Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development

Background  

The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC). Genetic studies in zebrafish and mice have established that the Hedgehog (Hh)-signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in CNCC development. Disruption of the Hh-signaling pathway in humans can lead to the spectral disorder of Holoprosencephaly (HPE), which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia [1, 2]. Previous work has uncovered a role for Hh-signaling in zebrafish dorsal neurocranium patterning and chondrogenesis, however Hh-signaling mutants have not been described with respect to the ventral pharyngeal arch (PA) skeleton. Lipid-modified Hh-ligands require the transmembrane-spanning receptor Dispatched 1 (Disp1) for proper secretion from Hh-synthesizing cells to the extracellular field where they act on target cells. Here we study chameleon mutants, lacking a functional disp1(con/disp1).     

Dose dependency of Disp1 and genetic interaction between Disp1 and other hedgehog signaling components in the mouse

Genetic analyses in Drosophila have demonstrated that a transmembrane protein Dispatched (Disp) is required for the release of lipid-modified Hedgehog (Hh) protein from Hh secreting cells. Analysis of Disp1 null mutant embryos has demonstrated that Disp1 plays a key role in hedgehog signaling in the early mouse embryo. Here we have used a hypomorphic allele in Disp1(Disp1(Delta)(2)), to extend our knowledge of Disp1 function in Hh-mediated patterning of the mammalian embryo. Through genetic combinations with null alleles of patched 1 (Ptch1), sonic hedgehog (Shh) and Indian hedgehog (Ihh), we demonstrate that Disp1 genetically interacts with Hh signaling components. As Disp1 activity is decreased we see a progressive increase in the severity of hedgehog-dependent phenotypes, which is further enhanced by reducing hedgehog ligand levels. Analysis of neural tube patterning demonstrates a progressive loss of ventral cell identities that most likely reflects decreased Shh signaling as Disp1 levels are attenuated. Conversely, increasing available Shh ligand by decreasing Ptch1 dosage leads to the restoration of ventral cell types in Disp1(Delta2/Delta2) mutants. Together, these studies suggest that Disp1 actively regulates the levels of hedgehog ligand that are available to the hedgehog target field. Further, they provide additional support for the dose-dependent action of Shh signaling in patterning the embryo. Finally, in-vitro studies on Disp1 null mutant fibroblasts indicate that Disp1 is not essential for membrane targeting or release of lipid-modified Shh ligand.     

Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates

Using zinc-finger nuclease-mediated mutagenesis, we have generated mutant alleles of the zebrafish orthologue of the chicken talpid3 (ta3) gene, which encodes a centrosomal protein that is essential for ciliogenesis. Animals homozygous for these mutant alleles complete embryogenesis normally, but manifest a cystic kidney phenotype during the early larval stages and die within a month of hatching. Elimination of maternally derived Ta3 activity by germline replacement resulted in embryonic lethality of ta3 homozygotes. The phenotype of such maternal and zygotic (MZta3) mutant zebrafish showed strong similarities to that of chick ta3 mutants: absence of primary and motile cilia as well as aberrant Hedgehog (Hh) signalling, the latter manifest by the expanded domains of engrailed and ptc1 expression in the somites, reduction of nkx2.2 expression in the neural tube, symmetric pectoral fins, cyclopic eyes and an ectopic lens. GFP-tagged Gli2a localised to the basal bodies in the absence of the primary cilia and western blot analysis showed that Gli2a protein is aberrantly processed in MZta3 embryos. Zygotic expression of ta3 largely rescued the effects of maternal depletion, but the motile cilia of Kupffer's vesicle remained aberrant, resulting in laterality defects. Our findings underline the importance of the primary cilium for Hh signaling in zebrafish and reveal the conservation of Ta3 function during vertebrate evolution.     

The zebrafish slow-muscle-omitted gene product is required for Hedgehog signal transduction and the development of slow muscle identity

Hedgehog proteins mediate many of the inductive interactions that determine cell fate during embryonic development. Hedgehog signaling has been shown to regulate slow muscle fiber type development. We report here that mutations in the zebrafish slow-muscle-omitted (smu) gene disrupt many developmental processes involving Hedgehog signaling. smu(-/-) embryos have a 99% reduction in the number of slow muscle fibers and a complete loss of Engrailed-expressing muscle pioneers. In addition, mutant embryos have partial cyclopia, and defects in jaw cartilage, circulation and fin growth. The smu(-/-) phenotype is phenocopied by treatment of wild-type embryos with forskolin, which inhibits the response of cells to Hedgehog signaling by indirect activation of cAMP-dependent protein kinase (PKA). Overexpression of Sonic hedgehog (Shh) or dominant negative PKA (dnPKA) in wild-type embryos causes all somitic cells to develop into slow muscle fibers. Overexpression of Shh does not rescue slow muscle fiber development in smu(-/-) embryos, whereas overexpression of dnPKA does. Cell transplantation experiments confirm that smu function is required cell-autonomously within the muscle precursors: wild-type muscle cells rescue slow muscle fiber development in smu(-/-) embryos, whereas mutant muscle cells cannot develop into slow muscle fibers in wild-type embryos. Slow muscle fiber development in smu mutant embryos is also rescued by expression of rat Smoothened. Therefore, Hedgehog signaling through Slow-muscle-omitted is necessary for slow muscle fiber type development. We propose that smu encodes a vital component in the Hedgehog response pathway.     

