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Title: Latent developmental potential to form limb-like structures in fish fins revealed by mutations in the Vav2/N-WASP pathway
Abstract: Despite descending from a common antecedent structure, the fins of teleost fishes and the limbs of tetrapods have evolved drastically divergent morphologies. These anatomical changes have been central in the success of these vertebrate lineages. One of the key differences seen in the evolution of limbs is the elaboration of the endoskeleton along the proximodistal axis, with multiple long bones that articulate end on end. Teleost fins, on the other hand, have no such articulations in their diminutive endoskeleton, and exhibit an extremely limited variation fin skeletal pattern. The ability to expand and elaborate a rudimentary appendage skeleton was a foundational early step in the evolution of limbs.
In this dissertation, I investigated the genetic basis of fin development and evolution. First, using a forward genetic approach in the zebrafish Danio rerio, I identified two mutants, rephaim and wanda, which due to single missense mutations develop novel long bones and distal articulations along the proximodistal axis of the pectoral fin. Importantly, these new bones are well patterned and integrated into the skeleton, form joints and integrate with the musculature. I discovered that the development of the new bones requires Hox11 paralogs, and loss of Hox13 paralogs results in an enhancement of the mutant phenotype, with even more new bones formed distally. These results suggest that the new bones are patterned with similar mechanisms used to specify the middle aspect of tetrapod limbs. Knockout of Wasl, the paralog of the affected gene in reph mutants, in the mouse limb mesenchyme results in a phenotype similar to that seen in Hoxa-11 mutants. Altogether, these results reveal that zebrafish have the developmental potential to form articulated fin skeletons, using limb-like patterning cues. As this potential was likely present in the bony fish ancestor, zebrafish retain this potential in a latent state, and it is able to be induced by a simple mutation.
Despite being the focus of a large number of studies, Wasl was not known to have a role in patterning or skeletal development prior to my findings. By generating zebrafish null mutants via CRISPR gene editing and Cre-based conditional knockout of Wasl in the mouse, I found that this gene is required for specific patterning of dermal and endochondral bones in both the axial and appendicular skeletons. The phenotypic effects observed are similar to different Hox null mutants, and suggest that Wasl may interact with Hox globally in skeletal patterning. In my analysis I identified a role for Wasl in lamellipodia formation within Hox responsive cells, and develop a mechanistic hypothesis for the function of this gene in the migration of osteoblast precursors.
As the identified zebrafish mutants expose ancestral atavistic traits, I analyzed genetic regulation of fin development in a basally-branching teleost relative, the bowfin Amia calva, which retains proximodistal elaboration of the pectoral fin. Using RNAseq analysis of developing fin bud transcriptomes, I identify developmental genetic features in common between fins and limbs, as well as Holostean-specific changes, including loss of fgf8 expression from the apical ectodermal ridge. These data act as a touchstone to link disparate structures to identify common mechanisms and causes of morphological change.
Committee: James Hanken and Matthew P. Harris (advisors), Hopi Hoekstra, George Lauder and Cliff Tabin