An in vivo CRISPR screen in chick embryos reveals a role for MLLT3 in specification of neural cells from the caudal epiblast

Developing tissues rely on the coordinated differentiation of stem cell populations in dynamically changing environments. The formation of the vertebrate neural tube is a well characterized example, where stem cells in the caudal epiblast differentiate to neural tissue while transitioning through a changing landscape of signals: rostro-caudally from Retinoic Acid (RA) to Wnt/FGF and dorsal-ventrally from sonic hedgehog to BMP. Despite an understanding of the signaling pathways involved in neural specification, the precise signal interpretation and gene regulatory mechanisms within an embryonic context remain poorly defined. To address this, we first developed an in vivo CRISPR screening approach in chick embryos. We then performed a multiplexed in vivo single-cell perturbation screen of transition genes from caudal epiblast to the neural tube. Our screen revealed a role for MLLT3, a component of the super elongation complex, in the specification of neural identity. MLLT3 is expressed in the epiblast and not the neural tube. Perturbation of MLLT3 disrupted caudal epiblast morphology, reduced neural tube identities, and resulted in the misregulation of genes involved in WNT and RA signaling. Neural specification was also disrupted by expressing mutant forms of Retinoic Acid Receptor A (RARα) lacking the MLLT3 binding domain or consisting only the MLLT3 binding region. Together, these findings validate an in vivo CRISPR screen in chick embryos for the first time and identify a previously unreported role for MLLT3 in caudal neural cell specification, mediated through an interaction with RARα. HH8 chicken embryos were electroporated ex ovo with CRISPR system marked by Citrine fluorescence, allowed to develop for 24 hours, and subsequently enriched for successful electroporation via disociation and FACS for Citrine expression. Finally cells were profiled by single cell RNA sequencing on the 10x platform.