks contain highly conserved noncoding elements and overlap with the genomic regulatory blocks of the transcription factor genes HHEX, SOX4, and IRX3. We report that human highly conserved noncoding elements in LD with the risk SNPs drive expression in endoderm or pancreas in transgenic mice and zebrafish. Both HHEX and SOX4 have recently been implicated in pancreas development and the regulation of insulin secretion, but IRX3 had no prior association with pancreatic function or development. Knockdown of its orthologue in zebrafish, irx3a, increased the number of pancreatic ghrelin-producing epsilon cells and decreased the number of insulin-producing beta-cells and glucagon-producing alpha-cells, thereby suggesting a direct link of pancreatic IRX3 function to both obesity and type 2 diabetes.

Mesodermal Wnt4a signaling regulates segmentation of head mesoderm and pharyngeal endoderm in zebrafish

Mesodermal Wnt4a signaling regulates segmentation of head mesoderm and pharyngeal endoderm in zebrafish

Endothelial Signals Modulate Hepatocyte Apicobasal Polarization in Zebrafish

Emerging evidence indicates that paracrine signals from endothelial cells play a role in tissue differentiation and organ formation [1-3]. Here, we identify a novel role for endothelial cells in modulating hepatocyte polarization during liver organogenesis. We find that in zebrafish, the apical domain of the hepatocytes predicts the location of the intrahepatic biliary network. The refinement of hepatocyte polarization coincides with the invasion of endothelial cells into the liver, and these endothelial cells migrate along the maturing basal surface of the hepatocytes. Using genetic, pharmacological, and transplantation experiments, we provide evidence that endothelial cells influence the polarization of the adjacent hepatocytes. This influence of endothelial cells on hepatocytes is mediated at least in part by the cell-surface protein Heart of glass and contributes to the establishment of coordinately aligned hepatocyte apical membranes and evenly spaced intrahepatic conduits.

Intra-endodermal interactions are required for pancreatic β-cell induction

The cellular origin of signals that regulate pancreatic β cell induction is not clearly defined. Here, we investigate the seeming paradox that Hedgehog/Smoothened signaling functions during gastrulation to promote pancreatic β cell development in zebrafish, yet has an inhibitory role during later stages of pancreas development in amniotes. Our cell transplantation experiments reveal that in zebrafish, Smoothened function is not required in β cell precursors. At early somitogenesis stages, when the zebrafish endoderm first forms a sheet, pancreatic β cell precursors lie closest to the midline; however, the requirement for Smoothened lies in their lateral neighbors, which ultimately give rise to the exocrine pancreas and intestine. Thus, pancreatic β cell induction requires Smoothened function cell-nonautonomously during gastrulation, to allow subsequent intra-endodermal interactions. These results clarify the function of Hedgehog signaling in pancreas development, identify an unexpected cellular source of factors that regulate β cell specification, and uncover complex patterning and signaling interactions within the endoderm.

Intra-Endodermal Interactions Are Required for Pancreatic β Cell Induction

The cellular origin of signals that regulate pancreatic beta cell induction is not clearly defined. Here, we investigate the seeming paradox that Hedgehog/Smoothened signaling functions during gastrulation to promote pancreatic beta cell development in zebrafish, yet has an inhibitory role during later stages of pancreas development in amniotes. Our cell transplantation experiments reveal that in zebrafish, Smoothened function is not required in beta cell precursors. At early somitogenesis stages, when the zebrafish endoderm first forms a sheet, pancreatic beta cell precursors lie closest to the midline; however, the requirement for Smoothened lies in their lateral neighbors, which ultimately give rise to the exocrine pancreas and intestine. Thus, pancreatic beta cell induction requires Smoothened function cell-nonautonomously during gastrulation, to allow subsequent intra-endodermal interactions. These results clarify the function of Hedgehog signaling in pancreas development, identify an unexpected cellular source of factors that regulate beta cell specification, and uncover complex patterning and signaling interactions within the endoderm.

Cdx4 is required in the endoderm to localize the pancreas and limitβ-cell number

Cdx transcription factors have crucial roles in anteroposterior patterning of the nervous system and mesoderm. Here we focus on the role of cdx4 in patterning the endoderm in zebrafish. We show that cdx4 has roles in determining pancreatic beta-cell number, directing midline convergence of beta-cells during early pancreatic islet formation, and specifying the anteroposterior location of foregut organs. Embryos deficient in cdx4 have a posteriorly shifted pancreas, liver and small intestine. The phenotype is more severe with knockdown of an additional Cdx factor, cdx1a. We show that cdx4 functions within the endoderm to localize the pancreas. Morpholino knockdown of cdx4 specifically in the endoderm recapitulates the posteriorly shifted pancreas observed in cdx4 mutants. Conversely, overexpression of cdx4 specifically in the endoderm is sufficient to shift the pancreas anteriorly. Together, these results suggest a model in which cdx4 confers posterior identity to the endoderm. Cdx4 might function to block pancreatic identity by preventing retinoic acid (RA) signal transduction in posterior endoderm. In support of this, we demonstrate that in cdx4-deficient embryos treated with RA, ectopic beta-cells are located well posterior to the normal pancreatic domain.

