Molecular Mechanisms Underlying Cardiac Neural Crest Development in Avian Embryos

Citation

Gandhi, Shashank (2021) Molecular Mechanisms Underlying Cardiac Neural Crest Development in Avian Embryos. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/y1e4-d090. https://resolver.caltech.edu/CaltechTHESIS:06012021-203020365

Abstract

The neural crest is a multipotent, vertebrate-specific stem cell population that gives rise to diverse cell types in the developing embryo, including craniofacial cartilage, enteric ganglia, and cardiac septa. Neural crest cells that originate from a given axial level in the embryo give rise to a characteristic array of progeny and follow distinct pathways from those arising at other levels. One of these subpopulations, called the cardiac neural crest, originates in the dorsal hindbrain and migrates into the developing heart, where it forms the aorticopulmonary septum, cardiac ganglion, and part of the interventricular septum. Mutations in or loss of these cells causes heart defects that are among the most common birth defects in the general population. For my thesis, I sought to identify the mechanisms that underlie the formation of neural crest cells, and confer cardiac neural crest cells with their unique developmental potential.

To enable interrogation of epistatic relationships between key neural crest genes during neural crest induction and crest specification, I first optimized the CRISPR-Cas9 system for genome editing in gastrula and neurula-stage chicken embryos. I then further improved the CRISPR toolbox by devising an all-in-one single-plasmid strategy that harnesses the self-cleavage properties of ribozymes for the simultaneous delivery of Cas9, gRNAs, and fluorescent reporters in transfected cells. This has enabled live tracking of wildtype and mutant neural crest cells as they migrate to their terminal locations.

Prior to their induction at the neural plate border, precursors in the neural plate border are transcriptionally primed toward multiple cell fates, including neural tube, neural crest, epidermis, and placode. While this priming has been thought to involve epigenetic regulation, chromatin remodeler genes have been overlooked in the context of neural crest formation given their concomitant expression in surrounding cell types. By combining single-cell transcriptional profiling of the early chick embryonic hindbrain with temporally-controlled knockouts, I uncovered a novel bimodal mechanism whereby the chromatin remodeler gene Hmga1 first regulates Pax7-dependent neural crest induction at the neural plate border, and later modulates Wnt signaling in the dorsal neural tube to control neural crest delamination. These results established Hmga1 as a direct regulator of neural crest induction and emigration.

Finally, given that amongst distinct neural crest subpopulations designated as cranial, cardiac/vagal, and trunk, only cardiac crest has the ability to contribute to heart development, and that neither trunk nor cranial neural crest subpopulations can rescue the loss of cardiac crest, I investigated the genetic logic that imbues cardiac crest with its unique ability to form cardiovascular derivatives. To this end, I combined surgical ablations, bulk and single-cell transcriptional profiling, RNA labeling, CRISPR-Cas9-mediated gene editing, transcription factor binding motif mutation analysis, and transgenic tissue grafting approaches to uncover and characterize a cardiac-neural-crest-specific subcircuit comprised of the transcription factors Sox8, Tgif1, and Ets1. I demonstrated that ectopic expression of this subcircuit in trunk neural crest cells reprogrammed them towards a cardiac-crest-like fate, and transplanting these reprogrammed cells in place of ablated cardiac crest restored cardiac-crest-like migration patterns and rescued outflow tract septation defects.

Taken together, my thesis work has not only built a genome engineering toolbox for a key model system in developmental biology, but has also expanded our understanding of the genetic circuits that govern the formation of the cardiac neural crest and underlie its unique ability to contribute to the heart.

Thesis Files