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.     

Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice

During embryogenesis, the pancreas arises from dorsal and ventral pancreatic protrusions from the primitive gut endoderm upon induction by different stimuli from neighboring mesodermal tissues. Recent studies have shown that Retinoic Acid (RA) signaling is essential for the development of the pancreas in non-mammalian vertebrates. To investigate whether RA regulates mouse pancreas development, we have studied the phenotype of mice with a targeted deletion in the retinaldehyde dehydrogenase 2 (Raldh2) gene, encoding the enzyme required to synthesize RA in the embryo. We show that Raldh2 is expressed in the dorsal pancreatic mesenchyme at the early stage of pancreas specification. RA-responding cells have been detected in pancreatic endodermal and mesenchymal cells. Raldh2-deficient mice do not develop a dorsal pancreatic bud. Mutant embryos lack Pdx 1 expression, an essential regulator of early pancreas development, in the dorsal but not the ventral endoderm. In contrast to Pdx 1-deficient mice, the early glucagon-expressing cells do not develop in Raldh2 knockout embryos. Shh expression is, as in the wild-type embryo, excluded from the dorsal endodermal region at the site where the dorsal bud is expected to form, indicating that the dorsal bud defect is not related to a mis-expression of Shh. Mesenchymal expression of the LIM homeodomain protein Isl 1, required for the formation of the dorsal mesenchyme, is altered in Raldh2--/-- embryos. The homeobox gene Hlxb9, which is essential for the initiation of the pancreatic program in the dorsal foregut endoderm, is still expressed in Raldh2--/-- dorsal epithelium but the number of HB9-expressing cells is severely reduced. Maternal supplementation of RA rescues early dorsal pancreas development and restores endodermal Pdx 1 and mesenchymal Isl 1 expression as well as endocrine cell differentiation. These findings suggest that RA signaling is important for the proper differentiation of the dorsal mesenchyme and development of the dorsal endoderm. We conclude that RA synthesized in the mesenchyme is specifically required for the normal development of the dorsal pancreatic endoderm at a stage preceding Pdx 1 function.     

Hedgehog signaling is required for differentiation of endocardial progenitors in zebrafish

Endocardial cells form the inner endothelial layer of the heart tube, surrounded by the myocardium. Signaling pathways that regulate endocardial cell specification and differentiation are largely unknown and the origin of endocardial progenitors is still being debated. To study pathways that regulate endocardial differentiation in a zebrafish model system, we isolated zebrafish NFATc1 homolog which is expressed in endocardial but not vascular endothelial cells. We further demonstrate that Hedgehog (Hh) but not VegfA or Notch signaling is required for early endocardial morphogenesis. Pharmacological inhibition of Hh signaling with cyclopamine treatment resulted in nearly complete loss of the endocardial marker expression. Simultaneous knockdown of the two zebrafish sonic hedgehog homologs, shh and twhh or Hh co-receptor smoothened (smo) resulted in similar defects in endocardial morphogenesis. Inhibition of Hh signaling resulted in the loss of fibronectin (fn1) expression in the presumptive endocardial progenitors as early as the 10-somite stage which suggests that Hh signaling is required for the earliest stages of endocardial specification. We further show that the endoderm plays a critical role in migration but not specification or differentiation of the endocardial progenitors while notochord-derived Hh is a likely source for the specification and differentiation signal. Mosaic analysis using cell transplantation shows that Smo function is required cell-autonomously within endocardial progenitor cells. Our results argue that Hh provides a critical signal to induce the specification and differentiation of endocardial progenitors.     

The zebrafish iguana locus encodes Dzip1, a novel zinc-finger protein required for proper regulation of Hedgehog signaling

