Ash1l loss-of-function results in structural birth defects and altered cortical development

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 Ash1l 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 Ash1l exon 4, which results in a frame shift and premature stop codon (p.V1693Afs*2). Our investigation evaluated the impact of Ash1l loss-of-function on survival and craniofacial skeletal development. Using a tamoxifen-inducible cre strain, we targeted Ash1l knockout early in cortical development [Emx1-Cre-ERT2; embryonic Day (e) 10.5]. 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 Ash1l germline knockout embryos appeared normal; however, no live Ash1l 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 Ash1l 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.