INVESTIGATION OF VOLVOX CARTERI CELL DIFFERENTIATION AND ITS EVOLUTION THROUGH FUNCTIONAL ANALYSIS OF REGA AND REGA HOMOLOGUES

Abstract

Multicellularity and cell differentiation have evolved multiple times in the history of life on this planet, but they evolved so long ago in most lineages that it has been difficult to learn the genetic mechanisms behind their evolution. The family of volvocine green algae comprise an excellent system to study this process, because it contains unicellular, colonial, and multicellular lineages with a relatively recent transition to multicellularity. V. carteri possess two different cell types, reproductive gonidia and motile somatic cells. The somatic regenerator A (regA) gene is essential for maintaining differentiation of the somatic cells, by repressing cell growth and reproduction. regA encodes a nuclear protein with a ~110-amino acid region known as the Volvocine Algal regA like (VARL) domain, which is present in several regA-like sequence (rls) genes in V. carteri and in other volvocine genomes. rlsA-D are the closest regA paralogs, but their functions are unknown. To help pursue my goal of understanding the evolution of cell differentiation in V. carteri, I developed new selectable markers for transformation and a new CRISPR/Cas9 genome editing system for V. carteri. To test the functional equivalency of VARL domains, I used chimeric regA genes encoding VARL domains derived from RegA paralogs�RlsA-D, or RegA homologs from Pandorina morum, Eudorina elegans (colonial) or Pleodorina japonica (cell differentiation). Only the chimeric protein with P. japonica RegA VARL domain rescued the cell differentiation phenotype of a regA mutant, suggesting a unique RegA VARL domain function in cell differentiation. To test the function of the close regA paralogs, I used RNAi (hairpin RNA) and genomic clones to manipulate expression of rlsA-D. rlsA-C RNAi transgene recipients did not express vegetative phenotypes. rlsD knockdown and overexpression have negative effects on growth during asexual life cycle. Interestingly, light deprivation increased levels of rlsD expression. This work reveals that the RegA VARL domain has a unique and specialized functions for somatic cell differentiation, and that rlsD might have a stress adaptation function, suggesting that cell differentiation might have evolved when a regulator of cell growth in response to stress was co-opted and modified to repress growth in somatic cells.