Members of the Hedgehog (Hh) family of intercellular signaling molecules play crucial roles in animal development. Aberrant regulation of Hh signaling in humans causes developmental defects, and leads to various genetic disorders and cancers. We have characterized a novel regulator of Hh signaling through the analysis of the zebrafish midline mutant iguana (igu). Mutations in igu lead to reduced expression of Hh target genes in the ventral neural tube, similar to the phenotype seen in zebrafish mutants known to affect Hh signaling. Contradictory at first sight, igu mutations lead to expanded Hh target gene expression in somites. Genetic and pharmacological analyses revealed that the expression of Hh target genes in igu mutants requires Gli activator function but does not depend on Smoothened function. Our results show that the ability of Gli proteins to activate Hh target gene expression in response to Hh signals is generally reduced in igu mutants both in the neural tube and in somites. Although this reduced Hh signaling activity leads to a loss of Hh target gene expression in the neural tube, the same low levels of Hh signaling appear to be sufficient to activate Hh target genes throughout somites because of different threshold responses to Hh signals. We also show that Hh target gene expression in igu mutants is resistant to increased protein kinase A activity that normally represses Hh signaling. Together, our data indicate that igu mutations impair both the full activation of Gli proteins in response to Hh signals, and the negative regulation of Hh signaling in tissues more distant from the source of Hh. Positional cloning revealed that the igu locus encodes Dzip1, a novel intracellular protein that contains a single zinc-finger protein-protein interaction domain. Overexpression of Igu/Dzip1 proteins suggested that Igu/Dzip1 functions in a permissive way in the Hh signaling pathway. Taken together, our studies show that Igu/Dzip1 functions as a permissive factor that is required for the proper regulation of Hh target genes in response to Hh signals.     

Retinoic acid-mediated patterning of the pre-pancreatic endoderm in Xenopus operates via direct and indirect mechanisms

Early patterning of the endoderm as a prerequisite for pancreas specification involves retinoic acid (RA) as a critical signalling molecule in gastrula stage Xenopus embryos. In extension of our previous studies, we made systematic use of early embryonic endodermal and mesodermal explants. We find RA to be sufficient to induce pancreas-specific gene expression in dorsal but not ventral endoderm. The differential expression of retinoic acid receptors (RARs) in gastrula stage endoderm is important for the distinct responsiveness of dorsal versus ventral explants. Furthermore, BMP signalling, that is repressed dorsally, prevents the formation of pancreatic precursor cells in the ventral endoderm of gastrula stage Xenopus embryos. An additional requirement for mesoderm suggests the production of one or more further pancreas inducing signals by this tissue. Finally, recombination of manipulated early embryonic explants, and also inhibition of RA activity in whole embryos, reveal that RA signalling, as it is relevant for pancreas development, operates simultaneously on both mesodermal and endodermal germ layers.     

Cardiac and CNS defects in a mouse with targeted disruption of suppressor of fused

The hedgehog (Hh) pathway is conserved from Drosophila to humans and plays a key role in embryonic development. In addition, activation of the pathway in somatic cells contributes to cancer development in several tissues. Suppressor of fused is a negative regulator of Hh signaling. Targeted disruption of the murine suppressor of fused gene (Sufu) led to a phenotype that included neural tube defects and lethality at mid-gestation (9.0-10.5 dpc). This phenotype resembled that caused by loss of patched (Ptch1), another negative regulator of the Hh pathway. Consistent with this finding, Ptch1 and Sufu mutants displayed excess Hh signaling and resultant altered dorsoventral patterning of the neural tube. Sufu mutants also had abnormal cardiac looping, indicating a defect in the determination of left-right asymmetry. Marked expansion of nodal expression in 7.5 dpc embryos and variable degrees of node dysmorphology in 7.75 dpc embryos suggested that the pathogenesis of the cardiac developmental abnormalities was related to node development. Other mutants of the Hh pathway, such as Shh, Smo and Shh/Ihh compound mutants, also have laterality defects. In contrast to Ptch1 heterozygous mice, Sufu heterozygotes had no developmental defects and no apparent tumor predisposition. The resemblance of Sufu homozygotes to Ptch1 homozygotes is consistent with mouse Sufu being a conserved negative modulator of Hh signaling.     

Retinoic acid signaling is required for a critical early step in zebrafish pancreatic development

The mechanisms that subdivide the endoderm into the discrete primordia that give rise to organs such as the pancreas and liver are not well understood. However, it is known that retinoic acid (RA) signaling is critical for regionalization of the vertebrate embryo: when RA signaling is either prevented or augmented, anteroposterior (AP) patterning of the CNS and mesoderm is altered and major developmental defects occur. We have investigated the role of RA signaling in regionalization of the zebrafish endoderm. Using a mutant that prevents RA synthesis and an antagonist of the RA receptors, we show that specification of both the pancreas and liver requires RA signaling. By contrast, RA signaling is not required for the formation of the endodermal germ layer or for differentiation of other endodermal organs. Timed antagonist and RA treatments show that the RA-dependent step in pancreatic specification occurs at the end of gastrulation, significantly earlier than the expression of known markers of pancreatic progenitors. In addition to being required for pancreatic specification, RA has the capacity to transfate anterior endoderm to a pancreatic fate.     

