Multicellular organisms arise from a single fertilized zygote, which must contain all information necessary to develop. As the embryo divides, the cells adopt distinct functional characteristics, and as they do so, they become committed to these fates, unable in most cases to convert from one identity to another. Though it has been well known and described for over a century now this year, this latter process, in this work referred to as cell fate restriction, is not well understood. In this thesis, I aim to contribute to the understanding of this developmental phenomenon. The tool I use is the ectopic expression of a terminal fate specifying transcription factor, CHE-1. This transcription factor normally functions to specify the fate of a pair of gustatory neurons in the nematode Caenorhabditis elegans. If ectopically expressed early in development, it is able to induce expression of its target genes, but by adulthood, most cells are refractory to its transcriptional activation, evidence of developmental cell fate restriction in most tissues of the animal. I first describe the work of Tulsi Patel to which I contributed, in which an RNAi screen revealed that PRC2 complex is responsible for preventing CHE-1 activity in the germline cells of C. elegans. I then describe a semi-clonal genetic screen in which I found many more mutants with a similar phenotype affecting germline cells, and cloned another gene that is able to induce expression specifically in the epidermis of the animals: usp-48, a highly conserved ubiquitous nuclear deubiquitinating enzyme. Next, I describe another screen where I ectopically express CHE-1 specifically in the adult epidermis, in which I found and cloned an additional six mutants: ogt-1, dot-1.1, pmk-1, sek-1, nhr-48, and C08A9.6, here named epco-1. In this screen I also isolated but was unable to clone an additional four mutants that likely represent an additional four genes. I discuss the nature of these genes and their potential roles in restricting cell fate. Lastly, I describe the optimization of a tissue-specific transcriptional profiling protocol, INTACT, for use in the characterization of the mutants. With this optimized protocol, I was able to perform detailed RNAseq on two individual neuron types from the animal, as well as wild-type epidermis. This optimized protocol will be used to characterize the mutants in the future. Together, these results tie unexpected genes to the function of cell fate restriction in the C. elegans epidermis, which will aid in our understanding of this fundamental developmental phenomenon.

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Eukaryotes use proteins to carefully package and compact their genomes to fit into the nuclei of their individual cells. Nucleosomes are the primary level of compaction. Nucleosomes are formed when DNA wraps around an octamer of histone proteins and a nucleosome's position can limit access to genetic regulatory elements. Therefore, nucleosomes represent a basic level of gene regulation. DNA and its associated proteins, called chromatin, is usually classified as euchromatin or heterochromatin. Euchromatin is transcriptionally active with loosely packed nucleosomes while heterochromatin is condensed with tightly packed nucleosomes and is transcriptionally silent. In order to become active, heterochromatin must first be remodeled. We have studied the effects of nucleosome positioning on transgene expression in vivo using Caenorhabditis elegans as a model. We show that both location and polarity of the DNA sequence can influence transgene expression. We also discuss some considerations for working with CRISPR/Cas9. A major reason for doing in vitro nucleosome reconstitutions is to determine the effects of DNA sequence on nucleosome formation and position. It has previously been implied that nucleosome reconstitutions are stochastic and not very reproducible. We show that nucleosome reconstitutions are highly reproducible under our reaction conditions. Our results also indicate that a minimum depth of 35X sequencing coverage be maintained for maximal gains in Pearson's correlation coefficients. Communicating science with others is an important skill for any researcher. The rising generation of scientists need mentors who can teach them how to be independent thinkers who can carry out scientific experiments and communicate their finding to others. With this goal in mind, we have devised a scaffolding pedagogical method to help transform undergraduates into confident independent thinkers and researchers.

Sperm play a pivotal role in determining the reproductive success of individuals whose sperm must compete directly with that of others. I used sperm precedence assays and experimental evolution to examine the role of sperm traits in the reproductive success of hermaphrodites and males in the androdioecious nematode Caenorhabditis elegans. First, sperm size and the rate of reproduction were analyzed, in the context of male-male sperm competition, for evidence of natural heritable variation. Sperm size proved to be a strong indicator of second-male sperm precedence in the genotypes examined. Second, I tested the theoretically predicted effect of larval development time on the number of self-sperm produced by hermaphrodites. I demonstrated that a short larval development period favored the evolution of fewer sperm, inline with theoretical predictions. These results provide important insights into C. elegans reproductive biology and more generally to our understanding of the evolution of reproductive systems.

Sperm play a pivotal role in determining the reproductive success of individuals whose sperm must compete directly with that of others. I used sperm precedence assays and experimental evolution to examine the role of sperm traits in the reproductive success of hermaphrodites and males in the androdioecious nematode Caenorhabditis elegans. First, sperm size and the rate of reproduction were analyzed, in the context of male-male sperm competition, for evidence of natural heritable variation. Sperm size proved to be a strong indicator of second-male sperm precedence in the genotypes examined. Second, I tested the theoretically predicted effect of larval development time on the number of self-sperm produced by hermaphrodites. I demonstrated that a short larval development period favored the evolution of fewer sperm, inline with theoretical predictions. These results provide important insights into C. elegans reproductive biology and more generally to our understanding of the evolution of reproductive systems.

Reproduction de : Thèse de doctorat : Biologie : Lyon 1 : 2006. / Titre provenant de l'écran titre. Bibliogr. f. 191-209.

Thèse doctorat : Aspects Moléculaires et Cellulaires de la Biologie : Strasbourg 1 : 2006. / Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 27 p.

Thesis (M.Sc.)--York University, 2008. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 83-89). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38831

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 58-65).

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 44-48).