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

M
Majid Alhomrani1,2
A
Abdulhakeem S. Alamri1,2
A
Ahmed Gaber2,3
M
Mohamed I. Saad4,5
A
Ashraf Albrakati6,*
W
Walaa F. Alsanie1,2,*
1Department of Clinical Laboratories Sciences, The Faculty of Applied Medical Sciences, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia.
2Research Centre for Health Sciences, Taif University, Taif, Saudi Arabia, P.O. Box 11099, Taif 21944, Saudi Arabia.
3Department of Biology, College of Science, Taif University, Taif, Saudi Arabia, P.O. Box 11099, Taif 21944, Saudi Arabia.
4Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Vic., Australia.
5Department of Molecular and Translational Sciences, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Vic., Australia.
6Department of Human Anatomy, College of Medicine, Taif University, Taif, Saudi Arabia, P.O. Box 11099, Taif 21944, Saudi Arabia.

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.

Cortical development disorders affect approximately 1-3% of the population and represent a major cause of intellectual disability and epilepsy (Guerrini and Dobyns, 2014). The human cerebral cortex, containing ~16 billion neurons organized into six distinct layers, requires precise molecular orchestration during development to establish proper connectivity and function (Libé-Philippot and Vanderhaeghen, 2021). This complex process involves coordinated neural progenitor proliferation, neuronal differentiation, radial migration and layer-specific fate specification (Cossart and Garel, 2022). Disruptions at any stage can result in severe neurodevelopmental disorders including autism spectrum disorder, schizophrenia and cortical malformations (Subramanian et al., 2020; Rajput et al., 2021; Wu et al., 2022; Salem et al., 2023). Understanding the molecular mechanisms governing cortical lamination is therefore critical for developing therapeutic interventions.
       
Neural cell adhesion molecules (CAMs) play fundamental roles in cortical development by mediating cell-cell interactions, guiding migration and modulating differentiation signals. Among these, Close Homolog of L1 (CHL1), a member of the L1 family of CAMs, has emerged as a critical regulator of neural development (Holm et al., 1996). CHL1 influences multiple developmental processes including axon guidance (Guseva et al., 2018), synapse formation (Andreyeva et al., 2010) and neuronal migration (Katic et al., 2014). Importantly, CHL1 exists in two functionally distinct forms: a soluble form [CHL(S)] that acts as a diffusible signal and an adherent form [CHL(B)] that mediates direct cell-cell adhesion (Hillenbrand et al., 1999). Previous studies suggest these isoforms exert differential effects on neuronal development (Zhou et al., 2012), but their specific roles in human cortical layer formation remain unexplored.
       
Despite extensive research on CHL1 in rodent models, critical knowledge gaps limit our understanding of its role in human cortical development. First, while both CHL1 isoforms have been characterized biochemically, their differential functions in regulating cortical neuron fate specification have not been investigated. Second, the temporal dynamics of CHL1 expression and function during human corticogenesis remain undefined. Third and most critically, how the spatial presentation of CHL1 (apical versus basal) influences cortical layer identity has never been examined, despite growing evidence that the subcellular localization of adhesion molecules determines their signaling outcomes and biological effects. These gaps are particularly significant given that human cortical development differs substantially from rodent models in timing, complexity and molecular signatures.
       
The clinical relevance of understanding CHL1 function is underscored by its association with multiple neuro developmental disorders. CHL1 mutations have been linked to intellectual disability (IQ range 50-70) and increased schizophrenia susceptibility (Angeloni et al., 1999). The CHL1 gene, located on chromosome 3p26.1, falls within a region frequently deleted in 3p deletion syndrome, which presents with severe cortical malformations and intellectual disability. Recent genome-wide association studies have identified CHL1 polymorphisms as risk factors for autism spectrum disorders, further highlighting its importance in human neurodevelopment (Paramasivam, 2021). However, the cellular and molecular mechanisms linking CHL1 dysfunction to these clinical phenotypes remain poorly understood, hampering development of targeted therapeutic approaches.
       
To address these knowledge gaps, we employed human embryonic stem cell (hESC)-derived cortical neurons, which faithfully recapitulate key aspects of human corticogenesis including temporal gene expression patterns, layer-specific marker expression and extended developmental timelines (Espuny et al., 2013). This model system offers unique advantages: (1) species-specific relevance for understanding human development, (2) precise control over CHL1 spatial presentation and (3) ability to track the same cell populations longitudinally across developmental stages. By combining this model with comprehensive transcriptional profiling and morphometric analyses, we can dissect how CHL1 isoforms differentially regulate cortical development.
       
