A forward genetics approach to identify novel core regulators of epithelial patte

Martindale, Mark

Abstract

This proposal aims to develop a forward genetic approach to identify core genetic components that regulate epithelial patterning and morphogenesis in the novel invertebrate cnidarian model system Nematostella vectensis. Epithelial morphogenesis is a critical component that underlies formation and proper development of structures ranging from the neural tube to internal organs. Invertebrate model systems are ideal tools for cost- effective forward approaches to identify genes that regulate similar biological processes to those in vertebrates. Nematostella is a unique system that has simple epithelial body plan, a genome more similar to vertebrates in gene content and organization than current invertebrate genetic systems, and is highly regenerative. During development of Nematostella, tentacle formation occurs around the mouth of the animal and is remarkably similar to the patterning and morphological changes observed in development of epithelial structures, such as the neural tube, limb buds, and organs, in vertebrate embryos. Though reverse genetic approaches are now well established allowing for investigations of candidate genes during Nematostella development, a forward non-biased approach to identify novel genes and pathways for universal biological processes is lacking. Development of forward approaches to study tentacle formation in this novel system will transform and enhance our understanding of epithelial patterning and morphogenesis. We have developed a protocol to induce mutations via treatment with the common mutagen ENU and describe a standard three generation crossing scheme to screen for recessive mutations that affect tentacle formation. We have generated a preliminary physical map, which we propose to expand in order to perform bulk segregant analysis to map mutant genomic regions linked to tentacle development phenotypes. Next-generation sequencing technology will be used to sequence the genomic region of interest in pooled mutant animals. The sequence data will be compared to the parental sequences and the sequenced Nematostella genome to identify the mutation(s) that lead to the observed tentacle phenotype. Mutations identified in this study will be used for future characterization and investigations focused on understanding the core components that control the initiation and execution of epithelial patterning and morphogenesis during animal development and regeneration. Upon completion of this study, we will have accomplished two goals that will promote our ability to investigate human health and development in invertebrate model systems. (1) We will have identified novel and core regulators of epithelial patterning and morphogenesis;and (2) We will have carried out the first genetic screen in a non-bilaterian marine invertebrate that has striking molecular similarity to vertebrates, thus providing a blueprint for future researchers to exploit this novel model system to improve our understanding of animal development as it relates to human health and regenerative medicine.

Public Health Relevance

This work is relevant to human health because it identifies core genes for future study that regulate patterning, growth, and morphological (shape) changes in sheets of cells called epithelial tissue. Morphological changes and controlled growth of epithelial tissue occurs in the development of nearly all body structures (neural tube, limbs and organs etc.) and defects in this process often lead to birth defects such as spina bifida or cleft palate. Identifying and studying genes that control epithelial tissue patterning, growth, and morphology will enhance our ability to develop detection and treatments for a wide range of birth defects.