Diversification of Dentate Gyrus Granule Cell Subtypes is Regulated by Nrg1 Nuclear Back-Signaling

Neuronal heterogeneity is a defining feature of the developing mammalian brain, but the mechanisms regulating the diversification of closely related cell types remain elusive. In this study, we investigated the heterogeneity of dentate gyrus (DG) granule cells (GCs) and the influence of a psychosis associated V321L mutation in Neuregulin1 (Nrg1) on GC subtype composition. Using morpho-electric characterization, single-nucleus gene expression, and chromatin accessibility profiling, we identified distinct morphological and molecular features of typical GCs and a rare subtype known as semilunar granule cells (SGCs). The V321L mutation disrupts Nrg1 nuclear back-signaling, resulting in an overabundance of SGC-like cells. We discovered pseudotime gene expression trajectories suggesting the potential for GC-to-SGC transitions, supported by the accessibility of SGC-specific genes in other GCs. Intriguingly, we found an increase in SGC-marker expression over the adolescence to adulthood transition window in wild-type mice, coinciding with a decline in Nrg1 nuclear back-signaling capacity. This suggests that intact Nrg1 signaling suppresses SGC-like fate acquisition, and that its natural downregulation may underlie the emergence of SGC-like cells during postnatal development. Similarly, a pathological block of nuclear back signaling by the V321L mutation in Nrg1, may result in acquisition of the SGC-like fate due to loss of the repressive mechanisms maintained by intact nuclear back signaling. Our findings reveal a novel role for Nrg1 in maintaining DG cell-type composition and suggest that disrupted subtype regulation may contribute to disease-associated changes in DG GC morphology and function. Understanding these mechanisms provides new insights into mechanisms of cell-type diversity and its potential role in psychiatric pathology. To investigate the heterogeniety of DG GC subtypes and influence of the V321L mutation in Nrg1, we prepared single nucleus suspensions from 4 WT and 4 mutant mouse (2M/2F) microdissected DGs. Nuclei were processed according to the 10x Genomics multiome kit to obtain single nucleus RNA and ATAC Seq libraries which were sequenced.神经元异质性（Neuronal heterogeneity）是发育中哺乳动物大脑的标志性特征，但调控密切相关细胞类型多样化的机制仍尚不明确。本研究针对齿状回（dentate gyrus, DG）颗粒细胞（granule cells, GCs）的异质性，以及神经调节蛋白1（Neuregulin1, Nrg1）中与精神病相关的V321L突变对GC亚型组成的影响展开了探究。我们通过形态电生理表征（morpho-electric characterization）、单细胞核基因表达与染色质可及性谱分析，明确了典型GC与稀有亚型——半月形颗粒细胞（semilunar granule cells, SGCs）的独特形态与分子特征。V321L突变会破坏Nrg1的核反向信号转导，导致类SGC细胞数量过剩。我们发现拟时基因表达轨迹提示GC向SGC转化的可能性，这一结论得到了其他GC中SGC特异性基因可及性数据的支持。有趣的是，我们观察到野生型小鼠在青春期至成年期的过渡窗口中，SGC标志物的表达水平有所上升，这与Nrg1核反向信号转导能力的下降相吻合。这表明完整的Nrg1信号转导会抑制类SGC命运的获得，而其天然的下调可能是产后发育过程中类SGC细胞出现的基础。同样，Nrg1的V321L突变对核反向信号转导的病理性阻断，可能会因完整核反向信号转导所维持的抑制机制丧失，导致细胞获得类SGC命运。本研究结果揭示了Nrg1在维持DG细胞类型组成方面的全新作用，并提示亚型调控异常可能参与了DG GC形态与功能的疾病相关改变。阐明这些机制为细胞类型多样性的产生机制及其在精神病理中的潜在作用提供了新见解。为探究DG GC亚型的异质性以及Nrg1的V321L突变的影响，我们从4只野生型与4只突变型小鼠（2只雄性、2只雌性）的显微切割齿状回中制备了单细胞核悬液。按照10x Genomics多组学试剂盒流程处理细胞核，以构建单细胞核RNA与ATAC测序文库并进行测序。