Multicellularity, genetics, and the discovery of a choanoflagellate sexual life cycle

The biology of the first animals provided the foundation upon which all animal diversity later evolved. Yet, we know surprisingly little about the first animals or how they arose. My doctoral research uses the choanoflagellate Salpingoeca rosetta, one of the closest living relatives of animals, to shed light on the cellular and molecular changes that led to the origin of multicellularity in animals. To this end, I have examined the genetic basis of choanoflagellate multicellular development and discovered the sexual life cycle of choanoflagellates./nMulticellular development is linked to sexual reproduction in many eukaryotes, including animals. Indeed, the single-celled bottleneck of the fertilized egg is thought to be critical for reducing genetic conflict and limiting the emergence of cheater cells in multicellular organisms. Choanoflagellates exhibit their own form of facultative multicellularity when they develop into rosette or chain colonies, but it was not known if this multicellularity was linked to a sexual life cycle, nor if these organisms possessed a sexual life cycle in the first place./nMy dissertation details the discovery of a sexual life cycle in S. rosetta, based on several independent lines of evidence. First, S. rosetta laboratory cultures can asexually reproduce as either haploid or diploid cells, and they can be induced to switch ploidy from haploid to diploid and back. Second, the presence of genome-wide haplotype blocks in laboratory isolates demonstrated a history of meiotic recombination in S. rosetta. Finally, during the haploid-to-diploid transition, time-lapse microscopy revealed instances of cell fusion, as would be expected during sexual reproduction. We concluded that S. rosetta has a sexual cycle. Future work will determine whether the sexual cycle is linked to the development of any of the morphological cell types, including the multicellular forms./nAlthough choanoflagellate and animal multicellular development bear morphological similarities and both evolved at similar points in evolutionary history, it is not known whether these traits evolved independently or had a single, ancient origin. Thus, I sought to identify the genes underlying choanoflagellate multicellular development to determine if they were related to genes used in animal development. Chapter three of my dissertation describes the first choanoflagellate forward genetic screen, which I used to study the genetic basis of rosette colony development. From 15,344 clonal lines screened, we isolated nine mutants with distinct defects in rosette development, including mutants with altered cell packing within rosettes, others that developed into large, amorphous multicellular structures, and one that rarely differentiated into multicellular forms./nI focused on a single mutant, Rosetteless, that was never observed to form rosette colonies but appeared to be otherwise wild type. A combination of genome re-sequencing and the first choanoflagellate mapping cross revealed that the Rosetteless phenotype was perfectly linked to a mutation that causes misregulated splicing in a predicted C-type lectin. Thus, the rosetteless lectin is the first gene to be linked to a role in rosette colony development. Additionally, the genotypes of the cross progeny allowed for the construction of a preliminary linkage map and revealed that S. rosetta inheritance fits the expectations of standard, Mendelian genetics. Further characterization of the other eight mutants promises to reveal additional genes in the rosette formation pathway./nWith the advent of choanoflagellate forward genetics, it should now be possible to fully elucidate the genetic basis of rosette formation, or other interesting aspect of choanoflagellate biology, even in the absence of transgenic tools. It is particularly intriguing that the Rosetteless lectin is related to an important class of animal genes but not one that we would have anticipated with a candidate gene approach. This demonstrates the utility of forward genetics for understanding rosette formation and hints at possible homology between the rosette development pathway and pathways in animals.

Advisor: King, Nicole