A high capacity for regeneration is present in a wide range of species throughout the animal kingdom, but is quite reduced in ourselves and other mammals. This discrepancy in ability has driven researchers to study how regeneration proceeds in a variety of species in order to uncover the underlying mechanisms that drive a regenerative response. At a genetic level, several genes have been detected to be important for successful regeneration to occur, one of which was identified as Kazald1 in the axolotl. This gene was heavily expressed in the blastema of the regenerating limb but not in the developing limb bud, and the knockdown of it significantly slowed and reduced the regenerative response. This resulted in a great interest in this gene as it appeared to both be specific to axolotl limb regeneration, and have a powerful effect on it. However, examination of regeneration in other species did not find a similar upregulation of their Kazald1. Furthermore, different studies in the axolotl were inconsistent in assigning a name to this gene, causing it to not always be labeled as Kazald1. All of this created confusion about just how similar the axolotl gene is to Kazald1 in other species, if the axolotl gene really is Kazald1, and if this gene exists and has a regenerative role in other species. My work thus aims to answer both of these questions.
In the first part of my thesis, I discover the presence of four genes in the axolotl with high similarity to Kazald1, and thus termed as Kazald genes. I then quantify their similarity to each other in terms of sequence similarity, contained protein domains, exon-intron structure, and predicted three-dimensional protein structure. These examinations uncover that all of the axolotl Kazald genes are highly similar to each other, and also to the mammalian Kazald1 gene, across all of these metrics.
In the second part of my thesis, I accurately determine the phylogeny of the axolotl Kazald genes and the Kazald genes of a diverse array of animal species, including both vertebrates and invertebrates. This analysis reveals the existence in all jawed vertebrates of a previously completely unknown gene family – the Kazald gene family – consisting of four members, which arose from the two rounds of whole genome duplication that are ancestral to this lineage. Thus, proper orthology is able to be determined between the Kazald genes in all vertebrates, and I propose names for each of the different members: Kazald1, Kazald2, Kazald3, and Kazald4. This enables the prediction of gene function for each specific Kazald gene and the examination of how novel or ancestral any discovered functions are.
In the third and final part of my thesis, I present the wide variety of tissues and developmental processes across many diverse animal species in which different Kazald genes are expressed, the majority of which are described here for the first time. I also determine how ancestral or novel these expression patterns are. Examples of these discoveries include ancestral roles of Kazald1 in skeletogenesis and odontogenesis and Kazald2 in regeneration, as well as novel roles that arose only in certain lineages, such as Kazald3 in teleost fish skeletogenesis and Kazald4 in the avian brain.
Altogether, my work provides a comprehensive study of a previously unknown gene family, uncovers its four members and when and how they arose, and finds in which species these gene family members are still maintained and in what tissues and biological processes they are expressed. It thus offers a holistic view of the entire gene family, its evolutionary history, and the important processes that they are involved in. This should supply a wide range of researchers with knowledge of these genes that they would otherwise likely overlook due to the large difficulties incurred by a lack of annotation and prior existing information. In this way, my work in this thesis also acts as a solid foundation for future studies focused on specific species or tissues, which can better describe the exact functions that the Kazald genes perform and the mechanisms through which they work.