Human-Specific NOTCH2NL Genes Affect Notch Signaling and Cortical Neurogenesis

Abstract

Genetic changes causing brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and is a determinant of neuronal number in the mammalian cortex. We find that three paralogs of human-specific NOTCH2NL are highly expressed in radial glia. Functional analysis reveals that 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 into cortical neurons. Furthermore, NOTCH2NL genes provide the breakpoints in 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism and deletions with microcephaly and schizophrenia. Thus, the emergence of human-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger human neocortex, accompanied by loss of genomic stability at the 1q21.1 locus and resulting recurrent neurodevelopmental disorders.

Keywords: 1q21.1; Notch signaling; autism; cortical organoids; human evolution; neural stem cells; neurodevelopment; neurodevelopmental disorders; segmental duplications; structural variation.

Figure 1. NOTCH2NL is located in a neurodevelopmental disease locus and exhibits variable gene and protein features

(A) Location of NOTCH2NL genes (red) and additional genes derived from human segmental duplication (light blue). TAR syn=Thrombocytopenia Absent Radius syndrome. (B) Gene and protein features of NOTCH2 and NOTCH2NL. (C) De novo assembly result of NOTCH2NL loci for H9 human ESCs and relative allele expression from week 5 cortical organoids. *Not enough nucleotide differences present to distinguish between the two NOTCH2NL^sh-2ntdel alleles. (D) Observed NOTCH2NL paratypes in 15 individuals. See also Fig. S1, S3, Table S1, S4.

Figure 2. Evolutionary analysis of NOTCH2NL-like genes reveals only human NOTCH2NL genes encode NOTCH-related proteins

(A) Coverage of genome sequencing reads mapped to the NOTCH2 locus (B) Schematic of NOTCH2NL-containing genes. (C) NOTCH2/NOTCH2NL immunoblot of human (“H”) and chimpanzee (“C”) cortical organoids. ECD: extracellular domain FL: full length. (D) Evolutionary history of NOTCH2NL genes in the great ape lineage. (E) Endocranial volume of archaic human fossils versus time (Holloway, et al., 2004). See also Figure S2.

Figure 3. Radial glia-specific expression of NOTCH2NL in human fetal brain samples

Scatterplot of 3466 fetal brain cells after single cell RNA-Seq principal components analysis and t-stochastic neighbor embedding (tSNE) as described in Nowakowski, et al., 2017. Cells are colored by annotated cell type clusters (A), NOTCH2NL expression (B) and NOTCH2 expression (C). See also Figure S3.

Figure 4. Ectopic expression of NOTCH2NL delays neuronal differentiation

(A) Overview of mouse cortical organoid differentiation protocol. Genes upregulated (B) or downregulated (C) in mouse organoids ectopically expressing NOTCH2NL^Sh,T197I compared to EV. Green asterisk; radial glia associated genes. Red asterisk; neuron differentiation genes. See also Figure S4 and Table S2.

Figure 5. Cortical organoids from hESCs lacking NOTCH2NL show premature neuronal maturation

(A,B) Schematic of NOTCH2NL alleles (A) and UCSC genome browser view of NOTCH2 and NOTCH2NL loci showing normalized genome sequencing coverage (B) in control (H9*) and NOTCH2NL mutant (H9^NOTCH2NLΔ) cell lines. Cortical organoid protocol schematic (C) with bright field images (D) and cell types generated (E). (F–I) Immunofluorescence staining of cortical organoids with markers of radial glia (PAX6, BLBP), intermediate progenitors (TBR2) and layer V cortical neurons (CTIP2). Scale bar=50 μm. (J) Spearman’s rank correlation using the top 250 upregulated and downregulated genes (H9^NOTCH2NLΔ / H9^*), and data from previously generated W3, W4 and W5 H9 organoids. (K) Heatmap showing expression for a selection of genes in the significantly enriched GO cluster ‘neuron differentiation’. See also Figure S5 and Table S2.

Figure 6. NOTCH2NL paratypes interact with NOTCH receptors and enhance Notch signaling

(A) Co-immunoprecipitation of NOTCH2 and NOTCH2NL analyzed by immunoblot. IP=Immunoprecipitation, N2NL=NOTCH2NL (B,C) Co-transfection of NOTCH2-GAL4 and NOTCH2NL in the pGL3-UAS reporter assay in co-culture with U2OS cells (B) or U2OS-JAG2 cells (C), n = 24 in 4 experiments. (D) Immunoblot showing immunoprecipitated NOTCH2NL in the medium of mouse ESCs ectopically expressing NOTCH2NL. (E) Effect of NOTCH2NL-conditioned medium in NOTCH reporter assay, n = 7 in 2 experiments. One-way anova with Tukey’s HSD (* p<10⁻⁴). (F–G) NOTCH reporter assay using the NOTCH1 receptor in conjunction with NOTCH2NL. (H–K) Assessment of common NOTCH2NL variants in reporter assay. (H–I) n = 12 in 2 experiments. (J–K) n = 6 in 1 experiment, t-test with Holm-Bonferroni correction (* p<0.05, ** p<10⁻³), error bars indicate SD. Data is analyzed using Two-way anova with Tukey’s HSD (* p<10⁻⁴, ** p<10⁻⁸, *** p<10⁻¹²). Error bars indicate SEM, unless otherwise specified. See also Figure S6.

Figure 7. Patients with 1q21.1 Distal Deletion/Duplication Syndrome show breakpoints and CNV in NOTCH2NLA and NOTCH2NLB

(A) UCSC Genome Browser screenshot from GRCh38. The duplication depth track indicates duplicated genome sequences as colored bars: white (single copy, N=1), orange (N=2–4), green (N=5), black (N>5). DNA sequence coverage tracks were generated in three ways. (1) Normalized read depth was calculated based on the entire region (red) or (2) by segmenting into 5 subregions: centromeric to NOTCH2NLA, NOTCH2NLA, between NOTCH2NLA and NOTCH2NLB, NOTCH2NLB, and telomeric to NOTCH2NLB (green) (STAR Methods). (3) Average coverage for breakpoints within NOTCH2NLA and NOTCH2NLB is shown as step function (blue). Where all models agree, the colors combine to black. (B) Schematic of the NOTCH2NL chromosomal configuration before and after a duplication or deletion event. See also Figure S7, Table S3–S4.