Therefore, this study aimed to investigate how CHL1 spatial presentation regulates human cortical neuron development. Specifically, we: (1) characterized temporal CHL1 expression during cortical differentiation (Days 28, 35, 42); (2) examined how apical (CHL(S)) versus basal (CHL(B)) presentation affects deep-layer (Tbr1, Ctip2) and upper-layer (Cux1, Satb2) markers; (3) determined whether CHL1 acts on progenitors or post-mitotic neurons and (4) assessed the relationship between transcriptional and morphological effects. This approach provides mechanistic insights into CHL1-mediated cortical patterning and its role in neurodevelopmental disorders.
HESC cell lines and culture conditions
 
Human embryonic stem cells (H9 line) were used to generate cortical neurons through in vitro differentiation. Cells were cultured in mTeSR1 medium at 37oC with 5% CO2. Differentiation was initiated once cultures reached approximately 95% confluence.
 
Differentiation of hESCs into cortical progenitors and neurons
 
Differentiation was initiated when cells reached approximately 95% confluence by replacing mTeSR1 with Cortical Mixture Media (CMM), comprising a 1:1 ratio of DMEM/F-12 and Neurobasal Medium, supplemented with L-Glutamine, Penicillin-Streptomycin, Non-Essential Amino Acids (NEAA), Insulin-Transferrin-Selenium (ITS-A), N-2 Supplement, B-27 Supplement and β-Mercaptoethanol. Unless otherwise specified, reagents were obtained from GIBCO. CMM was supplemented with SB431542 (1:1000, Cell Signaling, Cat. No. 14775) and LDN193189 (1:1000, Sigma, Cat. No. SML0559) during the neural induction phase (Days 1-11).
       
On Day 11, cells were passaged at a 1:2 ratio onto LN-521-coated wells using ReLeSR. After centrifugation (260 × g, 3 minutes), cells were resuspended in CMM containing 10 µM Y27632 and plated in 800-2000 µL volumes per well. From Days 12-17, CMM was supplemented with FGF2 (1:5000) to promote progenitor proliferation, with medium changes every other day. A second passage was performed on Day 17, after which cells entered the maturation phase (Days 18-34) and were maintained in CMM with medium refreshed every other day.
 
CHL1 presentation and treatment regimen
 
To assess the effects of soluble and adherent CHL1 on cortical differentiation, two treatment conditions were employed. For soluble CHL1, recombinant human CHL1 protein (R and D Systems, Cat. No. 2126-CH) was added to the culture medium (10 µg/mL) during the seeding step and maintained throughout the culture period with each medium change. For membrane-bound CHL1, LN-521-coated wells were first incubated overnight at 4oC with anti-His-tag antibody (Fc control), followed by PBS washes, blocking with 1% BSA (1 hour at 37oC) and incubation with recombinant CHL1 protein (overnight, 4oC). After washing, cells were seeded and cultured under standard differentiation conditions. Assessments were conducted at Day 28, Day 35 and Day 42. All treatments were performed with n= 3 independent biological replicates per condition.
 
Quantitative real-time PCR
 
Total RNA was extracted from differentiated cortical neurons using Trizol reagent (Sigma-Aldrich) following the manufacturer’s instructions. RNA quality and quantity were assessed using spectrophotometry and 1 µg of RNA was reverse transcribed into cDNA using SuperScript IV First-Strand Synthesis System (Invitrogen, Cat. No. 18091050). qRT-PCR was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Cat. No. A25742) on a QuantStudio 3 Real-time PCR System (Thermo Fisher Scientific). Primers targeting cortical development markers were used, including Tbr1, Ctip2, Satb2, CHL1, Tbr2, Brn2, Cux1 and Cux2. GAPDH was used as a housekeeping gene for normalization (Table 1). Reactions were run in triplicate and gene expression was analyzed using the 2-ΔΔCt method (Alsanie et al., 2017).

Table 1: List of primer sequences of the genes analyzed by qRT-PCR.


