Antagonistic interactions of hedgehog, Bmp and retinoic acid signals control zebrafish endocrine pancreas development

Pancreatic organogenesis is promoted or restricted by different signaling pathways. In amniotes, inhibition of hedgehog (Hh) activity in the early embryonic endoderm is a prerequisite for pancreatic specification. However, in zebrafish, loss of Hh signaling leads to a severe reduction of β-cells, leading to some ambiguity as to the role of Hh during pancreas development and whether its function has completely diverged between species. Here, we have employed genetic and pharmacological manipulations to temporally delineate the role of Hh in zebrafish endocrine pancreas development and investigate its relationship with the Bmp and retinoic acid (RA) signaling pathways. We found that Hh is required at the start of gastrulation for the medial migration and differentiation of pdx1-expressing pancreatic progenitors at later stages. This early positive role of Hh promotes β-cell lineage differentiation by restricting the repressive effects of Bmp. Inhibition of Bmp signaling in the early gastrula leads to increased β-cell numbers and partially rescued β-cell formation in Hh-deficient embryos. By the end of gastrulation, Hh switches to a negative role by antagonizing RA-mediated specification of the endocrine pancreas, but continues to promote differentiation of exocrine progenitors. We show that RA downregulates the Hh signaling components ptc1 and smo in endodermal explants, indicating a possible molecular mechanism for blocking axial mesoderm-derived Hh ligands from the prepancreatic endoderm during the specification stage. These results identify multiple sequential roles for Hh in pancreas development and highlight an unexpected antagonistic relationship between Hh and other signaling pathways to control pancreatic specification and differentiation.     

Bmp and Fgf signaling are essential for liver specification in zebrafish

Based on data from in vitro tissue explant and ex vivo cell/bead implantation experiments, Bmp and Fgf signaling have been proposed to regulate hepatic specification. However, genetic evidence for this hypothesis has been lacking. Here, we provide in vivo genetic evidence that Bmp and Fgf signaling are essential for hepatic specification. We utilized transgenic zebrafish that overexpress dominant-negative forms of Bmp or Fgf receptors following heat-shock induction. These transgenes allow one to bypass the early embryonic requirements for Bmp and Fgf signaling, and also to completely block Bmp or Fgf signaling. We found that the expression of hhex and prox1, the earliest liver markers in zebrafish, was severely reduced in the liver region when Bmp or Fgf signaling was blocked just before hepatic specification. However, hhex and prox1 expression in adjacent endodermal and mesodermal tissues appeared unaffected by these manipulations. Additional genetic studies indicate that the endoderm maintains competence for Bmp-mediated hepatogenesis over an extended window of embryonic development. Altogether, these data provide the first genetic evidence that Bmp and Fgf signaling are essential for hepatic specification, and suggest that endodermal cells remain competent to differentiate into hepatocytes for longer than anticipated.     

Fgf4 is required for left-right patterning of visceral organs in zebrafish

Fgf signaling plays essential roles in many developmental events. To investigate the roles of Fgf4 signaling in zebrafish development, we generated Fgf4 knockdown embryos by injection with Fgf4 antisense morpholino oligonucleotides. Randomized LR patterning of visceral organs including the liver, pancreas, and heart was observed in the knockdown embryos. Prominent expression of Fgf4 was observed in the posterior notochord and Kupffer's vesicle region in the early stages of segmentation. Lefty1, lefty2, southpaw, and pitx2 are known to play crucial roles in LR patterning of visceral organs. Fgf4 was essential for the expression of lefty1, which is necessary for the asymmetric expression of southpaw and pitx2 in the lateral plate mesoderm, in the posterior notochord, and the expression of lefty2 and lefty1 in the left cardiac field. Fgf8 is also known to be crucial for the formation of Kupffer's vesicle, which is needed for the LR patterning of visceral organs. In contrast, Fgf4 was required for the formation of cilia in Kupffer's vesicle, indicating that the role of Fgf4 in the LR patterning is quite distinct from that of Fgf8. The present findings indicate that Fgf4 plays a unique role in the LR patterning of visceral organs in zebrafish.     

Wnt Signaling Interacts with Bmp and Edn1 to Regulate Dorsal-Ventral Patterning and Growth of the Craniofacial Skeleton

Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC) cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D) and ventral (V) elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3), (Tg(hsp70I:tcf3-GFP), or the canonical Wnt inhibitor dickkopf1 (dkk1), (Tg(hsp70i:dkk1-GFP) after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe), and ventral cartilage differentiation (e.g. lower jaws). These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.     

