The zebrafish-secreted matrix protein you/scube2 is implicated in long-range regulation of hedgehog signaling

The Hedgehog (Hh) signal plays a pivotal role in induction of ventral neuronal and muscle cell types around the midline during vertebrate development [1]. We report that the gene disrupted in zebrafish you mutants, in which Hh signaling is impaired, encodes the secreted matrix protein Scube2. Consistently, epistasis analyses suggested that Scube2 functions upstream of Hh ligands or through a parallel pathway. In addition, overexpression analyses suggested that Scube2 is an essential, but a permissive, mediator of Hh signaling in zebrafish embryos. Surprisingly, the you gene is expressed in the dorsal neural tube, raising the possibility that Scube2 could indirectly act via a long-range regulator of Hh signaling. The dorsal Bmps have a long-range and opposing influence on Hh signaling [2-5]. We show that neural plate patterning is affected in you mutants in a way that is consistent with the aberrant long-range action of a Bmp-dependent signal. We further show that Bmp activity can be attenuated by the coexpression of Scube2. Our data support the idea that Scube2 can modulate the long-range action of Bmp-dependent signaling in the neural tube and somites.     

Expression profiling identifies novel Hh/Gli-regulated genes in developing zebrafish embryos

The Hedgehog (Hh) signaling pathway plays critical instructional roles during embryonic development. Misregulation of Hh/Gli signaling is a major causative factor in human congenital disorders and in a variety of cancers. The zebrafish is a powerful genetic model for the study of Hh signaling during embryogenesis, as a large number of mutants that affect different components of the Hh/Gli signaling system have been identified. By performing global profiling of gene expression in different Hh/Gli gain- and loss-of-function scenarios we identified known (e.g., ptc1 and nkx2.2a) and novel Hh-regulated genes that are differentially expressed in embryos with altered Hh/Gli signaling function. By uncovering changes in tissue-specific gene expression, we revealed new embryological processes that are influenced by Hh signaling. We thus provide a comprehensive survey of Hh/Gli-regulated genes during embryogenesis and we identify new Hh-regulated genes that may be targets of misregulation during tumorigenesis.     

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.     

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.     

Multiple roles for Hedgehog signaling in zebrafish pituitary development

The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog (Hh), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) families. To better understand when and where Hh signaling influences pituitary development, we have analyzed the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants). While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, our data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. We report that Hh signaling is necessary between 10 and 15 h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. We show that the Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (15-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. We also use the zebrafish Hh pathway mutants to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. Our results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland.