Evolutionary mechanisms of gastrulation and left-right patterning in amniotes. - PROJECT SUMMARY Although as humans we appear symmetrical on the outside, our internal organs are asymmetrically positioned along the left and right sides of our body. Left-right (L-R) patterning is a fundamental biological process that helps to ensure the correct positioning of our organs, and its perturbation is typically associated with congenital heart malformations and high mortality. In a typical deuterostome, including humans, proper L-R patterning involves motile cilia in the L-R organizing (LRO) region, which trigger a Ca2+ wave on the left side of the embryo. This results in asymmetric gene expression and ultimately asymmetric organogenesis. However, more than 65% of all tetrapods, including reptiles and even-toed ungulates, do not use motile cilia for L-R patterning. Instead, tilting of the LRO and asymmetric cellular movements somehow lead to molecular asymmetry. However, the mecha- nisms underpinning L-R asymmetry in these organisms are poorly understood. It is unclear how asymmetric cell movements originate, whether they trigger the asymmetrical Ca2+ wave, or if cilia are involved in any aspect of L-R patterning. Currently, the chicken embryo is used to represent the diversity of all 25,000 species of reptiles, and new models are required for a deeper evolutionary understanding of fundamental developmental events. Veiled chameleons (Chamaeleo calyptratus) are perfect for the study of early development and evolution in non- avian reptiles, since they lay large clutches of eggs at pre-gastrulation stages. Their LRO lacks motile cilia, and instead molecular asymmetry is established through large-scale morphological changes. Veiled chameleons have a sequenced and annotated genome, and are amenable to cell and embryo culture, as well as live imaging. The objective of this application therefore is to define the mechanisms governing L-R patterning in chameleons and thus expand our understanding of amniote development and evolution. The central hypothesis is that cellular flow and large-scale morphological changes trigger an evolutionarily conserved asymmetric Ca2+ wave, leading to molecular L-R asymmetry, which has undergone evolutionary change across amniotes. This hypothesis will be addressed in the following aims: (Aim 1) Determine the mechanics of establishing L-R patterning in veiled chameleon. (Aim 2) Evaluate genetic changes and conservation of the L-R patterning pathway across amniotes. The patterns of cell migration and the dynamics of Ca2+ signaling will be evaluated through live imaging, providing training in advanced microscopy. CRISPR/Cas9 gene editing will be adapted for use in chameleons and will include training in surgery and virus production. This study will result in the first scRNA-seq and scATAC-seq libraries for asymmetric gene expression between the left and right sides of chicken, chameleon, and mouse embryos, and will involve computational biology training. Successful completion of this project will be significant in the fields of L-R patterning and evo-devo, providing the first detailed study of the early steps of L-R patterning in a non-avian, non-mammalian amniote, which may revolutionize our current thinking about roles for cilia and Ca2+ signaling in L-R patterning. It will also lay the foundation for a successful independent career.
