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

ABSTRACT: 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.

ORGANISM(S): Gallus gallus

PROVIDER: GSE267653 | GEO | 2024/05/17

REPOSITORIES: GEO

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Project description:The spinal cord is generated progressively as cells leave the caudal region of the elongating body axis such that the temporal steps of neural differentiation become spatially separated along the head to tail axis. At key stages, it is therefore possible to isolate near-adjacent cell populations from the same embryo in distinct differentiation states. Cells in the caudal lateral epiblast adjacent to the primitive streak (also known as the stem zone, SZ, in the chick) express both early neural and mesodermal genes. Other cells in the stem zone will gastrulate to form the paraxial mesoderm or remain in the epiblast cell sheet and become neural progenitors. These latter cells form a new region called the preneural tube (PNT), which is flanked by unsegmented presomitic mesoderm and represents an early neural progenitor state that can be induced by FGF signalling to revert back to a multi-potent SZ state. Rostral to this, the closed caudal neural tube (CNT) is flanked by somites and is an early site of co-expression of genes characteristic of neural progenitors, and of ventral patterning genes (Diez del Corral et al., 2003). The CNT contains the first few neurons and exposure to FGF cannot revert this tissue to a multi-potent SZ state (Diez del Corral et al., 2002). The transition from the PNT to the CNT thus involves commitment to a neural fate that this is regulated by a switch from FGF to retinoid signalling. More advanced neuroepithelium is then located in more rostral neural tube (RNT), in which neuronal differentiation is ongoing and dorsoventral pattern is refined. This experiment uses the Affymetrix GeneChip chicken genome microarray to compare the transcriptomes of microdissections of these spatially distinct cell populations from the elongating neural axis of HH stage 10 chick embryos. Dissections were carried out in L15 medium at 4°C and explants pooled in TRIzol reagent (Gibco) for RNA extraction. Notochord was removed by controlled trypsin digestion that aimed to keep the neural ventral midline. For the microarrays, at least five tissue samples for each region were pooled to make each of three biological replicates for each (n>15 for each region).

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

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