This study examined the effects of soluble (
CHL(S)) and adherent (
CHL(B)) forms of
CHL1 on cortical neuron development at Days 28, 35 and 42.
Temporal regulation of CHL1 expression reveals a mid-developmental peak followed by rapid down regulation
To establish the temporal dynamics of
CHL1 during cortical development, we examined endogenous
CHL1 expression at three developmental stages (Fig 1). Expression was elevated at Day 28, peaked significantly at Day 35 (p<0.05), then declined dramatically by Day 42 (p<0.0001 versus both earlier timepoints). This temporal profile indicates that
CHL1 functions primarily during mid-stage cortical differentiation, with down regulation occurring as neurons mature.
CHL1 suppresses Tbr1 expression in both apical and basal configurations at day 28
We first examined whether
CHL1 affects deep-layer neuron specification at the early differentiation stage. At Day 28, both
CHL1(S) and
CHL1(B) significantly decreased
Tbr1 expression compared to their respective controls (Laminin and FC;
p<0.05 for both) (Fig 2A and B). This indicates that
CHL1, regardless of spatial orientation, initially suppresses this key layer VI marker during early cortical.
CHL1 does not alter the expression of early neurogenic transcription factors across surface conditions
To determine whether
CHL1’s effects extend to progenitor specification, we examined key neurogenic regulators at Day 28. Expression of
LHX2,
FOXG1,
TBR2, PAX6 and
TBR1 remained unchanged across all
CHL1 conditions (Fig 3). This indicates that
CHL1 does not interfere with core transcriptional machinery for progenitor identity, suggesting its regulatory effects are restricted to post-mitotic differentiation processes.
Orientation-sensitive regulation of Tbr1 and Ctip2 reveals selective bias toward deep-layer lineage at day 35
By Day 35, coinciding with peak
CHL1 expression,
CHL1 effects became orientation-specific (Fig 4). For
Tbr1,
CHL1(S) showed non-significant reduction versus Laminin (Panel A), while
CHL1(B) significantly suppressed expression versus FC (
p<0.05, Panel B). Conversely,
Ctip2 was significantly upregulated by
CHL1(S) versus Laminin (
p<0.05, Panel C), with no significant change in
CHL1(B) versus FC (Panel D). These divergent effects demonstrate that
CHL1 spatial presentation differentially regulates deep-layer markers during peak differentiation.
CHL1 modulates a broad set of cortical identity markers with spatial precision at day 35
Comprehensive profiling at Day 35 revealed complex orientation-dependent effects (Fig 5). Remarkably,
Tbr1 expression was significantly higher in
CHL1(B) compared to
CHL1(S) (
p<0.01, Panel A), representing a reversal from the uniform suppression observed at Day 28. This stage-specific switch suggests dynamic regulation of
CHL1 effects during development.
CHL1(B) also elevated
Satb2 versus FC (
p<0.05, Panel B), while
CHL1(S) significantly decreased
Tbr2 versus controls (
p<0.01, Panel C), indicating impaired intermediate progenitor expansion.
Ctip2 remained higher in
CHL1(B) than
CHL1(S) (Panel G), reinforcing orientation-specific effects on cortical fate determination.
CHL1 suppresses Cux1 while promoting ctip2, indicating layer-specific fate programming
Fig 6 further clarifies
CHL1’s dual role in fate specification. Panel A shows
Tbr1 expression remained stable across all substrate conditions. Panel B displays slight increases in
Tbr2, although no significance was achieved. Panel C reports no significant change in
Satb2, while Panel D (
Cux2) showed a mild but non-significant downward trend. Panel E (
Brn2) remained unchanged. Panel F, however, revealed a significant reduction in
Cux1 expression in
CHL1(S) compared to Laminin (p<0.05), suggesting a suppression of upper-layer identity. In contrast, Panel G showed that
Ctip2 was significantly elevated in
CHL1(B) relative to
CHL1(S) (p<0.05), highlighting an orientation-specific promotion of deep-layer fate. Together, these results underscore
CHL1’s ability to selectively repress superficial markers while enhancing deep-layer identities in a configuration-sensitive manner.
CHL1 does not significantly impact neurite morphology during cortical neuron development
Morphometric analysis was performed at Days 28, 35 and 42 across different substrate conditions (PDL,
CHL1(S), FC and
CHL1(B)) (Fig 7). At all timepoints, no significant differences were observed in total neurite length, dominant neurite length, number of primary branches, or percentage of cells lacking neurites between
CHL1-treated and control groups. While Day 35 showed minor trends in the
CHL1(B) group, these remained statistically insignificant. These findings demonstrate that
CHL1 does not affect neurite morphology, branching architecture, or neuritogenesis timing throughout cortical neuron development, contrasting with its pronounced effects on transcriptional programs.
This study comprehensively delineates the spatially segregated and temporally dynamic regulatory functions of
CHL1 on cortical neuronal differentiation. Using a panel of transcriptional markers and morphometric analysis across key developmental timepoints, we demonstrate that
CHL1 operates not as a broad modulator of early neurogenesis, but as a precise, isoform-specific effector of cortical fate identity and maturation. The spatial orientation of
CHL1 presentation on the surface (
CHL(S)) versus basally (
CHL(B)) emerges as a critical determinant of its regulatory role, differentially affecting the expression of laminar markers, the timing of neurogenic transitions and the balance of differentiation.
