Wnt signaling and divergent expression of duplicate genes in the leech Helobdella austinensis

Evolutionary developmental biology (Evo-Devo) studies the relationships between changes in the genome, development and body plan throughout evolutionary history. In Evo-Devo, the development of different species is compared to determine similarities and differences, which are interpreted in light of phylogenetic relationships. This helps us reconstruct ancestral states and understand the level of conservation and plasticity of developmental signaling pathways./nMolecular phylogenies have revealed three bilaterian superphyla: Lophotrochozoa , Ecdysozoa, and Deuterostomia. All three superphyla contain both segmented and non-segmented taxa, and the question of when and/or how often this trait evolved remains open. To understand this further we must compare species from all three superphyla, however, most developmental models are drawn from Ecdysozoa and Deuterostomia. Helobdella austinensis is a useful model for studying segmentation in Lophotrochozoa. They have large, identifiable blastomeres and develop via largely stereotyped cell lineages. All the segmental mesodermal and ectodermal tissues of the leech arise from a posterior growth zone (PGZ) made of five bilateral pairs of lineage restricted segmentation stem cells (teloblasts) and their mitotic progeny. Cell lineage analyses reveal that leeches undergo a distinct, lineage-driven process of segmentation, as opposed to a boundary-driven process operating in vertebrate and arthropod models./nThe goal of this work is to contribute to the understanding of the molecular process regulating the PGZ of Helobdella austinensis, focusing on the Wnt signaling pathway. With this knowledge, we can make comparisons with established model systems and draw inferences about the ancestry of this trait. This signaling pathway is conserved throughout bilaterian animals as a regulator of anterior-posterior patterning, segmentation, and stem cell regulation./nIt has previously been found that 10/13 of the Wnt ligands encoded in the Helobdella genome are expressed in the PGZ. I found activating the Wnt pathway using the small molecule lithium chloride (LiCl), causes the primary neurogenic stem cells (the N teloblasts) of the PGZ to divide symmetrically in a subset of embryos, whereas normally this cell only undergoes highly asymmetric divisions. In some cases, both resulting teloblast-like cells carry out stem cell-like divisions. I also tested for changes in the expression patterns of genes associated with this lineage, and obtained evidence suggesting that β-catenin, a key intermediate in the transduction of Wnt signaling, was upregulated in these embryos. This suggests the Wnt pathway plays a role in the formation of the stereotyped set of leech lineage-restricted stem cells./nGene duplication is a major contributing process to the diversification of genomes. I carried out two investigations aimed at exploring the consequences of this process in Helobdella. One dealt with duplication of an ancestral wnt16 gene, that appears to have occurred at some point in the lineage leading to the leech from a polychaete ancestor. Previous work showed that wnt16a is expressed in ventral ectoderm (N teloblast lineage) between stages 8-10. I show that wnt16b exists in at least two isoforms, and that these are expressed in lateral ectoderm (the O and P lineages). Moreover, the expression of at least one of these isoforms switches from ventrolateral ectoderm (O lineage) to dorsolateral ectoderm (P lineage) during embryogenesis. These results provide evidence that the wnt16 genes have undergone neofunctionalization or subfunctionalization since their duplication. These two genes may contribute to the segmental pattern in the leech by specifying fate and/or division patterns of primary blast cells./nI also investigated the consequences of gene duplication within a more rapidly evolving gene family, namely, the innexins, which encode invertebrate gap junctions. In particular, I compared expression patterns in Helobdella to those in the medicinal leech Hirudo verbana, to determine levels of conservation and divergence in the expression of innexin genes that have undergone duplication in the lineages leading to these modern leeches since their last common ancestor./nIn summary, the work presented here suggests that Wnt signaling plays a role in cell division and/or fate specification in multiple stages of embryogenesis in the leech, and thus supports the conclusion that the function of the Wnt signaling pathway is conserved across distantly related taxa. It also provides examples of divergent gene expression between duplicated genes in two different gene families.

Advisor: Weisblat, David A