 
Ethical approval
 
This study, along with all experimental procedures, was conducted according to the principles outlined by the ethics committee of the University of Taif (Approval No. TUZ-18-T-277).
 
Statistical analysis
 
All data are presented as mean ± standard error of the mean (SEM) from three independent biological replicates (n=3). Statistical analysis was conducted using GraphPad Prism (v9, GraphPad Software, San Diego, CA, USA). Data normality was assessed using the Shapiro-Wilk test. For comparisons between two groups, unpaired two-tailed Student’s t-test was used. For multiple group comparisons, one-way ANOVA followed by Tukey’s post hoc test was performed. All qRT-PCR reactions were run in technical triplicates. Statistical significance was set at p<0.05. For multiple comparisons within the same dataset, p-values were adjusted using the Benjamini-Hochberg method to control false discovery rate. Effect sizes were calculated using Cohen’s d for t-tests for ANOVA.
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.

Fig 1: Temporal expression of CHL1 during cortical differentiation.


 
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.

Fig 2: CHL1 suppresses Tbr1 expression in both orientations.



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.

Fig 3: CHL1 does not affect early neurogenic markers.


 
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.

Fig 4: Orientation-specific regulation of Tbr1 and Ctip2.


 
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.

Fig 5: CHL1 modulates cortical markers in orientation-dependent manner.


 
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.

Fig 6: Selective modulation of cortical markers by CHL1.


 
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.

Fig 7: CHL1 does not affect neurite morphology.


       
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.
In conclusion, CHL1 acts as an orientation-dependent molecular switch that precisely regulates cortical neuron fate without affecting morphology. The opposing effects of CHL(S) and CHL(B) suppressing versus promoting differentiation markers-combined with stage-specific regulation, reveal CHL1 as a critical determinant of cortical layer identity. These findings establish a new paradigm for understanding how spatial presentation of adhesion molecules controls neurodevelopment and offer therapeutic targets for CHL1-related.
 
Author contributions
 
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Ashraf Albrakati, Majid Alhomrani and Walaa F. Alsanie. The first draft of the manuscript was written by Majid Alhomrani and all authors commented on previous versions of the manuscript. All authors read and approved of the final manuscript.
 
Data availability statement
 
All the data supportive stated results exist in the manuscript.
 
Funding
 
This work was funded by the Deanship of Graduate Studies and Scientific Research, Taif University (project number 1-441-18).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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

M
Majid Alhomrani1,2
A
Abdulhakeem S. Alamri1,2
A
Ahmed Gaber2,3
M
Mohamed I. Saad4,5
A
Ashraf Albrakati6,*
W
Walaa F. Alsanie1,2,*
1Department of Clinical Laboratories Sciences, The Faculty of Applied Medical Sciences, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia.
2Research Centre for Health Sciences, Taif University, Taif, Saudi Arabia, P.O. Box 11099, Taif 21944, Saudi Arabia.
3Department of Biology, College of Science, Taif University, Taif, Saudi Arabia, P.O. Box 11099, Taif 21944, Saudi Arabia.
4Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Vic., Australia.
5Department of Molecular and Translational Sciences, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Vic., Australia.
6Department of Human Anatomy, College of Medicine, Taif University, Taif, Saudi Arabia, P.O. Box 11099, Taif 21944, Saudi Arabia.

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.

Cortical development disorders affect approximately 1-3% of the population and represent a major cause of intellectual disability and epilepsy (Guerrini and Dobyns, 2014). The human cerebral cortex, containing ~16 billion neurons organized into six distinct layers, requires precise molecular orchestration during development to establish proper connectivity and function (Libé-Philippot and Vanderhaeghen, 2021). This complex process involves coordinated neural progenitor proliferation, neuronal differentiation, radial migration and layer-specific fate specification (Cossart and Garel, 2022). Disruptions at any stage can result in severe neurodevelopmental disorders including autism spectrum disorder, schizophrenia and cortical malformations (Subramanian et al., 2020; Rajput et al., 2021; Wu et al., 2022; Salem et al., 2023). Understanding the molecular mechanisms governing cortical lamination is therefore critical for developing therapeutic interventions.
       
