Genomic Analysis of a Transcriptional Networks Directing Progression of Cell States During MGE development

Abstract

Background: Homeodomain (HD) transcription factor (TF) NKX2-1 critical for the regional specification of the medial ganglionic eminence (MGE) as well as promoting the GABAergic and cholinergic neuron fates via the induction of TFs such as LHX6 and LHX8. NKX2-1 defines MGE regional identity in large part through transcriptional repression, while specification and maturation of GABAergic and cholinergic fates is mediated in part by transcriptional activation via TFs such as LHX6 and LHX8. Here we analyze the signaling and TF pathways, downstream of NKX2-1, required for GABAergic and cholinergic neuron fate maturation.Methods: Differential ChIP-seq analysis was used to identify regulatory elements (REs) where chromatin state was sensitive to change in the Nkx2-1cKO MGE at embryonic day (E) 13.5. TF motifs in the REs were identified using RSAT.CRISPR-mediated genome editing was used to generate enhancer knockouts. Differential gene expression in these knockouts was analyzed through RT-qPCR and in situ hybridization. Functional analysis of motifs within hs623 was analyzed via site directed mutagenesis and reporter assays in primary MGE cultures. 6 Present address: Biogen. FIGURE LEGENDSFigure 1. Characterization of aREs and rREs in E13.5 MGE. (A) Proportion of aREs and rREs active in MGE, LGE and cortex. (B) Sections of transgenic embryos (from the VISTA browser) showing in vivo activity of rREs (hs848,

A Chromatin Accessibility Atlas of the Developing Human Telencephalon

“…TFs predicted to bind human pREs that specify pallial and subpallial structures include T-box family and Nkx family TFs, respectively, which have been shown in mice to specify cortical excitatory neuron and MGE-derived interneuron subtypes by binding at distal REs. Interestingly, the OCT4/SOX2 motif was enriched in basal ganglia specific pREs ( Figure 3D), and the function of this composite motif has been demonstrated in a mouse MGE enhancer of Tcf12 (Sandberg et al 2018) . Within pREs that were differentially accessible over cortical ages or cortical lamina, we also identified motifs for TFs that have been well-studied in mice, indicating their relevance to human brain development.…”

“…In order to explore potential upstream regulators of pREs, we looked for enrichment of TF binding motifs in these region-specific pREs. Basal ganglia specific pREs were enriched for several motifs including SOX TFs, with 9.9% containing the composite motif for OCT4-SOX2, a sequence that promotes MGE enhancer function in mice (Sandberg et al 2018) ( Figure 3D). Cortex specific pREs were enriched for motifs of cortical TFs that have well known functions in the developing cortex, including NEUROD1, OLIG2, NF1, and the T-BOX family members (e.g.…”

“…Postnatal day 2 mice were perfused with 4% paraformaldehyde, whole brain was dissected, and postfixed overnight in 4% paraformaldehyde, and transferred to 30% sucrose overnight. 20 micron thick cryosections were obtained and in situ hybridization using GFP RNA probe was performed as described (Sandberg et al 2018) . In situs were developed at 37C and were imaged two days later.…”

Hyperactive MEK1 signaling in cortical GABAergic neurons causes embryonic parvalbumin-neuron death and defects in behavioral inhibition

“…Recent scRNAseq analyses suggest the mature transcriptional signature of cardinal CIN subtypes is not fully specified until CINs migrate into the cortex (Mayer et al, 2018; Paul et al, 2017; Sandberg et al, 2018). MEF2C, a known substrate of ERK1/2 and p38 signaling, was identified as a transcription factor expressed early in the presumptive PV lineage, the deletion of which also leads to the selective reduction of PV-CINs (Mayer et al, 2018).…”

Brain regionalization by Polycomb‐group proteins and chromatin accessibility

“…[ 67 ] In particular, around 10% of the pREs with SOX2 motifs were the OCT4‐SOX2 composite motif, and OCT4‐SOX2 contributes to MGE enhancer function in mice. [ 92 ] This suggests that these pREs play a role in regulating region‐specific enhancers in the developing human brain.…”