Over the last several decades, the zebrafish has been developed into a powerful laboratory model organism. There is a burgeoning wealth of reverse genetic tools and conditional expression systems available to zebrafish researchers, although some of them have certain weaknesses and others are still not developed to their full potential. Because a reliable embryonic stem cell system has yet to be established, targeted knockouts and more subtle manipulations at endogenous loci have not been achieved in the zebrafish. To address this issue, I turned to zinc finger nucleases (ZFNs), which have been used for such manipulations in other systems. ZFNs are a chimeric fusion between zinc finger proteins (ZFPs), which can be engineered and designed to recognize specific DNA sequences, and the non-specific cleavage domain of the FokI endonuclease. Once the ZFPs bind to the targeted position, FokI will cleave the sequence and create a double strand break (DSB). The cell has two major pathways to repair DSBs: non-homologous end joining (NHEJ) and homology directed repair (HDR). In NHEJ, broken DNA ends are often misaligned, which leads to the incorporation of small insertions and deletions. HDR uses a homologous template to direct repair of the DSB. An exogenously supplied template, with significant regions of homology flanking the DSB, can also be used to direct repair and to "trick" the cell into incorporating novel sequence elements into endogenous loci. Both of these DSB repair pathways confer different ZFN-induced modifications: NHEJ for targeted mutagenesis, and HDR for a variety of manipulations including knock-ins and creation of conditional alleles. I have shown by proof-of-principle that ZFNs can be used efficiently for generating targeted knockouts in somatic cells and in the germline in zebrafish. Because there is a strong bias for DSB repair pathway choice in the zebrafish, ZFN-mediated HDR has proven to be a more challenging task. I have shown evidence of HDR working in the soma, and continue to screen for events in the germline, which is a much smaller population of cells.

Segmentation is a developmental program in animals that generates semi-repetitive structures along the body axis. In vertebrates, somites are formed sequentially from the mesoderm of the extending tailbud, eventually giving rise to structures such as axial muscles and vertebrae. Somites form with great regularity, every thirty minutes in zebrafish. The periodicity of somite formation is controlled by a set of oscillating genes known as the segmentation clock. To better understand the dynamics of the clock and its role in patterning somites, I have explored its behavior through an in vivo clock reporter, her1:her1-venus. I show that individual cells in the presomitic mesoderm express oscillating clock expression, consistent with predictions made by mathematical modeling and analysis in fixed embryos. I am able to rapidly track a large number of PSM cells using novel semi-automated cell tracking and fluorescence quantification programs. Through these methods, I find that the clock oscillations are coordinated through the Notch pathway, and a loss of Notch function causes slower and desynchonized clock oscillations. I also explore the interaction of mitosis and the segmentation clock, and find the two processes are connected. Finally, I investigate the slowing of the segmentation clock in real-time, and find that oscillations in the anterior PSM are about twice the periodicity of somitogenesis. This has interesting implications for the role of the segmentation clock in patterning somites, including the potential to polarize somites. By studying the segmentation clock in real-time, I am able to better investigate and understand the mechanisms driving somitogenesis.

T-box genes encode a family of transcription factors that contain a conserved DNA-binding domain, the T-box, and have been shown to play a crucial role in various developmental processes. Two zebrafish T-box genes, no tail (ntl) and spadetail (spt), as well as their orthologs in other vertebrates, have been shown to play important roles in the specification and patterning of posterior mesoderm. While wild-type zebrafish embryos develop ~30 somites that will later differentiate into muscle and vertebrae, spt mutants lack the anterior 15-17 trunk somites, and ntl mutants lack a notochord and the posterior 15 tail somites. Interestingly, embryos mutant for both ntl and spt lack all trunk and tail mesoderm, including tissues that form in both single mutants, indicating Ntl and Spt have overlapping functions in specifying these structures. Despite the obvious importance of T-box factors in development, relatively few of their transcriptional targets have been identified and tested to examine their role in mediating posterior mesoderm development. Recently, microarray results published by our lab and others have generated an extensive list of genes up- or down-regulated by Ntl and Spt. For my thesis I have chosen to focus on characterizing the role of several of these potential targets: mesogenin (msgn1), which belongs to the bHLH family of transcription factors; T-box gene 6-like (tbx6l), itself a T-box protein; RNA binding motif protein 38 (rbm38), an RNA-binding protein; and integrin beta 5 (itgb5), an adhesion and signaling molecule.

Through characterization of a null mutant, I have shown that msgn1 functions with spt to promote cell migration out of the tailbud, but is not essential for zebrafish development. Additionally, I have shown that depleting tbx6l by morpholino oligonucleotide results in perturbation of dorsal-ventral patterning during gastrulation as well as dose-dependent loss of tail mesoderm. I have also characterized the spatial and temporal expression patterns of rbm38 and itgb5 and shown that both genes are expressed in the presumptive mesoderm and tailbud, where they overlap with ntl and spt expression, consistent with a role in mesodermal development. Characterizing the functions of downstream targets will add to the gene regulatory network for specification of posterior mesoderm and help to detail the molecular mechanism of vertebrate posterior development in general.