Avila Justin A, Benthal Joseph T, Schafer Jenny C, Flaherty David K, Southard-Smith E Michelle
Vanderbilt Brain Institute, Vanderbilt University, Nashville, Tennessee.
Program in Human Genetics, Vanderbilt University, Nashville, Tennessee.
Cell Mol Gastroenterol Hepatol. 2025 Jul 21:101590. doi: 10.1016/j.jcmgh.2025.101590.
BACKGROUND & AIMS: Enteric nervous system (ENS) development requires migration, proliferation, and differentiation of progenitors for normal gastrointestinal (GI) motility. Sox10 deficit causes aganglionosis, modeling Hirschsprung disease (HSCR), and disrupts ratios of postnatal enteric neurons in proximal ganglionated bowel. How Sox10 deficiency alters enteric neuron ratios is unclear. Sox10's prominent expression in enteric neural crest-derived progenitors (ENCPs) and lack of this gene in mature enteric neurons led us to examine Sox10 effects in early ENS development.
Immunohistochemistry localized SOX10 in the developing ENS relative to HuC/D. ENS progenitors, developing neurons, and enteric glia were isolated from Sox10 and Sox10 littermates for single-cell RNA sequencing (scRNA-seq). scRNA-seq data was processed to identify cell type-specific markers, differentially expressed genes, cell fate trajectories, and gene regulatory network activity between genotypes. Hybridization chain reaction (HCR) coupled with immunohistochemistry validated expression changes.
SOX10 protein was detected in early ENS neurons. scRNA-seq profiles detected 3 neuronal trajectories emerging via 2 transition pathways accompanied by elevated activity of Hox gene regulatory networks (GRN). Sox10 scRNA-seq profiles exhibited a novel progenitor cluster, reduced numbers of cells in transitional states, and shifts in cell abundance between neuronal trajectories. Hoxa6 was differentially expressed in the neuronal trajectories impacted in Sox10 mutants, and HCR identified altered Hoxa6 expression in early developing neurons of Sox10 ENS.
Sox10 mutation shifts enteric neuron types by altering neuronal trajectories early in ENS development. Multiple neurogenic transcription factors are reduced in Sox10 scRNA-seq profiles. This work is the first to correlate changes in Hox expression, notably Hoxa6, with alterations in enteric neuron trajectories.
肠神经系统（ENS）的发育需要祖细胞的迁移、增殖和分化，以实现正常的胃肠（GI）蠕动。Sox10缺陷会导致神经节细胞缺失，模拟先天性巨结肠病（HSCR），并扰乱近端神经节化肠段中产后肠神经元的比例。Sox10缺乏如何改变肠神经元比例尚不清楚。Sox10在肠神经嵴衍生祖细胞（ENCPs）中显著表达，而在成熟肠神经元中缺乏该基因，这促使我们研究Sox10在ENS早期发育中的作用。
免疫组织化学法将SOX10定位到相对于HuC/D的发育中的ENS中。从Sox10和Sox10同窝小鼠中分离出ENS祖细胞、发育中的神经元和肠神经胶质细胞，进行单细胞RNA测序（scRNA-seq）。对scRNA-seq数据进行处理，以识别细胞类型特异性标记、差异表达基因、细胞命运轨迹以及不同基因型之间的基因调控网络活性。杂交链式反应（HCR）结合免疫组织化学验证表达变化。
在早期ENS神经元中检测到SOX10蛋白。scRNA-seq图谱检测到3条神经元轨迹通过2条过渡途径出现，同时伴有Hox基因调控网络（GRN）活性升高。Sox10的scRNA-seq图谱显示出一个新的祖细胞簇，过渡状态的细胞数量减少，以及神经元轨迹之间细胞丰度的变化。Hoxa6在Sox10突变体影响的神经元轨迹中差异表达，HCR确定Sox10 ENS早期发育神经元中Hoxa6表达改变。
Sox10突变通过在ENS发育早期改变神经元轨迹来改变肠神经元类型。在Sox10的scRNA-seq图谱中，多种神经源性转录因子减少。这项工作首次将Hox表达的变化，尤其是Hoxa6，与肠神经元轨迹的改变联系起来。
