Spatial dynamics and regulation of the zebrafish limb morphogenetic field

The main argument of this thesis is that the morphogenetic field is a functional unit of embryogenesis, and a paradigm to understand the self--differentiation and self--regulatory behaviors of the developmental system. This thesis can thus be viewed as an attempt to build a conceptual framework for the limb progenitor field, highlighting the spatial dynamics of limb field progenitors during limb bud initiation, as well as how the limb progenitor field copes with perturbations that disrupt limb rudiment formation.;First, to form a pectoral limb rudiment in the zebrafish, limb field LPM cells undergo anteroposterior convergence while migrating mediolaterally away from the midline. This process separates limb field progenitors from the adjacent cardiac and peritoneum progenitors. Two notable features exist in this convergence process: First, LPM cells largely preserve their relative AP positions during the AP convergence. Second, the center of limb field cell convergence is located within the posterior half of the limb field, such that anteriorly located limb field progenitors migrate further along the AP axis and merge there with the more posteriorly located limb field progenitors. On the cellular level, the epithelial morphology and cell-cell junctions maintained by limb field LPM cells during limb field cell convergence may account for their spatiotopic migration behaviors. On the gene level, the limb field selector gene Tbx5a activates an Fgf signaling ligand Fgf24, a potential convergence cue located within the posterior half field. The asymmetric limb field cell convergence along the AP axis is possibly a result of the asymmetric localization of the Fgf24 ligand, as when Tbx5a, Fgf24 function or the Fgf signaling activity is inhibited, limb field cell fail to converge, leading to failure of limb bud initiation and potential fate transformation of limb field progenitors into peritoneum precursors.;Second, a prepattern of the AP positional identities may exist within the limb progenitor field. The spatial dynamics of limb field progenitors can be perturbed in a quantitative manner with graded doses of Tbx5a or Fgf24 morpholinos. When limb field cell convergence is partially inhibited by intermediate dose of Tbx5a or Fgf24 morpholinos, the resultant pectoral limb endoskeleton can exhibit a spectrum of truncation phenotypes, ranging from wild-type--like morphology to complete limb absence. When a truncation takes place within the limb endoskeleton, the location of truncation is preferentially located within the anterior limb endoskeleton, in correlation with the biased disruption of convergence in anteriorly located limb field progenitors. This may suggest that a prepattern of AP positional identity exists prior to or during limb field cell convergence, which is faithfully transferred via the spatiotopic convergence to the limb bud primordium and finally to the AP axis of the limb endoskeleton.;Last but not least, the limb progenitor field can self-regulate. The limb progenitor field is robust against partial perturbations in limb field cell convergence with graded doses of Tbx5a morpholinos, as the intermediate limb endoskeleton truncation phenotype is suppressed compared to the expected outcome of an unregulated phenotype---morpholino dose---response. This robustness mechanism is at least partially due to the self---regulatory behaviors of limb field progenitors, which undergo clonal expansion to compensate for the suboptimal---sized limb rudiments and to achieve the wild-type---like limb endoskeleton morphology. This process may require the Fgf24 signaling---based feedback loop to remain intact, as when Fgf24 function is inhibited, the limb field loses its robust response against partial perturbations in limb field cell convergence.;Overall, the studies presented in the thesis have delineated a series of field behaviors such as border formation, tissue polarity, prepattern and self-regulation within the limb progenitor field. These observations have led to a model that describes the limb progenitor field as a system capable of self-differentiation and self-regulation. Future work will focus on elucidating the cellular and genetic mechanisms of tissue boundary, polarity and prepattern formation, as well as investigating the threshold factors that confer the capacity of self-regulation within the limb morphogenetic field.;Supplemental files include time-lapse movies of limb progenitor convergence in wild-type, Tbx5a morphant and Fgf24 morphant embryos, as well as list of scripts and equations used for cell tracking analyses.