Hominin-specific NOTCH2NL genes affect Notch signaling and cortical neurogenesis

Genetic changes causing brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and a determinant of neuronal number in the mammalian cortex. We find three paralogs of human-specific NOTCH2NL are highly expressed in radial glia. Functional analysis reveals different alleles of NOTCH2NL have varying potencies to enhance Notch signaling by interacting directly with NOTCH receptors. Consistent with a role in Notch signaling, NOTCH2NL ectopic expression delays differentiation of neuronal progenitors, while deletion accelerates differentiation. Furthermore, NOTCH2NL genes provide the breakpoints in typical cases of 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism, and deletions with microcephaly and schizophrenia. Thus, the emergence of hominin-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger hominin neocortex accompanied by loss of genomic stability at the 1q21.1 locus and a resulting recurrent neurodevelopmental disorder. H9 embryonic stem cells were used for directed differentiation towards cortical organoids. Using the CRISPR/Cas9 system, H9 control and H9 NOTCH2NL knockout lines were generated. Comparative analysis of RNA-seq at week 4 of differentiation was done to assess the effect of NOTCH2NL KO on cortical neurogenesis. Mouse 46C ES cell stable lines were generated, overexpressing either EV control, or NOTCH2NL-Sh_T197I cDNA. Mouse 46C stable cell lines were differentiated into cortical organoids, and gene expression level was compared by RNA-Seq at day 6 of differentiation.