At Day 28,
CHL1 orientation had no significant effect on cortical layer-specific gene expression or neurite morphology. This apparent lack of influence is consistent with previous findings that early stages of cortical development are primarily governed by intrinsic transcriptional programs and lineage-instructive signals, rather than substrate-mediated adhesion cues (
Chen et al., 1999;
Maness and Schachner, 2007;
Katic et al., 2017).
CHL1’s functional role appears to be temporally downstream of progenitor identity specification, becoming relevant during the subsequent stages of fate consolidation and cortical layer patterning (
Yang et al., 2017).
By day 35, the effects of
CHL1 presentation became significantly more pronounced and distinct between its apical and basal configurations.
CHL(S) consistently suppressed the expression of differentiation markers, including
Tbr1 and
Ctip2. These findings align with previous literature identifying
CHL1 as a key regulator of neuronal positioning and identity, though its presentation-specific inhibitory effect in this context reveals a novel functional layer (
Huang et al., 2011).
The reduction in Tbr2 under
CHL(S) further supports the notion that this isoform impedes the neurogenic-to-neuronal transition by constraining the amplification of intermediate progenitors, thereby limiting output to both deep and upper cortical layers. In contrast,
CHL(B) preserved or even enhanced the expression of deep-layer markers, particularly Ctip2, suggesting a supportive role in neuronal fate commitment. This effect likely reflects interactions with specific adhesion molecules such as NCAM or N-cadherin, both of which have been implicated in promoting deep-layer neuron differentiation and stabilizing their identity during cortical layer formation.
CHL(B)’s ability to preserve Ctip2 expression, a key transcription factor for deep-layer cortical neurons, suggests its role in stabilizing cortical populations during layer formation. These findings align with previous studies showing that adhesion molecules play critical roles in deep-layer cortical neuron differentiation and organization (
Demyanenko et al., 2004;
Togashi et al., 2009;
Klingler et al., 2023).
CHL(B) also elevated the expression of upper-layer markers such as
Satb2 and
Brn2, a finding not observed with
CHL(S). This dual promotion of both deep- and upper-layer markers under
CHL(B) points to its role in supporting a broad range of neuronal identities during the peak period of cortical differentiation. This could reflect its ability to act as a permissive signal rather than a directive one, allowing for regionally specified transcriptional programs to unfold in response to positional cues. The suppression of
Cux1 by
CHL(S), in contrast, highlights the isoform’s selective downregulation of superficial fate programs, reinforcing its role as a spatially localized suppressor of cortical identity.
By Day 42, both
CHL(S) and
CHL(B) began to influence the transition from proliferation to differentiation. Expression levels of Cux1 and Ctip2 were reduced across both configurations, consistent with previous reports that
CHL1 contributes to neuronal maturation and density regulation by facilitating cell cycle exit (
Hillenbrand et al., 1999;
Fernandes et al., 2023). However, the divergent effects on differentiation markers persisted.
CHL(S) suppressed
Tbr1 and
Ctip2 expression, especially at intermediate stages (Day 35), reinforcing its inhibitory influence on early deep-layer fate. By contrast,
CHL(B) exerted a milder effect on
Tbr1 and preserved Ctip2, suggesting support for cortical identity maintenance. This dual function-suppressing proliferation while sustaining fate markers positions
CHL(B) as a potential stabilizer of cortical architecture during late-stage neurodevelopment.
Despite
CHL1’s pronounced effects on transcriptional programs, morphometric analyses revealed no impact on neurite morphology across all developmental stages. Total neurite length, branching patterns and dominant neurite extension remained unchanged between
CHL1-treated and control conditions at Days 28, 35 and 42. This dissociation between molecular identity and structural features indicates that
CHL1 functions primarily as a transcriptional regulator rather than a morphogenic factor. Unlike other adhesion molecules that directly guide neurite extension (
Jiang et al., 2021;
Scapin et al., 2021),
CHL1 appears to modulate cortical fate specification without affecting neuronal architecture.
This isoform-specific and temporally regulated pattern was consistent throughout our analyses. The peak of
CHL1 expression at Day 35 (1) coincided with maximal transcriptional changes, where
CHL(S) suppressed deep-layer markers (
Tbr1,
Ctip2) while
CHL(B) maintained or enhanced their expression (5). Broader transcriptional profiling (5 and 6) confirmed that
CHL(B) supported both deep-and upper-layer markers (
Ctip2, Satb2, Brn2), whereas
CHL(S) selectively suppressed Cux1. The absence of effects at Day 28 (3) distinguished
CHL1 as a downstream regulator of neuronal identity rather than an initiator of neurogenesis. Critically, these molecular changes occurred independently of morphological alterations (7), reinforcing
CHL1’s selective role in transcriptional regulation.
Our findings provide mechanistic insights into
CHL1-associated neurodevelopmental disorders. The stage-specific and orientation-dependent effects explain how mutations affecting
CHL1 cleavage or membrane anchoring could disrupt cortical lamination, causing intellectual disability observed in patients. The temporal dynamics-peak expression at mid-differentiation-identify a therapeutic window for intervention.
The persistent effects on layer-specific markers after
CHL1 downregulation support the neurodevelopmental hypothesis of schizophrenia, explaining how early disruptions create lasting vulnerability.
CHL1 polymorphisms affecting spatial presentation could serve as disease biomarkers.
Therapeutically, these findings suggest biomaterial-based interventions controlling
CHL1 presentation. Promoting
CHL1(B) could enhance differentiation in developmental delays, while
CHL1(S) might benefit premature differentiation conditions. This precision approach, tailored to patient-specific genotypes, offers new strategies for treating neurodevelopmental disorders through targeted
CHL1 isoform manipulation.