Ash1l loss-of-function results in structural birth defects and altered cortical development [scRNA-seq]

The histone methyltransferase ASH1L is a critical developmental regulator of gene expression for many organ systems, but its role in brain development has not been deeply investigated. Over 130 autistic individuals have heterozygous loss-of-function ASH1L variants, and population studies confirm it is a high-risk autism gene. Several studies report autism-like behaviours in Ash1l deficient mice and characterised aspects of the underlying neuropathophysiology. 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). We assessed the impact of Ash1l loss-of-function on survival and craniofacial skeletal development. We used a tamoxifen-inducible cre strain to knockout Ash1l early in cortical development (Emx1cre-ERT2; e10.5). We used immunohistochemistry to assess cortical lamination, and IdU and EdU incorporation to birthdate cortical neurons. We utilised single cell RNA sequencing to compare cortical cell populations and identify differentially expressed genes. The proportion of homozygous Ash1l germline knockout embryos was normal at e18.5, however no live Ash1l null pups were present at birth (e18.5: n = 77, P = 0.90; p0: Ash1l+/+ n = 41, P = 0.00095). Ash1l-/- had shortened nasal bones at e18.5 (n = 31, P = 0.017). In the cortical-specific knockout, SATB2 neurons were increased and distributed through the cortical plate (n = 6/genotype, P = 0.0001). There were no differences in neuronal birthdating (n = 4-6/genotype, P = 0.40 for e14.5 and P = 0.057 for e15.5). Single cell RNA sequencing revealed numerous differences in gene expression that were sufficient to cluster control and mutant upper layer neurons separately. Pseudotime analysis revealed that the mutant cluster is on an altered cell differentiation trajectory. This study shows that Ash1l is a necessary gene for postnatal survival and normal craniofacial development. In the cortex, it has broad effects on gene expression and is necessary for normal upper layer neuronal cell fate. These findings suggest a basis for altered brain function in individuals with ASH1L variants. Male Emx1CreERT2;Ash1lfl/fl mouse was crossed with female Ash1lfl/fl mouse for timed pregnancy. At e10.5, the female mouse was injected with 100mg/kg tamoxifen. At e18.5, embryos were extracted and cortical tissue was dissected for scRNA-seq. The litter consisted of four mutant Emx1CreERT2; Ash1lfl/fl and three control Ash1lfl/fl, all used in subsequent analysis.