Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors

The Ras/MAPK pathway is critical for human development and plays a central role in the formation and progression of most cancers. Children born with germ-line mutations in BRAF, MEK1 or MEK2 develop cardio-facio-cutaneous (CFC) syndrome, an autosomal dominant syndrome characterized by a distinctive facial appearance, heart defects, skin and hair abnormalities and mental retardation. CFC syndrome mutations in BRAF promote both kinase-activating and kinase-impaired variants. CFC syndrome has a progressive phenotype, and the availability of clinically active inhibitors of the MAPK pathway prompts the important question as to whether such inhibitors might be therapeutically effective in the treatment of CFC syndrome. To study the developmental effects of CFC mutant alleles in vivo, we have expressed a panel of 28 BRAF and MEK alleles in zebrafish embryos to assess the function of human disease alleles and available chemical inhibitors of this pathway. We find that both kinase-activating and kinase-impaired CFC mutant alleles promote the equivalent developmental outcome when expressed during early development and that treatment of CFC-zebrafish embryos with inhibitors of the FGF-MAPK pathway can restore normal early development. Importantly, we find a developmental window in which treatment with a MEK inhibitor can restore the normal early development of the embryo, without the additional, unwanted developmental effects of the drug

Dosage of Fgf8 determines whether cell survival is positively or negatively regulated in the developing forebrain

FGF8 is known to be an important regulator of forebrain development. Here, we investigated the effects of varying the level of Fgf8 expression in the mouse forebrain. We detected two distinct responses, one that was proportionate with Fgf8 expression and another that was not. The latter response, which led to effects on cell survival, displayed a paradoxical relationship to Fgf8 dosage. Either eliminating or increasing Fgf8 expression increased apoptosis, whereas reducing Fgf8 expression had the opposite effect. To explain these counterintuitive observations, we suggest that an FGF8-dependent cell-survival pathway is negatively regulated by intracellular inhibitors produced in proportion to FGF8 concentration. Our data provide insight into the function of FGF8 in forebrain development and underscore the value of using multiple alleles and different experimental approaches to unravel the complexities of gene function in vertebrate development

Alternative splicing generates an isoform of the human Sef gene with altered subcellular localization and specificity

Receptor tyrosine kinases (RTKs) control a multitude of biological processes and are therefore subjected to multiple levels of regulation. Negative feedback is one of the mechanisms that provide an effective means to control RTK-mediated signaling. Sef has recently been identified as a specific antagonist of fibroblast growth factor (FGF) signaling in zebrafish and subsequently in mouse and human. Sef encodes a putative type I transmembrane protein that antagonizes the Ras/mitogen-activated protein kinase pathway in all three species. Mouse Sef was also shown to inhibit the phosphatidylinositol 3-kinase pathway. We show here that an alternative splicing mechanism generates an isoform of human Sef, hSef-b, which unlike the previously reported Sef (hSef-a) is a cytosolic protein. Contrary to hSef-a, which is ubiquitously expressed, hSef-b transcripts display a restricted pattern of expression in human tissues. hSef-b inhibits FGF-induced cell proliferation and prevents the activation of mitogen-activated protein kinase without affecting the upstream component MAPK kinase. Furthermore, hSef-b does not antagonize FGF induction of the phosphatidylinositol 3-kinase pathway. In addition to the effects on FGF signaling, hSef-b inhibited cellular response to platelet-derived growth factor but not other RTK ligands. Therefore, alternative splicing of the hSef gene expands the Sef feedback inhibition repertoire of RTK signaling

Mutation of the atrophin2 gene in the zebrafish disrupts signaling by fibroblast growth factor during development of the inner ear

The development of the vertebrate inner ear depends on the precise expression of fibroblast growth factors. In a mutagenesis screen for zebrafish with abnormalities of inner-ear development and behavior, we isolated a mutant line, ru622, whose phenotypic characteristics resembled those of null mutants for the gene encoding fibroblast growth factor 8 (Fgf8): an inconsistent startle response, circular swimming, fused otoliths, and abnormal semicircular canals. Positional cloning disclosed that the mutant gene encodes the transcriptional corepressor Atrophin2. Both the Fgf8 protein and zebrafish “similar expression to fgf genes” protein (Sef), an antagonist of fibroblast growth factors induced by Fgf8 itself, were found to be overexpressed in ru622 mutants. We therefore hypothesized that an excess of Sef eliminates Fgf8 signals and produces an fgf8 null phenotype in ru622 mutants. In support of this idea, we could rescue larvae whose atrophin2 expression had been diminished with morpholinos by reducing the expression of Sef as well. We propose that Atrophin2 plays a role in the feedback regulation of Fgf8 signaling. When mutation of the atrophin2 gene results in the overexpression of both Fgf8 and Sef, the excessive Sef inhibits Fgf8 signaling. The resultant imbalance of Fgf8 and Sef signals then underlies the abnormal aural development observed in ru622

Suppression of Alk8-mediated Bmp signaling cell-autonomously induces pancreatic β-cells in zebrafish

Bmp signaling has been shown to regulate early aspects of pancreas development, but its role in endocrine, and especially β-cell, differentiation remains unclear. Taking advantage of the ability in zebrafish embryos to cell-autonomously modulate Bmp signaling in single cells, we examined how Bmp signaling regulates the ability of individual endodermal cells to differentiate into β-cells. We find that specific temporal windows of Bmp signaling prevent β-cell differentiation. Thus, future dorsal bud-derived β-cells are sensitive to Bmp signaling specifically during gastrulation and early somitogenesis stages. In contrast, ventral pancreatic cells, which require an early Bmp signal to form, do not produce β-cells when exposed to Bmp signaling at 50 hpf, a stage when the ventral bud-derived extrapancreatic duct is the main source of new endocrine cells. Importantly, inhibiting Bmp signaling within endodermal cells via genetic means increased the number of β-cells, at early and late stages. Moreover, inhibition of Bmp signaling in the late stage embryo using dorsomorphin, a chemical inhibitor of Bmp receptors, significantly increased β-cell neogenesis near the extrapancreatic duct, demonstrating the feasibility of pharmacological approaches to increase β-cell numbers. Our in vivo single-cell analyses show that whereas Bmp signaling is necessary initially for formation of the ventral pancreas, differentiating endodermal cells need to be protected from exposure to Bmps during specific stages to permit β-cell differentiation. These results provide important unique insight into the intercellular signaling environment necessary for in vivo and in vitro generation of β-cells