@article{Development:1716,
      recid = {1716},
      author = {Boyle Anderson, Erin Alana Thorpe},
      title = {The Role of the Tbx5 Paralogues during Pectoral Fin  Development in Zebrafish},
      publisher = {The University of Chicago},
      school = {Ph.D.},
      address = {2019-03},
      pages = {238},
      abstract = {The transcription factor Tbx5 is necessary for development  of the forelimb and closely linked with the evolution of  this limb. Loss of just one copy of TBX5 in humans is  associated with Holt-Oram Syndrome (HOS), which manifests  as defects in both the forelimb and the heart. In  zebrafish, tbx5 was duplicated during the teleost specific  whole-genome duplication, and zebrafish therefore have two  copies of tbx5: tbx5a and tbx5b. In the pectoral fins of  wildtype embryos, the cells of the fin field migrate away  from the somites along the mediolateral (ML) axis and  converge along the anterio-posterior (AP) axis. Tbx5a  regulates the AP convergence of these cells during fin  field migration by controlling expression of fgf24 in a  subset of the fin field cues. Therefore, loss of Tbx5a  results in embryos lacking pectoral fins. Tbx5b-deficient  embryos form small pectoral fins, through unknown  mechanisms. In this dissertation, I aim to characterize the  role of tbx5b during zebrafish development, particularly  during pectoral fin development, and compare to the known  functions of tbx5a. These comparisons will provide insight  into the fate of duplicated genes in the zebrafish.

All  previous work on the role of tbx5b has been performed in  Tbx5b-deficient embryos generated via morpholino. In order  to confirm that the morpholino accurately recapitulates the  mutant phenotype, I first created and characterized a  mutant for tbx5b. The tbx5b -/- embryos phenocopy the  previously described Tbx5b-deficient embryos in both the  heart and the pectoral fin defects. Further analysis of  tbx5b -/- pectoral fins reveals that in addition to the  small size when compared to same-stage wildtype fins, tbx5b  -/- pectoral fins lack some anterior structures of the fin,  which is similar to the limb defects seen in HOS  patients.

In order to identify the different  transcriptional networks of embryos deficient in the tbx5  paralogues, whole-embryo RNA sequencing was performed in  wildtype embryos and embryos lacking the tbx5 paralogues  during the stages of pectoral fin development. Loss of the  tbx5 paralogues, in particular tbx5b, resulted in changes  to gene expression in the intermediate mesoderm, the  somites, and the yolk syncytial layer, which may be sources  of signaling cues during the migration processes of the  cells of the fin field, due to their proximity to the  migrating cells. Furthermore, loss of the tbx5 paralogues  produced morphological changes in both the vasculature and  the somites.

To understand how loss of tbx5b could result  in a small fin, I performed cell tracking analysis in the  fin field of Tbx5b-deficient embryos and compared this data  to previously collected data on the dynamics of the cells  of the fin field in both wildtype and Tbx5a- deficient  embryos. Normal fin field migration involves both an AP  convergence movement and a ML migration. In Tbx5a-deficient  embryos, the signaling molecule Fgf24 is no longer present,  resulting in a lack of AP convergence, although ML  migration is unaffected. In Tbx5b-deficient embryos, there  are mild defects in AP convergence, likely due to a  decrease in the levels of fgf24 expression. The anterior  cells are most strongly affected by loss of Tbx5b, which  may explain the anterior defects seen in Tbx5b-deficient  fins. Additionally, in Tbx5b-deficient embryos, there is no  net ML migration of the fin field. Furthermore, the  double-deficient embryos display defects in both AP  convergence and ML migration.

Overall, this dissertation  expands the knowledge on the role of the tbx5 paralogues in  zebrafish during migration of the cells of the fin field.  Loss of the tbx5 paralogues results in changes in  surrounding tissues, illustrating the complex interactions  and signaling that occur between tissues. Likewise, tissues  such as the intermediate mesoderm and yolk syn- cytial  layer have the ability to impact the development of the  fin. Additionally, both tbx5 paralogues regulate the  migration of the cells of the fin field, with tbx5a  primarily respon- sible for regulating AP convergence and  tbx5b primarily responsible for regulating the ML migration  movements. The genetic association of these movements along  orthogonal axes suggests that subfunctionalization of these  movements has occured. Furthermore, this data suggests that  in tetrapods, the ancestral Tbx5 regulates both migration  movements, such that loss of Tbx5 would result in cells  that fail to migrate, therefore explaining the lack of  forelimb in Tbx5-deficient tetrapods.},
      url = {http://knowledge.uchicago.edu/record/1716},
      doi = {https://doi.org/10.6082/uchicago.1716},
}