ventral and posterior mesoderm during trunk and tail outgrowth, we generated a transgenic zebrafish line containing a heatshock-inducible dominant-negative Bmp receptor-GFP fusion. Our data show that Bmps are important for tail organizer formation and for patterning the ventral mesoderm during early gastrulation. However, from mid-gastrulation to the early somitogenesis stages, Bmp signaling is important for ventral tail fin development and for preventing secondary tail formation. We conclude that the role of Bmp signaling in the ventral and posterior mesoderm changes as gastrulation proceeds.

Bone morphogenetic protein (Bmp) signaling has long been known to be important for the early development of the ventral mesoderm, including blood, vasculature and kidney cells. Although Bmp genes are continually expressed in the ventral cells throughout gastrulation and somitogenesis, previous studies in zebrafish have not addressed how the role of Bmp signaling changes over time to regulate ventral mesoderm development. Here, we describe the use of a transgenic inducible dominantnegative Bmp receptor line to examine the temporal roles of Bmp signaling in ventral mesoderm patterning. Surprisingly, we find that Bmp signaling from the mid-gastrula stage through early somitogenesis is important for excluding blood and vascular precursors from the extreme ventral mesoderm, and we show that this domain is normally required for development of the cloaca (the common gut and urogenital opening). Using a novel assay for cloacal function, we find that larvae with reduced mid-gastrula Bmp signaling cannot properly excrete waste. We show that the cloacal defects result from alterations in the morphogenesis of the cloaca and from changes in the expression of genes marking the excretory system. Finally, we show that HrT, a T-box transcription factor, is a Bmp-regulated gene that has an essential function in cloacal development. We conclude that sustained Bmp signaling plays an important role in specification of the zebrafish cloaca by maintaining the fate of extreme ventral cells during the course of gastrulation and early somitogenesis. Furthermore, our data suggest that alterations in Bmp signaling are one possible cause of anorectal malformations during human embryogenesis.

Convergent extension of the mesoderm is the major driving force of vertebrate gastrulation. During this process, mesodermal cells move toward the future dorsal side of the embryo, then radically change behavior as they initiate extension of the body axis. How cells make this transition in behavior is unknown. We have identified the scaffolding protein and tumor suppressor Gravin as a key regulator of this process in zebrafish embryos. We show that Gravin is required for the conversion of mesodermal cells from a highly migratory behavior to the medio-laterally intercalative behavior required for body axis extension. In the absence of Gravin, paraxial mesodermal cells fail to shut down the protrusive activity mediated by the Rho/ROCK/ Myosin II pathway, resulting in embryos with severe extension defects. We propose that Gravin functions as an essential scaffold for regulatory proteins that suppress the migratory behavior of the mesoderm during gastrulation, and suggest that this function also explains how Gravin inhibits invasive behaviors in metastatic cells.[Keywords: AKAP; convergence and extension; gastrulation; Gravin; Rho; zebrafish] Supplemental material is available at http://www.genesdev.org. Vertebrate gastrulation involves a tightly regulated series of cellular rearrangements that coordinate the movements of the three germ layers (endoderm, mesoderm, and ectoderm) to establish the final embryonic body plan (Keller 2002). This process requires a very precise regulation of cell polarity, cell migration, and cell division. One of the major driving forces of vertebrate gastrulation is convergent extension, a mechanism in which mesodermal cells move toward the future dorsal side of the embryo and then intercalate between neighboring cells, resulting in an overall dorsoventral narrowing (convergence) and anteroposterior lengthening (extension) of the embryo (Heisenberg and Tada 2002;Keller 2002; SolnicaKrezel 2006).Mesodermal cells in the zebrafish embryo involved in convergent extension undergo a series of changes starting with a slow dorsal-directed migration, a faster migration as they get closer to the future dorsal side, and then intercalation as they approach the dorsal midline (Concha and Adams 1998; Jessen et al. 2002;Solnica-Krezel 2006). As the mesodermal cells change their behaviors, they undergo a series of morphological changes in cell shape and protrusive activity (Concha and Adams 1998). During early gastrulation, mesodermal cells have an overall rounded phenotype and extend transient lamellipodia and bleb-like protrusions with random orientation (Concha and Adams 1998;. By mid-to late gastrulation, cells become more polarized with lamellipodia that become stabilized and mediolaterally oriented. These cells then migrate in a highly dorsally biased manner. Later in gastrulation, mesodermal cells become tightly packed on the dorsal side of the embryo. These cells, through contact inhibition of migratory behavior, become elongated and firmly attached to their neighboring cells, reduce protrusions,...

Here we investigate the function of zebrafish Bcl-2 family proteins and demonstrate important conservation of function across zebrafish and mammalian systems. We have isolated a zebrafish ortholog of mammalian BIM and show that it is the most toxic of the zebrafish BH3-only genes examined, sharing this characteristic with the mammalian BIM gene. The zebrafish bad gene shows a complete lack of embryonic lethality, but like mammalian BAD, its pro-apoptotic activity is regulated through phosphorylation of critical serines. We also found that the pattern of mitochondrial dysfunction observed by zebrafish BH3 domain peptides in a mammalian cytochrome c release assay recapitulates the pattern of embryonic lethality induced by the respective mRNA injections in vivo. In contrast to zebrafish Bim, Bid exhibited only weak binding to zebrafish Bcl-2 and moderate-to-weak overall lethality in zebrafish embryos and isolated mitochondria. Given that zebrafish Bcl-2 binds strongly to mammalian BID and BIM peptides and proteins, the protein identified as the zebrafish Bid ortholog has different properties than mammalian BID. Overall, our results demonstrate the high degree of functional conservation between zebrafish and mammalian Bcl-2 family proteins, thus validating the zebrafish as a model system to further dissect the molecular mechanisms that regulate apoptosis in future forward genetic and chemical modifier screens.

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