Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 82-95).

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct. 31, 2007). Includes bibliographical references.

The gastrula stage represents the point in development at which the three primary germ layers diverge. At this point the gene regulatory networks that specify the germ layers are established and the genes that define the differentiated states of the tissues have begun to be activated. These networks have been well characterized in sea urchins, but not in other echinoderms. Embryos of the brittle star Ophiocoma wendtii share a number of developmental features with sea urchin embryos, including the ingression of mesenchyme cells that give rise to an embryonic skeleton. Notable differences are that no micromeres are formed during cleavage divisions and no pigment cells are formed during development to the pluteus larva stage. More subtle changes in timing of developmental events also occur. To explore the molecular basis for the similarities and differences between these two echinoderms, the gastrula transcriptome of Ophiocoma wendtii was sequenced and characterized. I identified brittle star transcripts that correspond to 3385 genes in existing databases, including 1863 genes shared with the sea urchin Strongylocentrotus purpuratus gastrula transcriptome. I have characterized the functional classes of genes present in the transcriptome and compared them to those found in sea urchin. I then examined which members of the germ-layer specific gene regulatory networks (GRNs) of S. purpuratus are expressed in the O. wendtii gastrula. The results indicate that there is a shared "genetic toolkit" central to the echinoderm gastrula, a key stage in embryonic development, though there are also differences that reflect changes in developmental processes. The brittle star expresses genes representing all functional classes at the gastrula stage. Brittle stars and sea urchins have comparable numbers of each class of genes, and share many of the genes expressed at gastrula. Examination of the brittle star genes whose sea urchin orthologs are utilized in germ layer specification reveals a relatively higher level of conservation of key regulatory components compared to the overall transcriptome. I also identify genes that were either lost or whose temporal expression has diverged from that of sea urchins. Overall, the data suggest that embryonic skeleton formation in sea urchins and brittle stars represents convergent evolution by independent cooptation of a shared pathway utilized in adult skeleton formation. Transcription factors are of central importance to both development and evolution. Patterns of their expression and interactions form the gene regulatory networks which control the building of the embryonic body. Alterations in these patterns can result in the construction of altered bodies. To help increase understanding of this process, I compared the transcription factor mRNAs present in early gastrula-stage embryos of the brittle star Ophiocoma wendtii to those found in two species of sea urchins and a starfish. Brittle star homologs were found for one third of the transcription factors in the sea urchin genome and half of those that are expressed at equivalent developmental stages in sea urchins and starfish. Overall, the patterns of transcription factors found and not found in brittle star resemble those of other echinoderms, with the differences largely consistent with morphological differences. This study provides further evidence for the existence of deeply conserved developmental genetic processes, with various elements shared among echinoderms, deuterostomes, and metazoans.

The work in presented in this thesis identifies Drosophila Wnt8 as a novel zygotic target of the Dorsal/Twist/Snail network using a microarray analysis to identify differentially expressed genes in maternal dorsal mutant and gain-of-function Toll10b embryos as compared to wild type. In-situ hybridization with a Wnt8 antisense RNA probe revealed a fairly complex expression pattern in the early embryo. No maternal expression was observed and the first zygotic expression appeared at stage 4 at the poles. This was followed by patchy and relatively weak expression in the presumptive mesoderm with stronger expression in the mesectoderm and later the neuroectoderm. These expression required the Dorsal/Twist/Snail network with some input from Delta in the neuroectoderm. All embryonic Wnt8 expression ceased after late stage 10. Snail was found to repress Wnt8 in the presumptive mesoderm. The relevance of Wnt8 as a Snail target was tested by bypassing this repression in wild type embryos using a maternal Gal4 driver to drive UAS-Wnt8. This led to a loss of ventral furrow formation and a phenocopy of the snail mutant phenotype, thereby indicating that the repression of Wnt8 by Snail is important for gastrulation. Further investigation into the mechanism revealed a reduction in the expression of multiple target genes of Dorsal (including snail) in these Wnt8 overexpressing embryos. Ventral nuclear Dorsal protein was reduced as compared to wild type, suggesting that high levels of Wnt8 can antagonize Dorsal nuclear localization. Deficiency embryos lacking Wnt8 showed the opposite phenotype of expanded anterior and posterior snail RNA staining, as well as an expanded nuclear Dorsal signal in the posterior. This could be phenocopied using dsRNA against Wnt8, and was fully rescueable in the deficiency background using a Wnt8 genomic fragment. It has been reported that loss-of-function snail embryos lose the sharp lateral boundaries and high levels of snail expression more rapidly as compared to wild type. We hypothesize that this loss is due to derepressed Wnt8 antagonizing Dorsal and consequently its target, snail. In support of our hypothesis, double mutants of snail and the Wnt8 deficiency show a rescue of the snail pattern, though not a rescue of ventral furrow formation. Western blot analysis reveals a decrease in the levels of phosphorylation of Dorsal in Wnt8 overexpressing embryos as compared to wild type. Phosphorylation of Dorsal is required for its nuclear translocation. Hence, these data corroborate the observation of reduced nuclear Dorsal in embryos overexpressing Wnt8. Together, these data point to Wnt8 being an important target and a feedback inhibitor of the Dorsal/Twist/Snail pathway that achieves its effect by the inhibition of Dorsal.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 3 uppsatser.

Fibroblast growth factor (Fgf) and Retinoic acid (RA) are known to be involved in patterning the posterior embryo. Work has shown that Fgf can convert anterior tissue into posterior fates and that embryos deficient in Fgf signaling lack posterior trunk and tail structures. Likewise, studies performed on RA have shown that overexpression of RA posteriorizes anterior tissue, while disrupting RA signaling yields a loss of posterior fates. While it appears these signals are necessary for posterior development, the role Fgf and RA play in development of the hindbrain is still enigmatic. A detailed study of the requirements for Fgf and RA in the early vertebrate hindbrain are lacking, namely due to a deficiency in gene markers for the presumptive hindbrain at early developmental stages. In this study, we make use of recently isolated genes, which are expressed in the presumptive hindbrain region at early developmental stages, to explore Fgf and RA regulation of the early vertebrate hindprain. We employed both overexpression and loss of function approaches to explore the role of Fgf in early vertebrate development with an emphasis on the presumptive hindbrain region in zebrafish embryos. By loss of function analysis, we show that Fgf regulates genes expressed exclusively in the hindbrain region (meis3 and hoxbla) as well as genes whose expression domains encompass both the hindbrain and more caudal regions (nlz and hoxb1b), thus demonstrating a requirement for Fgf signaling throughout the anteroposterior axis of the hindbrain (rostral to caudal hindbrain) by mid-gastrula stages. To further characterize early gene regulation by Fgf, we utilized an in vitro system and found that Fgf is sufficient to induce nlz directly and hoxb1b indirectly, while it does not induce meis3 or hoxb1a. Furthermore, in vivo work demonstrates that Fgf soaked beads can induce nlz and hoxb1b adjacent to the bead and meis3at a distance. Given the regulation of these genes in vitro and in vivo by Fgf and their position along the rostrocaudal axis of the embryo, our results suggest an early acting Fgf resides in the caudal end of the embryo and signals at a distance to the hindbrain. We detect a similar regulation of hindbrain genes by RA at gastrula stages as well, suggesting that both factors are essential for early hindbrain development. Interestingly however, we find that the relationship between Fgf and RA is dynamic throughout development. Both signals are required at gastrula stages as disruption of either pathway alone disrupts hindbrain gene expression, but a simultaneous disruption of both pathways at later stages is required to disrupt the hindbrain. We suggest that Fgf and RA are present in limiting concentrations at gastrula stages, such that both factors are required for gene expression or that one factor is necessary for activation of the other. Our results also reveal a changing and dynamic relationship between Fgf and RA in the regulation of the zebrafish hindbrain, suggesting that at segmentation stages, Fgf and RA may no longer be limiting or that they are no longer interdependent. As we have demonstrated that an early Fgf signal is required for gastrula stage hindbrain development, we next questioned which Fgf performed this function. We have demonstrated that the early Fgf signal required for hindbrain development is not Fgf3 or Fgf8, two Fgfs known to be involved in signaling centers at the mid-hindbrain boundary (MHB) and rhombomere (r) 4. We further show that two recently identified Fgfs, Fgf4 and Fgf24 are also insufficient alone or in combination with other known Fgfs to regulate hindbrain gene expression. However, as Fgfs may act combinatorially, we do not rule out the possibility of their involvement in early hindbrain gene regulation. However, as time passes and additional Fgfs are isolated and cloned, the elusive Fgf signal required for early hindbrain development will likely be identified. Taken together, we propose that an early acting Fgf residing in the caudal end of the embryo regulates hindbrain genes together with RA at gastrula stages. We suggest that both Fgf and RA are required for gene expression at gastrula stages, but this requirements changes over time as Fgf and RA become redundant. We also demonstrate that the Fgf required for gastrula stage hindbrain development has yet to be identified.