Mouse Dispatched homolog1 is required for long-range,but not juxtacrine,Hh signaling

Precise patterning of cell types along the dorsal-ventral axis of the spinal cord is essential to establish functional neural circuits. In order to prove the feasibility of studying a single biological process through random mutagenesis in the mouse, we have identified recessive ENU-induced mutations in six genes that prevent normal specification of ventral cell types in the spinal cord. We positionally cloned the genes responsible for two of the mutant phenotypes, smoothened and dispatched, which are homologs of Drosophila Hh pathway components. The Dispatched homolog1 (Disp1) mutation causes lethality at midgestation and prevents specification of ventral cell types in the neural tube, a phenotype identical to the Smoothened (Smo) null phenotype. As in Drosophila, mouse Disp1 is required to move Shh away from the site of synthesis. Despite the existence of a second mouse disp homolog, Disp1 is essential for long-range signaling by both Shh and Ihh ligands. Our data indicate that Shh signaling is required within the notochord to maintain Shh expression and to prevent notochord degeneration. Disp1, unlike Smo, is not required for this juxtacrine signaling by Shh.     

Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of dispatched

The dispatched (disp) gene is required for long-range Hedgehog (Hh) signaling in Drosophila. Here, we demonstrate that one of two murine homologs, mDispA, can rescue disp function in Drosophila and is essential for all Hh patterning activities examined in the early mouse embryo. Embryonic fibroblasts lacking mDispA respond normally to exogenously provided Sonic hedgehog (Shh) signal, but are impaired in stimulation of other responding cells when expressing Shh. We have developed a biochemical assay that directly measures the activity of Disp proteins in release of soluble Hh proteins. This activity is disrupted by alteration of residues functionally conserved in Patched and in a related family of bacterial transmembrane transporters, thus suggesting similar mechanisms of action for all of these proteins.     

Scube2 mediates Hedgehog signalling in the zebrafish embryo

The Hedgehog family of secreted morphogens specifies the fate of a large number of different cell types within invertebrate and vertebrate embryos, including the muscle cell precursors of the embryonic myotome of zebrafish. Formation of Hedgehog-sensitive muscle fates is disrupted within homozygous zebrafish mutants of the "you"-type class, the majority of which disrupt components of the Hedgehog (HH) signal transduction pathway. We have undertaken a phenotypic and molecular characterisation of one of these mutants, you, which we show results from mutations within the zebrafish orthologue of the mammalian gene scube2. This gene encodes a member of the Scube family of proteins, which is characterised by several protein motifs including EGF and CUB domains. Epistatic and molecular analyses position Scube2 function upstream of Smoothened (Smoh), the signalling component of the HH receptor complex, suggesting that Scube2 may act during HH signal transduction prior to, or during, receipt of the HH signal at the plasma membrane. In support of this model we show that scube2 has homology to cubilin, which encodes an endocytic receptor involved in protein trafficking suggesting a possible mode of function for Scube2 during HH signal transduction.     

Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells

Members of the Hedgehog (Hh) family of secreted signaling proteins function as potent short-range organizers in animal development. Their range of action is limited by a C-terminal cholesterol tether and the upregulation of Patched (Ptc) receptor levels. Here we identify a novel segment-polarity gene in Drosophila, dispatched (disp), and demonstrate that its product is required in sending cells for normal Hh function. In the absence of Disp, cholesterol-modified but not cholesterol-free Hh is retained in these cells, indicating that Disp functions to release cholesterol-anchored Hh. Despite their opposite roles, Disp and Ptc share structural homology in the form of a sterol-sensing domain, suggesting that release and sequestration of cholesterol-modified Hh may be based on related molecular pathways.     

