Human-specific NOTCH2NLB gene, similar to NOTCH2NLA and NOTCH2NLC genes, localizes to the chromosomal band 1q21.1 and originates from the partial duplication of the NOTCH2 gene, which localizes to the chromosomal band 1p12 (Fiddes et al. 2018) or from duplication of the NOTCH2NLR gene (Fiddes et al. 2019). Fiddes et al. 2018 and Suzuki et al. 2018 reported that the duplicated segment of the NOTCH2 gene in NOTCH2NLA, NOTCH2NLB, NOTCH2NLC, and NOTCH2NLR includes the promoter region of NOTCH2 and exons 1-4 of NOTCH2 that encode EGF repeats 1-6. The duplicated segment does not include the NOTCH2 gene regions that encode the transmembrane and cytoplasmic domains (Fiddes et al. 2018). NOTCH2 introns 1-4 are also included in the duplicated segment (Suzuki et al. 2018). The fifth exon of NOTCH2NLA, NOTCH2NLB, NOTCH2NLC, and NOTCH2NLR is derived from the NOTCH2 intronic sequence (intron 4 of NOTCH2) and encodes a C-terminus of ~20 amino acids unique to NOTCH2NL proteins (Fiddes et al. 2018, Suzuki et al. 2018). Several polymorphic alleles (paratypes) of NOTCH2NLA, NOTCH2NLB and NOTCH2NLC genes exist in humans that mainly differ in their start and stop codons, thus producing polymorphic protein isoforms of different lengths (Fiddes et al. 2018). Occasionally, a protein product of an identical sequence can be produced by paratypes of two different NOTCH2NL genes (Fiddes et al. 2018). While all NOTCH2NL paratypes are transcribed, paratypes containing premature stop codons may not be translated (Fiddes et al. 2018). In addition to the specific combination of NOTCH2NL paratypes (called NOTCH2NL haplotype), individuals can also differ by the total copy number of NOTCH2NL genes, due to variability in the number of the copies of NOTCH2NLC (Fiddes et al. 2018).

NOTCH2NLB is secreted (Fiddes et al. 2018).

Mouse cortical organoids engineered to express ectopic human NOTCH2NLB construct show increased expression of genes involved in negative regulation of neuronal differentiation, such as Foxg1, Id4, Fezf2, Sox3, and Six3, and decreased expression of genes that promote neuronal differentiation, such as Cntn2, Nefl, Gap43, and Sox10 (Fiddes et al. 2018). Id4 has been previously reported as a Notch target gene during mouse brain development (Li et al. 2012, Boareto et al. 2017).

NOTCH2 is highly expressed throughout human embryonic corticogenesis, reaching a peak at gestational week 9, while NOTCH2NLA and NOTCH2NLB are expressed at lower levels between gestational weeks 7 and 9 and their expression increases at later stages, in gestational week 21, including in the germinal zones of the cortical wall, such as the outer subventricular zone (oSVZ) (Suzuki et al. 2018). Overall, during human embryonic corticogenesis, levels of NOTCH2NLB mRNA are the highest of the four NOTCH2NLs (Suzuki et al. 2018). By RNA in situ hybridization, NOTCH2 mRNA is expressed mostly along the apical part of the ventricular zone of the developing human brain, while mRNAs of NOTCH2NLs are detected throughout the ventricular zone (Suzuki et al. 2018).

In a human embryonic stem cell (hESC)-based in vitro model of human cortical neurogenesis, ectopic expression of NOTCH2NLB from a lentiviral vector during early stages of corticogenesis leads to increased clonal expansion of cortical progenitors accompanied by prolonged expression of SOX2, the latter indicative of a slower exhaustion of the progenitor pool, and generation of a higher number of neurons (Suzuki et al. 2018). After seven days of NOTCH2NLB overexpression, hESC-derived cortical progenitors show increased PAX6 expression (indicative of progenitor fate) and decreased TUBB3 expression (indicative of neuronal fate), decrease in cell cycle exit and increase in cell cycle re-entry, suggesting that NOTCH2NLB stimulates self-renewal and symmetric proliferative divisions (Suzuki et al. 2018). Overexpression of NOTCH1 intracellular domain (NICD1) in hESC-derived cortical progenitors has a similar effect as NOTCH2NLB overexpression (Suzuki et al. 2018), and both overexpressed NICD1 and overexpressed NOTCH2NLB lead to upregulation of the NOTCH target gene HES1 (Suzuki et al. 2018). The EGF repeats of NOTCH2NLB are critical for its role in cortical development (Suzuki et al. 2018).

In utero electroporation of human NOTCH2NLB expression construct into mouse embryonic cortex mainly affects Pax6-positive apical radial glia (aRG) cells in the ventricular zone, with no effect on Pax6-positive progenitors in more basal compartments, which may correspond to outer radial glia (oRG) progenitors, or on Eomes (Tbr2)-positive basal progenitors (Suzuki et al. 2018). The activity of NOTCH transcriptional reporter construct in the mouse cortex in vivo is increased by ectopic overexpression of NICD1 or NOTCH2NLB (Suzuki et al. 2018).