Pancreatic Development and Disease

Pancreatic Development and Disease

A look at life from the homeodomain

​ThisThis workshop brought together developmental biologists and oncologists for three intense days of discussions about the recent progress that has been made on the roles of homeodomain proteins in development, haematopoiesis and leukaemogenesis. The homeo-domain is a 61-amino-acid DNA-binding motif with specific sequence and structural characteristics (Gehring & Hiromi, 1986). Homeodomain proteins were initially identified in Drosophila as being responsible, when mutated, for homeotic transformations—that is, the conversion of a part of the body into the likeness of another. Since their initial discovery, the number of homeodomain proteins has increased enormously, and they comprise several different families of transcription factors. The main stars of the meeting were members of the Hox family and of the three amino-acid loop extension (TALE) families, with some other homeodomain proteins making guest appearances. TALE proteins include the PBC family (Exd in Drosophila and Pbx proteins in vertebrates) and the Hth/Meis superfamily (Hth in Drosophila, and Meis and Prep proteins in vertebrates). The link between Hox and TALE proteins is their functional interactions, which were initially revealed in flies by genetic analyses and later supported by biochemical data (Mann & Affolter, 1998; Moens & Selleri, 2006). PBC proteins have been shown to act as Hox cofactors that mainly determine Hox DNA-binding specificity and selectivity. Hth/Meis proteins interact with PBC proteins, participating in ternary complexes with Hox proteins, or modulating PBC protein stability and subcellular localization. This European Molecular Biology Organization/European School of Molecular Medicine workshop on Homeodomain Proteins, Hematopoietic Development and Leukemias took place in Riva del Garda, Italy, between 23 and 25 March 2006, and was organized by F. Blasi, ... Functional specificities and redundancies A large part of the meeting was devoted to the presentation and discussion of the consequences of the complete or partial inactivation of different TALE products in several vertebrates. These studies arrived at two main conclusions: first, TALE proteins are essential for the development of most areas of the organism; and second, they have both individual functions and a large degree of redundancy. This was clearly shown in the Pbx gene family, for which an advanced phenotypic analysis of single and compound mutants was presented. In mice, the individual contribution of each member of the family seemed to be different because, whereas the Pbx1 single mutant had a wide range of malformations, only minor or no phenotypes were obvious in Pbx2 and Pbx3 mutants. However, when a reduced Pbx2 and/or Pbx3 dosage was added to the Pbx1−/− background, strong exacerbations of the malformations were detected, as reported by L. Selleri (New York, NY, USA) and M. Cleary (Stanford, CA, USA). In particular, the cardiovascular system, the axial skeleton, the limbs and the craniofacial area were shown to be strongly affected. A.J. Waskiewicz (Edmonton, Canada) also described redundancy among Pbx genes in zebrafish, in which the functions of the Pbx2 and Pbx4 gene products seem to overlap in a range of tissues, including the hindbrain and tectum, and during early haematopoiesis. Hox gene products are known to be largely redundant, especially within paralogous groups. In the case of the Meis and Prep gene families, pleiotropic functions in patterning and differentiation processes were also described (see below); however, genetic analyses designed to evaluate the possible extent of redundancy among the different members of these gene families are still at an early stage. At the meeting, several tissues were reported to require the activity of homeodomain proteins for proper development; we highlight some of these data below.

Zebrafish endoderm formation is regulated by combinatorial Nodal, FGF and BMP signalling

In the zebrafish embryo, the mesoderm and endoderm originate from common precursors and segregate during gastrulation by mechanisms that are largely unknown. Understanding how the signalling pathways that regulate endoderm and mesoderm formation interact is crucial to understanding how the germ layers are established. Here, we have analysed how the FGF and BMP pathways interact with Nodal signalling during the process of endoderm formation. We found that activation of the FGF/ERK pathway disrupts endoderm formation in the embryo and antagonizes the ability of an activated form of Tar/Acvr1b to induce endoderm at the animal pole. By contrast, inhibition of FGF signalling increases the number of endodermal precursors and potentiates the ability of Tar*/Acvr1b to induce endoderm at the animal pole. Using a pharmacological inhibitor of the FGF receptor, we show that reducing FGF signalling partially rescues the deficit of endoderm precursors in bon mutant embryos. Furthermore, we found that overexpression of BMPs compromises endoderm formation, suggesting that formation of endoderm precursors is negatively regulated by BMPs on the ventral side. We show that simultaneous inhibition of the FGF/Ras and BMP pathways results in a dramatic increase in the number of endoderm precursors. Taken together, these data strongly suggest that BMP and FGF-ERK pathways cooperate to restrict the number of endodermal progenitors induced in response to Nodal signalling. Finally, we investigated the molecular basis for the FGF-MAPK-dependent repression of endoderm formation. We found that FGF/ERK signalling causes phosphorylation of Casanova/Sox32, an important regulator of endoderm determination, and provide evidence that this phosphorylation attenuates its ability to induce sox17. These results identify a molecular mechanism whereby FGF attenuates Nodal-induced endodermal transcription factors and highlight a potential mechanism whereby mesoderm and endoderm fates could segregate from each other.