Essential role of spi-1-like (spi-1l) in zebrafish myeloid cell differentiation.

The ETS protein Spi-1/Pu.1 plays a pivotal and widespread role throughout hematopoiesis in many species. This study describes the identification, characterization, and functional analysis of a new zebrafish spi transcription factor spi-1-like (spi-1l) that is expressed in primitive myeloid cells, erythro-myelo progenitor cells, and in the adult kidney. Spi-1l functions genetically downstream of etsrp, scl, and spi-1/pu.1 in myeloid differentiation. Spi-1l is coexpressed in a subset of spi-1/pu.1 cells and its function is necessary and sufficient for macrophage and granulocyte differentiation. These results establish a critical role for spi-1l in zebrafish myeloid cell differentiation.

One-Eyed Pinhead and Spadetail are essential for heart and somite formation.

Mutant analysis in the zebrafish Danio rerio has demonstrated distinct developmental roles for the T-box transcription factor Spadetail (Spt) and the Nodal-receptor cofactor One-Eyed Pinhead (Oep) in the formation of mesoderm and endoderm. Here, we show that spt and oep genetically interact and are together essential for the formation of cardiac and somitic mesoderm. These two mesodermal defects are dependent on different effectors of Nodal signalling; cardiac mesoderm formation involves the mix-like transcription factor Bonnie and Clyde (Bon), whereas somitogenesis is dependent on a different pathway. Analysis of the somite defect in Zoep;spt embryos has provided insights into the control of somitic mesoderm formation by Spt, which was previously implicated in the regulation of cell adhesion and motility. We show that the failure to form somites in Zoep;spt embryos is independent of this and that Spt must have an additional function. We propose that the major role of Spt in somitogenesis is to promote the differentiation of presomitic mesoderm from tailbud progenitors by antagonizing progenitor-type gene expression and behaviour.

Eomesodermin is a localized maternal determinant required for endoderm induction in zebrafish.

In zebrafish, endoderm induction occurs in marginal blastomeres and requires Casanova (Cas), the first endoderm-specific factor expressed in the embryo. Whereas the transcription factors Gata5 and Bon are necessary and sufficient for cas expression in marginal blastomeres, Bon and Gata5 are unable to induce cas in animal pole cells, suggesting that cas expression requires an additional, unidentified factor(s). Here, we show that cas expression depends upon the T box transcription factor Eomesodermin (Eomes), a maternal determinant that is localized to marginal blastomeres. Eomes synergizes potently with Bon and Gata5 to induce cas, even in animal pole blastomeres. We show that Eomes is required for endogenous endoderm induction, acting via an essential binding site in the cas promoter. Direct physical interactions between Eomes, Bon, and Gata5 suggest that Eomes promotes endoderm induction in marginal blastomeres by facilitating the assembly of a transcriptional activating complex on the cas promoter.

The zebrafish genome contains two inducible, functional cyclooxygenase-2 genes.

Cyclooxygenase is a key enzyme in prostanoid biosynthesis. Mammalian species have two cyclooxygenases, constitutively expressed cyclooxygenase-1 (Cox-1) and inducible cyclooxygenase-2 (Cox-2). Cox-1 and/or Cox-2 have been also identified in other vertebrates, including fish. We identified a second zebrafish Cox-2 gene orthologue, Cox-2b. All of the functionally important amino acids for cyclooxygenase enzymes are conserved in Cox-2b. The 3' untranslated region of the Cox-2b message contains AU rich elements characteristic of regulation at the level of mRNA stability. Constitutive tissue expression patterns for Cox-2a and Cox-2b are distinct, but overlap. Both Cox-2a and Cox-2b expression are inducible in the kidney when fish are exposed to tetradecanoylphorbol acetate. Like Cox-2a, Cox-2b protein, expressed in COS cells is functionally active. Thus, the zebrafish genome contains two functional, inducible Cox-2 genes. Database searching demonstrates that some fish genomes contain multiple Cox-1 or Cox-2 cyclooxygenase genes, suggesting alternate duplication and retention of this gene.

Interplay between FGF, one-eyed pinhead, and T-box transcription factors during zebrafish posterior development.

The zebrafish T-box transcription factors spadetail (spt) and the brachyury ortholog no tail (ntl) are together essential for posterior mesoderm formation. In addition to being functionally redundant, spt and ntl also genetically interact with zygotic mutant alleles of one-eyed pinhead (Zoep), leading to synergistic mesodermal defects. Here we have used genetic and pharmacological assays to address the mechanism of these interactions. We show that Zoep and ntl are together required upstream of spt expression, accounting for the severity of the mesodermal defects in Zoep;ntl embryos. Since Xenopus brachyury is proposed to regulate fgf expression, and FGF signaling is required for spt expression, we analyzed the involvement of the FGF signaling pathway in these genetic interactions. Using a specific inhibitor of FGFR activity to indirectly assay the strength of FGF signaling in individual embryos, we found that spt and ntl mutant embryos were both hypersensitive to the FGFR inhibitor. This hypersensitivity is consistent with the possibility that Spt and Ntl function upstream of FGF signaling. Furthermore, we show that minor pharmacological or genetic perturbations in FGF signaling are sufficient to dramatically enhance the Zoep mutant phenotype, providing a plausible explanation for why Zoep genetically interacts with spt and ntl. Finally, we show that Zoep and ace/fgf8 function are essential for the formation of all posterior tissues, including spinal cord. Taken together, our data provide strong in vivo support for the regulation of FGF signaling by T-box transcription factors, and the cooperative activity of Oep and FGF signaling during the formation of posterior structures.

HrT is required for cardiovascular development in zebrafish.

The recently identified zebrafish T-box gene hrT is expressed in the developing heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT are expressed in cardiovascular cells from Drosophila to mouse, suggesting that the function of hrT is evolutionarily conserved. The role of hrT in cardiovascular development, however, has not thus far been determined in any animal model. Using morpholino antisense oligonucleotides, we show that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation were normal in hrT morphant embryos, subsequently the hearts failed to undergo looping, and late onset defects in chamber morphology and gene expression were observed. In particular, we found that the loss of hrT function led to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, we show that overexpression of hrT causes a significant downregulation of tbx5, indicating that one key role of hrT is to regulate the levels of tbx5. Secondly, we found that HrT is required to inhibit the expression of the blood lineage markers gata1 and gata2 in the most posterior lateral plate mesoderm. Finally, we show that HrT is required for vasculogenesis in the trunk, leading to similar vascular defects to those observed in midline mutants such as floating head. hrT expression in the vascular progenitors depends upon midline mesoderm, indicating that this expression is one important component of the response to a midline-derived signal during vascular morphogenesis.