In humans and all chordates the notochord plays an indispensable role in supporting and patterning the developing body plan. During embryogenesis, defects in notochord formation are the cause of birth defects such as spina bifida and vertebral deformities, while during post-natal development notochord remnants can cause painful discopathy and, more rarely, chordomas, malignant and potentially life-threatening tumors. Despite the considerable biomedical interest in the role of the notochord in spine development and in tumorigenesis, both the comprehension of the specific functions of evolutionarily conserved transcription factors in notochord development and the knowledge of the cis-regulatory mechanisms controlling notochord gene expression are still fragmentary. These gaps in knowledge greatly limit our ability to pinpoint the genes and regulatory sequences responsible for notochord-derived defects and to potentially rectify their expression. We use the invertebrate chordate Ciona (tunicate, or sea squirt) to investigate the role of evolutionarily conserved transcription factors in notochord formation and to uncover the regulatory mechanisms that ensure their proper deployment during the development of this crucial structure. Ciona was selected for these studies because it provides a fast-developing model system with an experimentally accessible notochord and a compact, fully sequenced genome, and because several genes expressed in the Ciona notochord are evolutionarily conserved across all chordates. The objective of this project is to establish in our laboratory the CRISPR/Cas9-mediated genome editing technique for systematically knocking down notochord transcription factors of interest and for editing their regulatory regions and determine the effects on gene expression. These resources will advance our research on the evolutionarily conserved genetic toolkit that is employed by widely different chordates to properly form the notochord and guide the correct formation of the body plan. The objective of this proposal will be obtained through the following approaches: the identification of the cis- regulatory modules (CRMs) that control notochord gene expression of 10 evolutionarily conserved transcription factors (Aim 1); the elucidation of the function of these transcription factors in specific steps of notochord morphogenesis (Aim 2). Completion of these experiments will shed light on the components of the notochord GRN and their reciprocal regulatory interactions and will inform and accelerate studies of notochord-derived birth defects in other chordates.
relevance to public health. The notochord is a structure essential for the support and patterning of the body of all chordates, including humans, during their embryonic development; its malformations are responsible for a variety of birth defects, including spina bifida and vertebral deformities, and in adults remnants of the notochord form the central-most region of the intervertebral discs, but can also give rise to bone cancers called chordomas. We will use the invertebrate chordate Ciona to reconstruct the genetic toolkit of the notochord, to identify regulatory mechanisms shared with other chordates, and to study the consequences of the inactivation of regulatory regions of notochord genes within their global genomic context. Completion of this project will propel research on notochord defects and will inform the identification of elusive regulatory mutations.
