Somites are fundamental structures within the paraxial mesoderm of the vertebrate embryo that give rise to the vertebrae and muscle of the trunk and tail. Studies of knockout mice and gene expression analyses have shown that the Notch pathway is crucial in establishing the reiterative pattern of somites. A large-scale screen in zebrafish previously identified five mutants that show abnormalities in somite boundary formation. Four have essentially the same phenotype, with posterior somite defects and neuronal hyperplasia; recent work has suggested that genes affected in these mutants encode components of the Notch signaling cascade. The fifth mutant, fused somites (fss), shows a different phenotype characterized by complete lack of somite formation along the entire antero-posterior axis. Gene expression and phenotypic analyses in mutant embryos have implicated Fss in somite formation independent of Notch signaling, suggesting the presence of a new pathway regulating somite boundary formation. We show here that the fss gene encodes a T-box transcription factor that is expressed in intermediate to anterior presomitic mesoderm (PSM) and is involved in PSM maturation.
To investigate the conservation of mechanisms for mesodermal patterning between zebrafish and Xenopus, we isolated two cDNA clones encoding bone morphogenetic protein (BMP)-related proteins from a zebrafish cDNA library. Based on their predicted amino acid sequences, these two clones were designated as zbmp-2 and zbmp-4. Whole-mount in situ hybridization analysis revealed that in gastrula embryo, both genes were localized in the ventral part of the embryo, consistent with the proposed function of Xenopus BMP-4 in ventral mesoderm specification. zbmp-4 expression, however, was also seen in the embryonic shield, the most dorsal mesodermal structure. To examine the ability of zbmp-2 to ventralize mesoderm, we injected synthetic mRNA into zebrafish embryos and found that overexpression of this gene eliminated dorsal structures including notochord at both morphological and molecular level. In contrast, expression of ventral marker gene eve1 was expanded to the dorsal side. These effects are analogous to the ventralization of embryos caused by ectopic xBMP-4 expression. Taken together, one may conclude that the developmental mechanisms for mesodermal patterning regulated by BMPs are evolutionarily conserved between amphibians and teleosts.
The mechanisms generating stably differentiated cell-types from multipotent precursors are key to understanding normal development and have implications for treatment of cancer and the therapeutic use of stem cells. Pigment cells are a major derivative of neural crest stem cells and a key model cell-type for our understanding of the genetics of cell differentiation. Several factors driving melanocyte fate specification have been identified, including the transcription factor and master regulator of melanocyte development, Mitf, and Wnt signalling and the multipotency and fate specification factor, Sox10, which drive mitf expression. While these factors together drive multipotent neural crest cells to become specified melanoblasts, the mechanisms stabilising melanocyte differentiation remain unclear. Furthermore, there is controversy over whether Sox10 has an ongoing role in melanocyte differentiation. Here we use zebrafish to explore in vivo the gene regulatory network (GRN) underlying melanocyte specification and differentiation. We use an iterative process of mathematical modelling and experimental observation to explore methodically the core melanocyte GRN we have defined. We show that Sox10 is not required for ongoing differentiation and expression is downregulated in differentiating cells, in response to Mitfa and Hdac1. Unexpectedly, we find that Sox10 represses Mitf-dependent expression of melanocyte differentiation genes. Our systems biology approach allowed us to predict two novel features of the melanocyte GRN, which we then validate experimentally. Specifically, we show that maintenance of mitfa expression is Mitfa-dependent, and identify Sox9b as providing an Mitfa-independent input to melanocyte differentiation. Our data supports our previous suggestion that Sox10 only functions transiently in regulation of mitfa and cannot be responsible for long-term maintenance of mitfa expression; indeed, Sox10 is likely to slow melanocyte differentiation in the zebrafish embryo. More generally, this novel approach to understanding melanocyte differentiation provides a basis for systematic modelling of differentiation in this and other cell-types.
The expression patterns of region-specific neuroectodermal genes and fate-map analyses in zebrafish gastrulae suggest that posterior neural development is initiated by nonaxial signals, distinct from organizer-derived secreted bone morphogenetic protein (BMP) antagonists. This notion is further supported by the misexpression of a constitutively active form of zebrafish BMP type IA receptor (CA-BRIA) in the zebrafish embryos. It effectively suppressed the anterior neural marker, otx2, but not the posterior marker, hoxb1b. Furthermore, we demonstrated that the cells in the presumptive posterior neural region lose their neural fate only when CA-BRIA and Xenopus dominant-negative fibroblast growth factor (FGF) receptors (XFD) are coexpressed. The indications are that FGF signaling is involved in the formation of the posterior neural region, counteracting the BMP signaling pathway within the target cells. We then examined the functions of Fgf3 in posterior neural development. Zebrafish fgf3 is expressed in the correct place (dorsolateral margin) and at the correct time (late blastula to early gastrula stages), the same point that the most precocious posterior neural marker, hoxb1b, is first activated. Unlike other members of the FGF family, Fgf3 had little mesoderm-inducing activity. When ectopically expressed, Fgf3 expands the neural region with suppression of anterior neural fate. However, this effect was mediated by Chordino (zebrafish Chordin), because Fgf3 induces chordino expression in the epiblast and Fgf3-induced neural expansion was substantially suppressed in dino mutants with mutated chordino genes. The results obtained in the present study reveal multiple actions of the FGF signal on neural development: it antagonizes BMP signaling within posterior neural cells, induces the expression of secreted BMP antagonists, and suppresses anterior neural fate.
DNA photolyases are enzymes which mediate the light-dependent repair (photoreactivation) of UV-induced damage products in DNA by direct reversal of base damage rather than via excision repair pathways. Arabidopsis thaliana contains two photolyases specific for photoreactivation of either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4)pyrimidones (6-4PPs), the two major UV-B-induced photoproducts in DNA. Reduced FADH and a reduced pterin were identified as cofactors of the native Arabidopsis CPD photolyase protein. This is the first report of the chromophore composition of any native class II CPD photolyase protein to our knowledge. CPD photolyase protein levels vary between tissues and with leaf age and are highest in flowers and leaves of 3-5-week-old Arabidopsis plants. White light or UV-B irradiation induces CPD photolyase expression in Arabidopsis tissues. This contrasts with the 6-4PP photolyase protein which is constitutively expressed and not regulated by either white or UV-B light. Arabidopsis CPD and 6-4PP photolyase enzymes can remove UV-B-induced photoproducts from DNA in planta even when plants are grown under enhanced levels of UV-B irradiation and at elevated temperatures although the rate of removal of CPDs is slower at high growth temperatures. These studies indicate that Arabidopsis possesses the photorepair capacity to respond effectively to increased UV-B-induced DNA damage under conditions predicted to be representative of increases in UV-B irradiation levels at the Earth's surface and global warming in the twenty-first century.
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