E-cadherin is required for gastrulation cell movements in zebrafish

E-cadherin is a member of the classical cadherin family and is known to be involved in cell-cell adhesion and the adhesion-dependent morphogenesis of various tissues. We isolated a zebrafish mutant (cdh1(rk3)) that has a mutation in the e-cadherin/cdh1 gene. The mutation rk3 is a hypomorphic allele, and the homozygous mutant embryos displayed variable phenotypes in gastrulation and tissue morphogenesis. The most severely affected embryos displayed epiboly delay, decreased convergence and extension movements, and the dissociation of cells from the embryos, resulting in early embryonic lethality. The less severely affected embryos survived through the pharyngula stage and showed flattened anterior neural tissue, abnormal positioning and morphology of the hatching gland, scattered trigeminal ganglia, and aberrant axon bundles from the trigeminal ganglia. Maternal-zygotic cdh1(rk3) embryos displayed epiboly arrest during gastrulation, in which the enveloping layer (EVL) and the yolk syncytial layer but not the deep cells (DC) completed epiboly. A similar phenotype was observed in embryos that received antisense morpholino oligonucleotides (cdh1MO) against E-cadherin, and in zebrafish epiboly mutants. Complementation analysis with the zebrafish epiboly mutant weg suggested that cdh1(rk3) is allelic to half baked/weg. Immunohistochemistry with an anti-beta-catenin antibody and electron microscopy revealed that adhesion between the DCs and the EVL was mostly disrupted but the adhesion between DCs was relatively unaffected in the MZcdh1(rk3) mutant and cdh1 morphant embryos. These data suggest that E-cadherin-mediated cell adhesion between the DC and EVL plays a role in the epiboly movement in zebrafish.     

Zebrafish aussicht mutant embryos exhibit widespread overexpression of ace (fgf8) and coincident defects in CNS development

During the development of the zebrafish nervous system both noi, a zebrafish pax2 homolog, and ace, a zebrafish fgf8 homolog, are required for development of the midbrain and cerebellum. Here we describe a dominant mutation, aussicht (aus), in which the expression of noi and ace is upregulated. In aus mutant embryos, ace is upregulated at many sites in the embryo, while noi expression is only upregulated in regions of the forebrain and midbrain which also express ace. Subsequent to the alterations in noi and ace expression, aus mutants exhibit defects in the differentiation of the forebrain, midbrain and eyes. Within the forebrain, the formation of the anterior and postoptic commissures is delayed and the expression of markers within the pretectal area is reduced. Within the midbrain, En and wnt1 expression is expanded. In heterozygous aus embryos, there is ectopic outgrowth of neural retina in the temporal half of the eyes, whereas in putative homozygous aus embryos, the ventral retina is reduced and the pigmented retinal epithelium is expanded towards the midline. The observation that aus mutant embryos exhibit widespread upregulation of ace raised the possibility that aus might represent an allele of the ace gene itself. However, by crossing carriers for both aus and ace, we were able to generate homozygous ace mutant embryos that also exhibited the aus phenotype. This indicated that aus is not tightly linked to ace and is unlikely to be a mutation directly affecting the ace locus. However, increased Ace activity may underly many aspects of the aus phenotype and we show that the upregulation of noi in the forebrain of aus mutants is partially dependent upon functional Ace activity. Conversely, increased ace expression in the forebrain of aus mutants is not dependent upon functional Noi activity. We conclude that aus represents a mutation involving a locus normally required for the regulation of ace expression during embryogenesis.     

Ectopic Wnt signal determines the eyeless phenotype of zebrafish masterblind mutant

masterblind (mbl) is a zebrafish mutation characterised by the absence or reduction in size of the telencephalon, optic vesicles and olfactory placodes. We show that inhibition of Gsk3beta in zebrafish embryos either by overexpression of dominant negative dn gsk3beta mRNA or by lithium treatment after the midblastula transition phenocopies mbl. The loss of anterior neural tissue in mbl and lithium-treated embryos is preceded by posteriorization of presumptive anterior neuroectoderm during gastrulation, which is evident from the anterior shift of marker genes Otx2 and Wnt1. Heterozygous mbl embryos showed increased sensitivity to inhibition of GSK3beta by lithium or dn Xgsk3beta that led to the loss of eyes. Overexpression of gsk3beta mRNA rescued eyes and the wild-type fgf8 expression of homozygous mbl embryos. emx1 that delineates the telencephalon is expanded and shifted ventroanteriorly in mbl embryos. In contrast to fgf8, the emx1 expression domain was not restored upon overexpression of gsk3beta mRNA. These experiments place mbl as an antagonist of the Wnt pathway in parallel or upstream of the complex consisting of Axin, APC and Gsk3beta that binds and phosphorylates beta-catenin, thereby destabilising it. mbl maps on LG 3 close to a candidate gene axin1. In mbl we detected a point mutation in the conserved minimal Gsk3beta-binding domain of axin1 leading to a leucine to glutamine substitution at position 399. Overexpression of wild-type axin1 mRNA rescued mbl completely, demonstrating that mutant axin1 is responsible for the mutant phenotype. Overexpression of mutant L399Q axin1 in wild-type embryos resulted in a dose-dependent dominant negative activity as demonstrated by the loss of telencephalon and eyes. We suggest that the function of Axin1/Mbl protein is to antagonise the Wnt signal and in doing so to establish and maintain the most anterior CNS. Our findings provide new insights into the mechanisms by which the Wnt pathway generates anteroposterior polarity of the neural plate.     

