Aberrant medial ganglionic eminence (MGE) GABAergic neurogenesis contributes to Huntington's disease pathogenesis

This study used single-cell RNA sequencing to characterize early transcriptional alterations in medial ganglionic eminence (MGE) development caused by mutant huntingtin expression in a Huntington's disease mouse model. Single-cell transcriptomes were generated from fluorescence-activated cell sorted (FACS) Dlx1-eGFP–positive MGE cells isolated from embryonic day 12.5 (E12.5) BACHD and wild-type control embryos. To control for biological variability, MGE tissue from eight embryos per genotype was collected and pooled into two independent biological replicates per condition, with each replicate consisting of four paired MGEs. Following dissociation and sorting, each replicate was processed independently for single-cell library preparation using the Chromium Next GEM Single Cell 3' v3.1 platform (10x Genomics). Libraries were sequenced on an Illumina NextSeq 2000, yielding an average depth of approximately 26,000 reads per cell. Single-cell transcriptomic profiling was used to (i) resolve the cellular composition of the E12.5 MGE, (ii) define major progenitor and precursor populations along the MGE neurogenic trajectory, and (iii) assess genotype-dependent transcriptional changes using pseudobulk differential expression approaches. Particular emphasis was placed on basal GABAergic progenitors and postmitotic precursor cells. Together, this scRNA-seq dataset provides a high-resolution framework for interrogating how mutant huntingtin perturbs early progenitor states and downstream lineage programs during ventral telencephalic development, particularly the MGE. Overall design: This study was designed to define how mutant huntingtin (mHTT) alters early medial ganglionic eminence (MGE) neurogenesis at single-cell resolution, with particular emphasis on progenitor cell states, lineage trajectories, and cell-cycle–associated transcriptional regulation. Single-cell RNA sequencing (scRNA-seq) was performed on fluorescence-activated cell sorted (FACS) Dlx1-eGFP–positive cells isolated from the MGEs of embryonic day 12.5 (E12.5) mouse embryos. Two experimental conditions were analyzed: (i) BACHD embryos expressing full-length human mutant huntingtin and (ii) wild-type (WT) littermate controls. The Dlx1-eGFP reporter was used in both genotypes to enrich for MGE-derived GABAergic progenitors and early postmitotic precursors, enabling selective interrogation of ventral telencephalic lineages. To generate biological replication while minimizing litter-specific bias, eight embryos per genotype were harvested by cesarean section and pooled into two independent biological replicates per condition, each consisting of four pairs of MGEs. Thus, the experimental design comprised four scRNA-seq libraries in total (two BACHD pools and two WT pools), with each pool representing an independent biological replicate derived from multiple embryos. Following enzymatic dissociation, Dlx1-eGFP? cells were isolated by high-speed FACS and processed independently for scRNA-seq library preparation using the 10x Genomics Chromium Next GEM Single Cell 3' v3.1 platform. Libraries were sequenced to comparable depth across samples, yielding between ~8,000 and ~13,000 cells per library. Raw sequencing data were generated as multiplexed FASTQ files and processed uniformly across all samples. The primary experimental variable under investigation was genotype (BACHD vs WT). Secondary analytical variables included cell type, developmental lineage, and cell-cycle state, inferred computationally from gene-expression profiles. After quality control, ambient RNA correction, and doublet removal, datasets were integrated to mitigate batch effects while preserving biological differences between genotypes. Unsupervised clustering resolved transcriptionally distinct cell populations spanning apical radial glia, basal intermediate progenitors, newly postmitotic progenitors, interneuron precursors, projection neuron precursors, and minor non-MGE populations. Differential gene-expression analyses were performed using a pseudobulk strategy, aggregating cells by cluster, biological replicate, genotype, and—where indicated—cell-cycle phase. Cell-cycle state was inferred using Seurat's CellCycleScoring method, generating continuous S-phase and G2/M-phase transcriptional scores and assigning cells to G1-like, S, or G2/M categories. This enabled explicit testing of whether genotype-associated transcriptional changes reflected altered cell-cycle composition or phase-intrinsic dysregulation. Phase-restricted pseudobulk analyses were performed to assess genotype effects within matched cell-cycle states. Overall, this experimental design combines controlled genetic comparison, biological replication, lineage-restricted cell selection, and cell-cycle–resolved single-cell transcriptomics to interrogate early developmental mechanisms by which mutant huntingtin perturbs MGE progenitor biology