From Genome to Morphology: The Dissection of a Developmental Gene Regulatory Network

• Main Author: Shashikant, Tanvi
• Format: Others
• Published: Research Showcase @ CMU 2017
• Online Access: http://repository.cmu.edu/dissertations/944; http://repository.cmu.edu/cgi/viewcontent.cgi?article=1983&context=dissertations

As development proceeds, cells acquire specialized properties and functions that are critical for the formation of a complex multicellular organism. Despite having the same genome, groups of embryonic cells perform varied developmental functions due to the precise regulation of gene expression that enables specialized genes to be expressed in the right place at the right time. The expression of these specialized genes drives morphogenesis, and enables the formation of complex tissues and body parts. A key question in developmental biology is: how do cells decode instructions from the genome to carry out morphogenetic processes? Gene regulatory networks (GRNs) are powerful tools for elucidating the genomic control of morphology. GRNs depict interactions between regulatory genes such as transcription factors and signaling molecules and effector genes that carry out morphogenetic functions. The GRN underlying the skeletogenic lineage in the sea urchin embryo has emerged as a model network to study how the genome directs the specification of a cell lineage during development. The morphological process of skeletogenesis has been studied extensively in the sea urchin embryo, and several lineage-specific regulatory genes have been identified and linkages among them have been elucidated. The initial activation of this network specifically in the skeletogenic lineage has been dissected, and most functional regulatory linkages among TFs have been elucidated. Some functional regulatory linkages between skeletogenic regulators and effectors have been mapped. I have identified a handful of novel regulatory genes and hundreds of novel effector genes in the skeletogenic lineage in a high-throughput manner, resulting in a much more comprehensive view of the regulatory and effector genes involved in skeletogenesis. We also uncovered functional interactions between two TFs and a set of over 200 effector genes, greatly expanding the number of regulatory connections between TFs and effector genes in the network. The large majority of regulatory connections in the network have been uncovered by perturbing the function of regulatory genes and assaying the effect of this perturbation on other genes. Direct regulatory connections cannot be differentiated from indirect regulatory connections using this method. Only a handful of direct interactions between skeletogenic regulators and effectors have been mapped by conventional cis-regulatory analysis. I have been able to identify over 3,000 putative cis-regulatory modules (CRMs) mediating skeletogenic gene expression using genome-wide techniques. I have inferred some regulatory connections into these CRMs and demonstrated the value of using differential chromatin accessibility to identify cell-type-specific CRMs in a high-throughput manner in early embryos. This thesis work has greatly expanded the number of skeletogenic effector genes in the network and enabled the identification of thousands of regulatory connections between upstream TFs and downstream effector genes. This effort to construct a detailed and comprehensive skeletogenic GRN will enable a detailed understanding of how instructions from the genome are decoded during the establishment of a cell lineage during development. Several discrete GRN subcircuits elucidated in the skeletogenic GRN can be dissected in greater detail and used to understand the fundamental principles of GRN architecture. This detailed GRN can be used to obtain a deeper understanding of the evolutionary mechanisms that enable the acquisition of novel morphological structures during speciation. The network includes biomineralization genes that are conserved across vertebrates, and further dissection of the regulation of these genes will aid in the discovery of a common biomineralization toolkit likely used by diverse animal lineages.