THE EVOLUTION OFHOX [sic] PARALOG GROUP 2 GENE EXPRESSION AND REGULATION IN THE JAPANESE MEDAKA

Abstract

Hox paralog group 2 (PG2) genes are evolutionarily conserved developmental regulatory genes that function to specify rhombomere (r) and pharyngeal arch (PA) identities in animal embryos. Several rounds of whole genome duplications in animals, including one specific to ray-finned fishes, and post-genome duplication independent gene loss have resulted in divergent Hox PG2 gene complements across evolutionarily divergent teleost fishes. Divergence in gene complements may have, in part, been responsible for generating divergent Hox PG2 gene expression patterns and specification of hindbrain and PA-derived structures during the evolution of osteichthyan embryogenesis. In this dissertation, I describe the cDNA cloning and expression analysis of Japanese medaka (Oryzias latipes) Hox PG2 genes. I show that there are only two functional canonical genes, hoxa2a and b2a, and that a previously identified hoxa2b gene is a transcribed pseudogene, [psi]hoxa2b. The canonical genes, hoxa2a and b2a, were expressed in developing rhombomeres and PAs in a manner that was generally conserved throughout the osteichthyans. By contrast, [psi]hoxa2b was expressed at detectable levels only in noncanonical Hox PG2 gene expression domains, including the ventral-most aspect of the neural tube, the pectoral fin buds and the caudal-most region of the embryonic trunk, indicative that regulatory control elements needed for spatiotemporal specification of expression have diverged from the canonical orthologs. In order to understand whether sequence divergence within cis-regulatory control elements are linked to the divergent expression patterns of the medaka hoxa2 paralogs, conserved genomic sequences upstream of the medaka hoxa2a and [psi]hoxa2b genes were tested functionally using a transgenic GFP reporter system. The medaka hoxa2a r3/5 enhancer region (r3/5ER) was shown to direct reporter gene expression in r4, PA2 and the posterior PAs, while the r3/5ER of [psi]hoxa2b directed reporter gene expression in r3-7, PA2 and the posterior PAs, which is different from transgenic mapping studies of the orthologous regions tested in chick and mouse embryos. These analyses provide evidence for significant post-genome duplication divergence in cis-regulatory element function in the r3/5ER of osteichthyans. Further, they question the ancestral nature of the r3/5ER prior to the evolutionary split of sarcopterygians (lobe-finned fishes) from the actinopterygians (ray-finned fishes).