| In vertebrate embryology, the formation of three distinct cellular populations during gastrulation, termed germ layers, is critical for the proper patterning of the embryo as well as the formation and placement of most of the tissues of the adult animal. Understanding the genetics of the initial specification of these three germ layers, the ectoderm, mesoderm, and endoderm, has been a goal for developmental biologists since the concept arose in the late 1800's. In zebrafish, just prior to the onset of gastrulation, the cells that will give rise to mesodermal tissues are located near the equator of the embryo, at the junction of the embryo proper and the vegetal extraembryonic yolk, where they are heavily intermingled with endoderm precursors. Signaling in- and between these germ layer precursors as well as signals emanating from the extraembryonic tissue are important for the proper specification of both mesoderm and endoderm.;This dissertation describes a novel technique for the isolation of user-defined populations of cells from developing zebrafish embryos. This technique was employed to isolate and examine newly specified mesoderm and endoderm precursor cells of the late blastula zebrafish embryo. Our study was the first to isolate this particular cell population without genetically altering the whole embryo and led to the discovery of genes with previously undescribed expression in mesoderm and endoderm.;This dissertation also addresses open questions regarding mesoderm induction signals emanating from an extraembryonic nuclear syncytium termed the YSL, which is unique to teleosts. Although the YSL is a fish-specific structure, it has been proposed as an evolutionary relative of mouse primitive endoderm, which contributes primarily to extraembryonic lineages. Previously, we described a combinatorial role for Nodal signaling and mxtx2, a transcription factor expressed in the YSL, in the induction of ntla, a zebrafish ortholog of the pan-mesodermal transcription factor Brachyury/T . Ntla is required for proper posterior development, as zebrafish Ntla mutants lack a significant amount of posterior tissue and do not form a fully differentiated notochord. I identified Bmp2b and Bmp7a as Mxtx2-dependent secreted signals that are necessary, along with Nodal signaling, for ntla induction. These data provide a mechanism for understanding how a transcription factor, acting cell autonomously in the extraembryonic YSL, can exert its effects on cells of the embryo proper. It has been well established that BMP signaling plays a critical role in ventral cell fate determination, but this work identifies a novel extraembryonic-specific role for BMPs in mesoderm induction.;Taken together, the work presented here provides insight into germ layer specification, a critical step in early embryonic development. Defects in germ layer induction and/or gastrulation result in spontaneous abortion or severe birth defects. An example of this is caudal dysgenesis, whose phenotypic spectrum is thought to be a result of a mesoderm deficiency. As the pathways that are involved in the early steps of mesoderm formation unfold, with the aid of studies such as those presented in this dissertation, a better understanding of this class of birth defects should arise. |