Regulation of Hex gene expression and initial stages of avian hepatogenesis by Bmp and Fgf signaling

The vertebrate liver and heart arise from adjacent cell layers in the anterior lateral (AL) endoderm and mesoderm of late gastrula embryos, and the earliest stages of liver and heart development are interrelated through reciprocal tissue interactions. Although classical embryological studies performed several decades ago in chick and quail defined the timing of hepatogenic induction in birds and the important role for cardiogenic mesoderm in this process, almost nothing is known about the molecular aspects of avian liver development. Here we use in vivo and explantation assays to investigate tissue interactions and signaling pathways regulating Hex, a homeobox gene required for liver development, and the earliest stages of hepatogenesis in the chick embryo. We find that explants of late gastrula anterior lateral endoderm plus mesoderm, which have been used extensively for studies relating to heart development, also produce albumin-expressing hepatoblasts. Expression of Hex, the earliest known molecular marker for the hepatogenic endoderm, and albumin, indicative of early committed hepatoblasts, requires both autocrine Bmp signaling and a specific paracrine signal from the cardiogenic (anterior lateral) mesoderm. Endodermal expression of Fox2a, in contrast, requires the mesoderm but is independent of Bmp signaling. In vivo induction assays show that the ability of BMP2 to activate Hex expression in the endoderm is restricted to a region that is only slightly larger than the endogenous domain of Hex expression. Although Fgfs can substitute for the cardiogenic mesoderm to support the expression of Hex and albumin in the endoderm, several Fgf genes are expressed in the anterior lateral endoderm but an Fgf expressed predominantly in the mesoderm was not identified. Studies also showed that Fgf gene expression in the endoderm does not require a signal from the mesoderm. Mechanisms regulating endodermal signaling pathways activated by Fgfs may therefore be more complex than previously appreciated.     

Bmp7 functions via a polarity mechanism to promote cloacal septation

Background

During normal development in human and other placental mammals, the embryonic cloacal cavity separates along the axial longitudinal plane to give rise to the urethral system, ventrally, and the rectum, dorsally. Defects in cloacal development are very common and present clinically as a rectourethral fistula in about 1 in 5,000 live human births. Yet, the cellular mechanisms of cloacal septation remain poorly understood.

Methodology/Principal Findings

We previously detected Bone morphogenetic protein 7 (Bmp7) expression in the urorectal mesenchyme (URM), and have shown that loss of Bmp7 function results in the arrest of cloacal septation. Here, we present evidence that cloacal partitioning is driven by Bmp7 signaling in the cloacal endoderm. We performed TUNEL and immunofluorescent analysis on cloacal sections from Bmp7 null and control littermate embryos. We found that loss of Bmp7 results in a dramatic decrease in the endoderm survival and a delay in differentiation. We used immunological methods to show that Bmp7 functions by activating the c-Jun N-terminal kinase (JNK) pathway. We carried out confocal and 3D imaging analysis of mitotic chromosome bundles to show that during normal septation cells in the cloacal endoderm divide predominantly in the apical-basal direction. Loss of Bmp7/JNK signaling results in randomization of mitotic angles in the cloacal endoderm. We also conducted immunohistochemical analysis of human fetal sections to show that BMP/phospho-SMAD and JNK pathways function in the human cloacal region similar as in the mouse.

Conclusion/Significance

Our results strongly indicate that Bmp7/JNK signaling regulates remodeling of the cloacal endoderm resulting in a topological separation of the urinary and digestive systems. Our study points to the importance of Bmp and JNK signaling in cloacal development and rectourethral malformations.     

