טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentWagner Florian
SubjectAn Integrative Genomic Perspective on Gene Regulation and
Tissue Specification in C. elegans Development
DepartmentDepartment of Biology
Supervisor Professor Itai Yanai
Full Thesis textFull thesis text - English Version


Abstract

The development of multicellular organisms is governed at the molecular level by gene regulatory networks (GRN), which comprise the regulatory relationships of all signaling molecules and transcription factors involved. Understanding the structure and function of these GRN is a principle goal in developmental biology. The functional output of GRNs can be probed by analyzing gene expression, which provides essential information for the elucidation of network structures.  To date, however, no species has its complete GRN described, as classical methods for probing gene expression are low-throughput and biased towards candidate genes. Newer methods that allow high-throughput expression profiling lack either spatial or temporal resolution. This work demonstrates the applicability of CEL-Seq, a new RNA-Seq protocol designed to analyze gene expression at the single-cell level. RNA-Seq has allowed for unprecedented detail in gene expression analysis, yet its efficient application to single cells is challenged by the small starting amounts of RNA. CEL-Seq overcomes this problem by barcoding and pooling samples before linearly amplifying mRNA using one round of in-vitro transcription. This work presents an analysis pipeline for analyzing CEL-Seq data, together with a high-level user interface that allows the analysis to be performed efficiently and without expert computer knowledge. It further characterizes the genomic characteristics of the method, and evaluates the method’s sensitivity and accuracy based on spike-in data and dilution experiments, as a function of starting material and mRNA copy number. CEL-Seq-derived single-cell gene expression patterns from early C. elegans embryos allow the analysis of asymmetrical localization between sister blastomeres. Genes with localization to somatic lineages are enriched for genes known to be involved in embryonic patterning, suggesting that for the first time, cells-specific C. elegans GRN components were identified in an unbiased manner. In constrast, a large fraction of chitin-related genes involved in eggshell synthesis are shown to be preferentially localized to the germ cell lineage, presumably via a yet unidentified regulatory mechanism acting in oocytes and early embryos, highlighting the potential of unbiased single-cell expression studies for discovering novel expression signatures of developmental processes. CEL-Seq’s potential for trascriptome-based blastomere identification is explored using cross-validation techniques, showing that only a few replicates are required for accurately distinguishing sister blastomeres, opening up new avenues of research in developmental biology.