CHL1 Regulates Cortical Neuron Identity and Laminar Formation during Stem Cell-derived Neurogenesis

Background: Close Homolog of L1 (CHL1), a neural cell adhesion molecule, plays a critical role in cortical development, but its isoform-specific and stage-dependent functions remain poorly defined. This study examines the differential effects of CHL1 presented either apically (CHL(S)) or basally (CHL(B)) on cortical neuron differentiation and maturation at key developmental stages. Methods: Using human embryonic stem cell-derived cortical neurons, we performed quantitative gene expression analyses and morphometric assessments at Days 28, 35 and 42 to evaluate how CHL1 presentation affects neuronal differentiation. Result: At early differentiation (Day 28), CHL1 showed minimal effects on gene expression. By Day 35, CHL(S) suppressed deep-layer markers (Tbr1, Ctip2) and Tbr2, suggesting inhibition of intermediate progenitor expansion. In contrast, CHL(B) maintained or elevated Ctip2, Satb2 and Brn2, indicating support for laminar identity stabilization. By Day 42, orientation-specific effects persisted with CHL(S) suppressing Cux1 and CHL(B) promoting Ctip2. Notably, CHL1 did not affect neurite morphology at any stage examined. These findings position CHL1 as a transcriptional regulator of cortical neuron identity, with orientation- and stage-specific effects on gene expression but not morphogenesis. The results highlight its role in cortical layering and support further investigation into CHL1-mediated signaling pathways in neuro developmental disorders.