BMP signalling regulates anteroposterior endoderm patterning in zebrafish

In vertebrates, the embryonic dorsoventral asymmetry is regulated by the bone morphogenetic proteins (Bmp) activity gradient. In the present study, we have used dorsalized swirl (bmp2b) and ventralized chordino (chordin) zebrafish mutants to investigate the effects of dorsoventral signalling on endoderm patterning and on the differentiation and positioning of its derivatives. Alterations of dorsoventral Bmp signalling do not perturb the induction of endodermal precursors, as shown by normal amounts of cells expressing cas and sox17 in swirl and chordino gastrulae, but affect dramatically the expression pattern of her5, a regulator of endoderm anteroposterior patterning in zebrafish. In particular, increased levels of Bmp signalling in chordino gastrulae are associated with a markedly reduced her5 expression domain, that may be abolished by injecting bmp2b mRNA. Conversely, in swirl mutants, lacking Bmp2b, the her5 expression domain is expanded. Thus, a gradient of Bmp2b signalling defines the extension of the her5 expression domain at gastrulation and the allocation of anterior endodermal precursors. A balanced Bmp2b signalling is also required for the normal development of the pancreas, as shown by the sharp reduction of the pancreatic primordium in swirl embryos and its expansion in chordino mutants. In the latter, at 3 days post-fertilization, the increased Bmp signalling does not compromise the endocrine/exocrine pancreas compartmentalization, but the right/left positioning of the pancreas and liver is randomized. Our results suggest that by regulating the expression of her5, the Bmp2b/Chordin gradient directs the anteroposterior patterning of endoderm in zebrafish embryos.     

Regulation of avian cardiogenesis by Fgf8 signaling

The avian heart develops from paired primordia located in the anterior lateral mesoderm of the early embryo. Previous studies have found that the endoderm adjacent to the cardiac primordia plays an important role in heart specification. The current study provides evidence that fibroblast growth factor (Fgf) signaling contributes to the heart-inducing properties of the endoderm. Fgf8 is expressed in the endoderm adjacent to the precardiac mesoderm. Removal of endoderm results in a rapid downregulation of a subset of cardiac markers, including Nkx2.5 and Mef2c. Expression of these markers can be rescued by supplying exogenous Fgf8. In addition, application of ectopic Fgf8 results in ectopic expression of cardiac markers. Expression of cardiac markers is expanded only in regions where bone morphogenetic protein (Bmp) signaling is also present, suggesting that cardiogenesis occurs in regions exposed to both Fgf and Bmp signaling. Finally, evidence is presented that Fgf8 expression is regulated by particular levels of Bmp signaling. Application of low concentrations of Bmp2 results in ectopic expression of Fgf8, while application of higher concentrations of Bmp2 result in repression of Fgf8 expression. Together, these data indicate that Fgf signaling cooperates with Bmp signaling to regulate early cardiogenesis.     

Reciprocal endoderm-mesoderm interactions mediated by fgf24 and fgf10 govern pancreas development

In amniotes, the pancreatic mesenchyme plays a crucial role in pancreatic epithelium growth, notably through the secretion of fibroblast growth factors. However, the factors involved in the formation of the pancreatic mesenchyme are still largely unknown. In this study, we characterize, in zebrafish embryos, the pancreatic lateral plate mesoderm, which is located adjacent to the ventral pancreatic bud and is essential for its specification and growth. We firstly show that the endoderm, by expressing the fgf24 gene at early stages, triggers the patterning of the pancreatic lateral plate mesoderm. Based on the expression of isl1, fgf10 and meis genes, this tissue is analogous to the murine pancreatic mesenchyme. Secondly, Fgf10 acts redundantly with Fgf24 in the pancreatic lateral plate mesoderm and they are both required to specify the ventral pancreas. Our results unveil sequential signaling between the endoderm and mesoderm that is critical for the specification and growth of the ventral pancreas, and explain why the zebrafish ventral pancreatic bud generates the whole exocrine tissue.     

Effects of specific and prolonged expression of zebrafish growth factors,Fgf2 and Lif in primordial germ cells in vivo

Primordial germ cells (PGCs), specified early in development, proliferate and migrate to the developing gonad before sexual differentiation occurs in the embryo and eventually give rise to spermatogonia or oogonia. In this study, we discovered that nanos3 3′UTR, a common method used to label PGCs, not only directed PGC-specific expression of DsRed but also prolonged this expression up to 26 days post fertilization (dpf) when DsRed-nanos3 3′UTR hybrid mRNAs were introduced into 1- to 2-cell-stage embryos. As such, we employed this knowledge to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and bone morphogenetic protein 4 (Bmp4) in the PGCs and evaluate their effects on PGC development in vivo for over a period of 3 weeks. The results show that expression of Fgf2 significantly increased PGC number at 14- and 21-dpf while Bmp4 resulted in severe ventralization and death of the embryos by 3 days. Expression of Lif resulted in a significant disruption of PGC migration. Mopholino knockdown experiments indicated that Lif illicited its effect on PGC migration through Lif receptor a (Lifra) but not Lifrb. The general approach described in this study could be used to achieve prolonged PGC-specific expression of other proteins to investigate their roles in germ cell and gonad development. The results also indicate that zebrafish PGCs have a mechanism to stabilize and prolong the expression of mRNA that carries nanos3 3′UTR. Understanding this mechanism may make it possible to achieve prolonged RNA expression in other cell types.     

