The Architecture and Evolution of a Model Developmental Gene Regulatory Network Controlling Sea Urchin Skeletogenesis

Abstract

A key challenge in developmental biology is to determine how anatomy is encoded in the genome. During development, cell types are specified by networks of differentially expressed regulatory genes and signaling pathways, structured as modular gene regulatory networks (GRNs), which in turn regulate downstream effector genes that encode for specialized proteins involved in carrying out morphogenetic processes. GRNs are powerful tools for providing insights into the genomic control of morphogenesis. GRNs provide a causal explanation of development and can be used to answer fundamentalquestions on how changes in the genome can drive the evolution of morphological novelties. Furthermore, there is significant evidence that evolutionary changes indevelopmental programs and GRNs that give rise to anatomy, at both trans and cis levels, have played an important role in morphological evolution. To elucidate the architecture and evolution of a GRN, I focused on the sea urchin skeletogenic GRN, one of the mostcomprehensive GRN of any embryo. Alx1, a Paired-class homeodomain transcription factor, has a conserved andessential role in skeletogenesis throughout the echinoderm phylum. To explore the role of protein-level, trans-regulatory changes in transcription factors in driving GRN evolution,I performed a detailed structure-function analysis of the sea urchin Alx1 protein. Using a rescue assay, I identified a specific, novel domain (which I termed the D2 domain) that is essential for skeletogenic function. The paralogue of Alx1, Alx4, was not functionally interchangeable with Alx1, but insertion of the D2 domain imparted skeletogenic functionon Alx4. Using cross-species protein experiments, I showed that Alx1 proteins from closely related sea urchin species are functionally interchangeable, while Alx1 proteinsfrom more distantly related echinoderms are not, mainly due to protein-level differences within the C-terminus but outside the highly conserved Domain 2. In addition, I determinedthat D2 domain is subject to alternative splicing in echinoderms. I also provided evidence that a gene duplication event permitted the functional specialization of Alx1 through changes in exon-intron organization.To elucidate the evolutionary history of GRNs that control morphological features at the cis-regulatory level, comprehensive information of direct transcriptional inputs into CRMs would be required. Developmental GRNs like the sea urchin skeletogenic GRN, however, have mostly been deduced based on gene expression studies. Hence, it isdifficult to discriminate between direct or indirect interactions. To identify regions of the genome directly bound by Alx1 during skeletogenesis, I carried out chromatin immunoprecipitation sequencing (ChIP-seq) using a custom rabbit polyclonal antibody raised against a peptide contained within the D2 domain of Alx1. I found that manyterminal differentiation genes receive direct transcriptional inputs from Alx1, suggesting a shallow network. Additionally, my analysis revealed that intermediate transcription factors previously shown to be downstream of Alx1 all receive direct inputs from Alx1, signifyingthat Alx1 regulates effector genes via indirect and direct mechanisms. I also validated 23 high-confidence ChIP-seq peaks using GFP reporters and identified 18 active CRMs,which represents a high success rate for CRM discovery. Furthermore, I confirmed that a conserved, palindromic Alx1 binding site was essential for expression by performing adetailed analysis of a representative CRM. To determine direct transcriptional inputs and to explore differences in utilization of CRMs between adult and larval skeletogenic cells, I performed a detailed dissection of a representative CRM belonging to Sp-KirrelL, an effector gene that encodes for a transmembrane protein required for sea urchin primarymesenchyme cell fusion. Taken together, this thesis work deepens our understanding of how transcriptionfactors can evolve through protein-level changes and how such changes can underlie important morphological innovations. In addition, my work greatly improves our current understanding of the sea urchin skeletogenic gene regulatory circuitry, which can be further utilized for comparative evolutionary studies.