Abstract：
The histone methyltransferase ASH1L plays a crucial role in regulating gene expression across various organ systems during development, yet its role in brain development remains largely unexplored. Over 130 individuals with autism harbour heterozygous loss-of-function ASH1L variants, and population studies confirm it as a high-risk autism gene. Previous studies on Ash1 l deficient mice have reported autistic-like behaviours and provided insights into the underlying neuropathophysiology. In this study, we used mice with a cre-inducible deletion of Ash1 l exon 4, which results in a frame shift and premature stop codon (p.V1693Afs*2). Our investigation evaluated the impact of Ash1 l loss-of-function on survival and craniofacial skeletal development. Using a tamoxifen-inducible cre strain, we targeted Ash1 l knockout early in cortical development (Emx1-Cre-ERT2; e10.5). Immunohistochemistry was utilized to assess cortical lamination, while EdU incorporation aided in birthdating cortical neurons. Additionally, single-cell RNA sequencing was employed to compare cortical cell populations and identify genes with differential expression. At e18.5, the proportion of homozygous Ash1 l germline knockout embryos appeared normal; however, no live Ash1 l null pups were present at birth (e18.5: n = 77, P = 0.90; p0: n = 41, P = 0.00095). Notably, Ash1l-/- exhibited shortened nasal bones (n = 31, P = 0.017). In the cortical-specific knockout model, SATB2 neurons showed increased numbers (n = 6/genotype, P = 0.0001) and were distributed through the cortical plate. Birthdating revealed generation of ectopically placed deep layer neurons that express SATB2 (e13.5 injection: n = 4/genotype, P = 0.0126). Single cell RNA sequencing revealed significant differences in gene expression between control and mutant upper layer neurons, leading to distinct clustering. Pseudotime analysis indicated that the mutant cluster followed an altered cell differentiation trajectory. This study underscores the essential role of Ash1 l in postnatal survival and normal craniofacial development. In the cortex, ASH1L exerts broad effects on gene expression and is indispensable for determining the fate of upper layer cortical neurons. These findings provide valuable insights into the potential mechanisms of ASH1L neuropathology, shedding light on its significance in neurodevelopmental disorders like autism.
摘要：
组蛋白甲基转移酶ASH1L在发育过程中调节各种器官系统的基因表达中起着至关重要的作用。然而，它在大脑发育中的作用在很大程度上仍未被探索。超过130名自闭症患者携带杂合功能丧失ASH1L变体，人口研究证实它是一种高风险的自闭症基因。先前对Ash1l缺陷小鼠的研究报告了自闭症样行为，并提供了对潜在神经病理生理学的见解。在这项研究中,我们使用了cre诱导的Ash1l外显子4缺失的小鼠，这导致了移码和过早的终止密码子（p。V1693Afs*2）。我们的调查评估了Ash1l功能丧失对生存和颅面骨骼发育的影响。使用他莫昔芬诱导的cre菌株，我们在皮质发育早期针对Ash1l基因敲除(Emx1-Cre-ERT2;e10.5)。免疫组织化学用于评估皮质分层，而EdU掺入有助于出生皮质神经元。此外,单细胞RNA测序用于比较皮质细胞群体并鉴定具有差异表达的基因.在e18.5时，纯合的Ash1l种系敲除胚胎的比例似乎正常；然而，出生时没有活的Ash1l空幼崽（e18.5：n=77，P=0.90；p0：n=41，P=0.00095）。值得注意的是,Ash1l-/-显示鼻骨缩短（n=31，P=0.017）。在皮质特异性敲除模型中,SATB2神经元显示数量增加（n=6/基因型，P=0.0001），并分布在皮质板上。出生日期揭示了表达SATB2的异位放置的深层神经元的产生（e13.5注射：n=4/基因型，P=0.0126)。单细胞RNA测序显示对照和突变上层神经元之间的基因表达存在显着差异，导致不同的聚类。伪时间分析表明，突变簇遵循改变的细胞分化轨迹。这项研究强调了Ash1l在出生后生存和正常颅面发育中的重要作用。在大脑皮层,ASH1L对基因表达具有广泛的影响，对于确定上层皮质神经元的命运是必不可少的。这些发现为ASH1L神经病理学的潜在机制提供了有价值的见解，阐明其在自闭症等神经发育障碍中的重要性。