Forebrain and hindbrain development in zebrafish is sensitive to ethanol exposure involving agrin,Fgf, and sonic hedgehog function

BACKGROUND: Ethanol is a teratogen that affects numerous developmental processes in the nervous system, which includes development and survival of GABAergic and glutamatergic neurons. Possible molecular mechanisms accounting for ethanol's effects on nervous system development include perturbed fibroblast growth factor (Fgf) and Sonic hedgehog (Shh) signaling. In zebrafish, forebrain GABAergic neuron development is dependent on Fgf19 and Shh signaling. The present study was conducted to test the hypothesis that ethanol affects GABAergic and glutamatergic neuron development by disrupting Fgf, Shh, and agrin function. METHODS: Zebrafish embryos were exposed to varying concentrations of ethanol during a range of developmental stages, in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin or Shh function. In situ hybridization was used to analyze glutamic acid decarboxylase (GAD1) gene expression, as well as markers of glutamatergic neurons. RESULTS: Acute ethanol exposure results in marked reduction in GAD1 gene expression in forebrain and hindbrain, and reduction of glutamatergic neuronal markers in hindbrain. Subthreshold ethanol exposure, combined with agrin or Shh MO treatment, produces a similar diminution in expression of markers for GABAergic and glutamatergic neurons. Consistent with the ethanol effects on Fgf and Shh pathways, Fgf19, Fgf8, or Shh mRNA overexpression rescues ethanol‐induced decreases in GAD1 and Atonal1a gene expression. CONCLUSIONS: These studies demonstrate that GABAergic and glutamatergic neuron development in zebrafish forebrain or cerebellum is sensitive to ethanol exposure, and provides additional evidence that a signaling pathway involving agrin, Fgfs and Shh may be a critical target of ethanol exposure during zebrafish embryogenesis. Birth Defects Research (Part A), 2013. © 2012 Wiley Periodicals, Inc.     

A conserved role for retinoid signaling in vertebrate pancreas development

Retinoic acid (RA) signaling plays critical roles in the regionalization of the central nervous system and mesoderm of all vertebrates that have been examined. However, to date, a role for RA in pancreas and liver development has only been demonstrated for the teleost zebrafish. Here, we demonstrate that RA signaling is required for development of the pancreas but not the liver in the amphibian Xenopus laevis and the avian quail. We disrupted RA signaling in Xenopus tadpoles, using both a pharmacological and a dominant-negative strategy. RA-deficient quail embryos were obtained from hens with a dietary deficiency in vitamin A. In both species we found that pancreas development was dependent on RA signaling. Furthermore, treatment of Xenopus tadpoles with exogenous RA led to an expansion of the pancreatic field. By contrast, liver development was not perturbed by manipulation of RA signaling. Taken together with our previous finding that RA signaling is necessary and sufficient for zebrafish pancreas development, these data support the hypothesis that a critical role for RA signaling in pancreas development is a conserved feature of the vertebrates.     

Restraint of Fgf8 signaling by retinoic acid signaling is required for proper heart and forelimb formation

Cardiomelic or heart–hand syndromes include congenital defects affecting both the forelimb and heart, suggesting a hypothesis where similar signals may coordinate their development. In support of this hypothesis, we have recently defined a mechanism by which retinoic acid (RA) signaling acts on the forelimb progenitors to indirectly restrict cardiac cell number. However, we still do not have a complete understanding of the mechanisms downstream of RA signaling that allow for the coordinated development of these structures. Here, we test the hypothesis that appropriate Fgf signaling in the cardiac progenitor field downstream of RA signaling is required for the coordinated development of the heart and forelimb. Consistent with this hypothesis, we find that increasing Fgf signaling can autonomously increase cardiac cell number and non-autonomously inhibit forelimb formation over the same time period that embryos are sensitive to loss of RA signaling. Furthermore, we find that Fgf8a, which is expressed in the cardiac progenitors, is expanded into the posterior in RA signaling-deficient zebrafish embryos. Reducing Fgf8a function in RA signaling-deficient embryos is able to rescue both heart and forelimb development. Together, these results are the first to directly support the hypothesis that RA signaling is required shortly after gastrulation in the forelimb field to temper Fgf8a signaling in the cardiac field, thus coordinating the development of the heart and forelimb.