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.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/d8-knsq-7y91 |
| Date | January 2019 |
| Creators | Rahe, Dylan Parker |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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