The spt-1 mutation alters segmental arrangement and axonal development of identified neurons in the spinal cord of the embryonic zebrafish

We examined the arrangement and development of identified neurons in zebrafish embryos homozygous for the mutation spt-1, which acts autonomously and specifically to alter the development of precursors of trunk segmented mesoderm, resulting in muscle-deficient myotomes. We found that the mutation alters the morphology, number, and arrangement of identified motoneurons. By transplanting identified motoneurons between wild-type and mutant embryos, we found that the effect of the mutation was nonautonomous. We suggest that the segmental arrangement and proper axonal development of motoneurons may result from interactions with segmented mesoderm.     

Dispatched and scube mediate the efficient secretion of the cholesterol-modified hedgehog ligand

The Hedgehog (Hh) signaling pathway plays critical roles in metazoan development and in cancer. How the Hh ligand is secreted and spreads to distant cells is unclear, given its covalent modification with a hydrophobic cholesterol molecule, which makes it stick to membranes. We demonstrate that Hh ligand secretion from vertebrate cells is accomplished via two distinct and synergistic cholesterol-dependent binding events, mediated by two proteins that are essential for vertebrate Hh signaling: the membrane protein Dispatched (Disp) and a member of the Scube family of secreted proteins. Cholesterol modification is sufficient for a heterologous protein to interact with Scube and to be secreted in a Scube-dependent manner. Disp and Scube recognize different structural aspects of cholesterol similarly to how Niemann-Pick disease proteins 1 and 2 interact with cholesterol, suggesting a hand-off mechanism for transferring Hh from Disp to Scube. Thus, Disp and Scube cooperate to dramatically enhance the secretion and solubility of the cholesterol-modified Hh ligand.     

Laminins, via heparan sulfate proteoglycans, participate in zebrafish myotome morphogenesis by modulating the pattern of Bmp responsiveness

In zebrafish, Hedgehog-induced Engrailed expression defines a muscle fibre population that includes both slow and fast fibre types and exhibits an organisational role on myotome and surrounding tissues, such as motoneurons and lateral line. This Engrailed-positive population is restricted in the myotome to a central domain. To understand how this population is established, we have analysed the phenotype of the sly/lamc1 mutation in the Laminin γ1 chain that was shown to specifically affect Engrailed expression in pioneers. We find that the sly mutation affects Engrailed expression in the entire central domain and that Hedgehog signalling does not mediate this effect. We show that Bmp-responding cells are excluded from the central domain and that this pattern is modulated by laminins, but not by Hedgehog signalling. Knockdown of Bmp signalling rescues Engrailed expression in the sly mutant and ectopically activates Engrailed expression in slow and fast lineages in wild-type embryos. Last, extracellular matrix-associated heparan sulfate proteoglycans are absent in sly and their enzymatic removal mimics the sly phenotype. Our results therefore show that laminins, via heparan sulfate proteoglycans, are instrumental in patterning Bmp responsiveness and that Bmp signalling restricts Engrailed expression to the central domain. This study underlines the importance of extracellular cues for the precise spatial modulation of cell response to morphogens.     

The you gene encodes an EGF-CUB protein essential for Hedgehog signaling in zebrafish

Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed you-class mutations, that share common defects in somite shape and in most cases disrupt Hedgehog signaling. These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you-class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.     

A yolk protein mutant leads to defects in the secretion machinery of Drosophila melanogaster.

The three yolk proteins of Drosophila melanogaster are synthesized in the fat body and ovarian follicle cells. A mutation in yolk protein 3, YP3S1, has been described in which the leader sequence is not cleaved from the protein. We describe here ultrastructural and molecular studies on the YP3S1 mutant and show that the mutant protein enters the secretory pathway and forms precipitates, often as electron dense material in excessive elaborations of the plasma membrane. Females homozygous for YP3S1 lay fewer eggs than wild type flies and these embryos are less viable. The abnormal ultrastructure of the yolk spheres observed suggests that whilst YP3 is not completely essential for viability, it is required for normal yolk sphere morphogenesis.