Mys Protein Regulates Protein Kinase A Activity by Interacting with Regulatory Type I�� Subunit during Vertebrate Development

During embryonic development, protein kinase A (PKA) plays a key role in cell fate specification by antagonizing the Hedgehog (Hh) signaling pathway. However, the mechanism by which PKA activity is regulated remains unknown. Here we show that the Misty somites (Mys) protein regulates the level of PKA activity during embryonic development in zebrafish. We isolate PKA regulatory type Iα subunit (Prkar1a) as a protein interacting with Mys by pulldown assay in HEK293 cells followed by mass spectrometry analysis. We show an interaction between endogenous Mys and Prkar1a in the zebrafish embryo. Mys binds to Prkar1a in its C terminus region, termed PRB domain, and activates PKA in vitro. Conversely, knockdown of Mys in zebrafish embryos results in reduction in PKA activity. We also show that knockdown of Mys induces ectopic activation of Hh target genes in the eyes, neural tube, and somites downstream of Smoothened, a protein essential for transduction of Hh signaling activity. The altered patterning of gene expression is rescued by activation of PKA. Together, our results reveal a molecular mechanism of regulation of PKA activity that is dependent on a protein-protein interaction and demonstrate that PKA activity regulated by Mys is indispensable for negative regulation of the Hh signaling pathway in Hh-responsive cells.     

Hedgehog signaling induces arterial endothelial cell formation by repressing venous cell fate

In vertebrate embryos, the dorsal aorta and the posterior cardinal vein form in the trunk to comprise the original circulatory loop. Previous studies implicate Hedgehog (Hh) signaling in the development of the dorsal aorta. However, the mechanism controlling specification of artery versus vein remains unclear. Here, we investigated the cell-autonomous mechanism of Hh signaling in angioblasts (endothelial progenitor cells) during arterial-venous specification utilizing zebrafish mutations in Smoothened (Smo), a G protein-coupled receptor essential for Hh signaling. smo mutants exhibit an absence of the dorsal aorta accompanied by a reciprocal expansion of the posterior cardinal vein. The increased number of venous cells is equivalent to the loss of arterial cells in embryos with loss of Smo function. Activation of Hh signaling expands the arterial cell population at the expense of venous cell fate. Time-lapse imaging reveals two sequential waves of migrating progenitor cells that contribute to the dorsal aorta and the posterior cardinal vein, respectively. Angioblasts deficient in Hh signaling fail to contribute to the arterial wave; instead, they all migrate medially as a single population to form the venous wave. Cell transplantation analyses demonstrate that Smo plays a cell-autonomous role in specifying angioblasts to become arterial cells, and Hh signaling-depleted angioblasts differentiate into venous cells instead. Collectively, these studies suggest that arterial endothelial cells are specified and formed via repressing venous cell fate at the lateral plate mesoderm by Hh signaling during vasculogenesis.     

Regulator of G-Protein Signaling – 5 (RGS5) Is a Novel Repressor of Hedgehog Signaling

Hedgehog (Hh) signaling plays fundamental roles in morphogenesis, tissue repair, and human disease. Initiation of Hh signaling is controlled by the interaction of two multipass membrane proteins, patched (Ptc) and smoothened (Smo). Recent studies identify Smo as a G-protein coupled receptor (GPCR)-like protein that signals through large G-protein complexes which contain the Gα_i subunit. We hypothesize Regulator of G-Protein Signaling (RGS) proteins, and specifically RGS5, are endogenous repressors of Hh signaling via their ability to act as GTPase activating proteins (GAPs) for GTP-bound Gα_i, downstream of Smo. In support of this hypothesis, we demonstrate that RGS5 over-expression inhibits sonic hedgehog (Shh)-mediated signaling and osteogenesis in C3H10T1/2 cells. Conversely, signaling is potentiated by siRNA-mediated knock-down of RGS5 expression, but not RGS4 expression. Furthermore, using immuohistochemical analysis and co-immunoprecipitation (Co-IP), we demonstrate that RGS5 is present with Smo in primary cilia. This organelle is required for canonical Hh signaling in mammalian cells, and RGS5 is found in a physical complex with Smo in these cells. We therefore conclude that RGS5 is an endogenous regulator of Hh-mediated signaling and that RGS proteins are potential targets for novel therapeutics in Hh-mediated diseases.     

Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

Chondrocyte hypertrophy is an essential process required for endochondral bone formation. Proper regulation of chondrocyte hypertrophy is also required in postnatal cartilage homeostasis. Indian hedgehog (Ihh) and PTHrP signaling play crucial roles in regulating the onset of chondrocyte hypertrophy by forming a negative feedback loop, in which Ihh signaling regulates chondrocyte hypertrophy by controlling PTHrP expression. To understand whether there is a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy, we have both activated and inactivated Ihh signaling in the absence of PTHrP during endochondral skeletal development. We found that upregulating Ihh signaling in the developing cartilage by treating PTHrP(-/-) limb explants with sonic hedgehog (Shh) protein in vitro, or overexpressing Ihh in the cartilage of PTHrP(-/-) embryos or inactivating patched 1 (Ptch1), a negative regulator of hedgehog (Hh) signaling, accelerated chondrocyte hypertrophy in the PTHrP(-/-) embryos. Conversely, when Hh signaling was blocked by cyclopamine or by removing Smoothened (Smo), a positive regulator of Hh signaling, chondrocyte hypertrophy was delayed in the PTHrP(-/-) embryo. Furthermore, we show that upregulated Hh signaling in the postnatal cartilage led to accelerated chondrocyte hypertrophy during secondary ossification, which in turn caused reduction of joint cartilage. Our results revealed a novel role of Ihh signaling in promoting chondrocyte hypertrophy independently of PTHrP, which is particularly important in postnatal cartilage development and homeostasis. In addition, we found that bone morphogenetic protein (Bmp) and Wnt/beta-catenin signaling in the cartilage may both mediate the effect of upregulated Ihh signaling in promoting chondrocyte hypertrophy.     

The dual regulator Sufu integrates Hedgehog and Wnt signals in the early Xenopus embryo

Hedgehog (Hh) and Wnt proteins are important signals implicated in several aspects of embryonic development, including the early development of the central nervous system. We found that Xenopus Suppressor-of-fused (XSufu) affects neural induction and patterning by regulating the Hh/Gli and Wnt/β-catenin pathways. Microinjection of XSufu mRNA induced expansion of the epidermis at the expense of neural plate tissue and caused enlargement of the eyes. An antisense morpholino oligonucleotide against XSufu had the opposite effect. Interestingly, both gain- and loss-of-function experiments resulted in a posterior shift of brain markers, suggesting a biphasic effect of XSufu on anteroposterior patterning. XSufu blocked early Wnt/β-catenin signaling, as indicated by the suppression of XWnt8-induced secondary axis formation in mRNA-injected embryos, and activation of Wnt target genes in XSufu-MO-injected ectodermal explants. We show that XSufu binds to XGli1 and Xβ-catenin. In Xenopus embryos and mouse embryonic fibroblasts, Gli1 inhibits Wnt signaling under overexpression of β-catenin, whereas β-catenin stimulates Hh signaling under overexpression of Gli1. Notably, endogenous Sufu is critically involved in this crosstalk. The results suggest that XSufu may act as a common regulator of Hh and Wnt signaling and contribute to intertwining the two pathways.     

Hedgehog signaling plays a cell-autonomous role in maximizing cardiac developmental potential

Elucidation of the complete roster of signals required for myocardial specification is crucial to the future of cardiac regenerative medicine. Prior studies have implicated the Hedgehog (Hh) signaling pathway in the regulation of multiple aspects of heart development. However, our understanding of the contribution of Hh signaling to the initial specification of myocardial progenitor cells remains incomplete. Here, we show that Hh signaling promotes cardiomyocyte formation in zebrafish. Reduced Hh signaling creates a cardiomyocyte deficit, and increased Hh signaling creates a surplus. Through fate-mapping, we find that Hh signaling is required at early stages to ensure specification of the proper number of myocardial progenitors. Genetic inducible fate mapping in mouse indicates that myocardial progenitors respond directly to Hh signals, and transplantation experiments in zebrafish demonstrate that Hh signaling acts cell autonomously to promote the contribution of cells to the myocardium. Thus, Hh signaling plays an essential early role in defining the optimal number of cardiomyocytes, making it an attractive target for manipulation of multipotent progenitor cells.