Neural cell adhesion molecules (CAMs) play fundamental roles in cortical development by mediating cell-cell interactions, guiding migration and modulating differentiation signals. Among these, Close Homolog of L1 (CHL1), a member of the L1 family of CAMs, has emerged as a critical regulator of neural development (Holm et al., 1996). CHL1 influences multiple developmental processes including axon guidance (Guseva et al., 2018), synapse formation (Andreyeva et al., 2010) and neuronal migration (Katic et al., 2014). Importantly, CHL1 exists in two functionally distinct forms: a soluble form [CHL(S)] that acts as a diffusible signal and an adherent form [CHL(B)] that mediates direct cell-cell adhesion (Hillenbrand et al., 1999). Previous studies suggest these isoforms exert differential effects on neuronal development (Zhou et al., 2012), but their specific roles in human cortical layer formation remain unexplored.
       
Despite extensive research on CHL1 in rodent models, critical knowledge gaps limit our understanding of its role in human cortical development. First, while both CHL1 isoforms have been characterized biochemically, their differential functions in regulating cortical neuron fate specification have not been investigated. Second, the temporal dynamics of CHL1 expression and function during human corticogenesis remain undefined. Third and most critically, how the spatial presentation of CHL1 (apical versus basal) influences cortical layer identity has never been examined, despite growing evidence that the subcellular localization of adhesion molecules determines their signaling outcomes and biological effects. These gaps are particularly significant given that human cortical development differs substantially from rodent models in timing, complexity and molecular signatures.
       
The clinical relevance of understanding CHL1 function is underscored by its association with multiple neuro developmental disorders. CHL1 mutations have been linked to intellectual disability (IQ range 50-70) and increased schizophrenia susceptibility (Angeloni et al., 1999). The CHL1 gene, located on chromosome 3p26.1, falls within a region frequently deleted in 3p deletion syndrome, which presents with severe cortical malformations and intellectual disability. Recent genome-wide association studies have identified CHL1 polymorphisms as risk factors for autism spectrum disorders, further highlighting its importance in human neurodevelopment (Paramasivam, 2021). However, the cellular and molecular mechanisms linking CHL1 dysfunction to these clinical phenotypes remain poorly understood, hampering development of targeted therapeutic approaches.
       
To address these knowledge gaps, we employed human embryonic stem cell (hESC)-derived cortical neurons, which faithfully recapitulate key aspects of human corticogenesis including temporal gene expression patterns, layer-specific marker expression and extended developmental timelines (Espuny et al., 2013). This model system offers unique advantages: (1) species-specific relevance for understanding human development, (2) precise control over CHL1 spatial presentation and (3) ability to track the same cell populations longitudinally across developmental stages. By combining this model with comprehensive transcriptional profiling and morphometric analyses, we can dissect how CHL1 isoforms differentially regulate cortical development.
       
Therefore, this study aimed to investigate how CHL1 spatial presentation regulates human cortical neuron development. Specifically, we: (1) characterized temporal CHL1 expression during cortical differentiation (Days 28, 35, 42); (2) examined how apical (CHL(S)) versus basal (CHL(B)) presentation affects deep-layer (Tbr1, Ctip2) and upper-layer (Cux1, Satb2) markers; (3) determined whether CHL1 acts on progenitors or post-mitotic neurons and (4) assessed the relationship between transcriptional and morphological effects. This approach provides mechanistic insights into CHL1-mediated cortical patterning and its role in neurodevelopmental disorders.
HESC cell lines and culture conditions
 
Human embryonic stem cells (H9 line) were used to generate cortical neurons through in vitro differentiation. Cells were cultured in mTeSR1 medium at 37oC with 5% CO2. Differentiation was initiated once cultures reached approximately 95% confluence.
 
Differentiation of hESCs into cortical progenitors and neurons
 
Differentiation was initiated when cells reached approximately 95% confluence by replacing mTeSR1 with Cortical Mixture Media (CMM), comprising a 1:1 ratio of DMEM/F-12 and Neurobasal Medium, supplemented with L-Glutamine, Penicillin-Streptomycin, Non-Essential Amino Acids (NEAA), Insulin-Transferrin-Selenium (ITS-A), N-2 Supplement, B-27 Supplement and β-Mercaptoethanol. Unless otherwise specified, reagents were obtained from GIBCO. CMM was supplemented with SB431542 (1:1000, Cell Signaling, Cat. No. 14775) and LDN193189 (1:1000, Sigma, Cat. No. SML0559) during the neural induction phase (Days 1-11).
       
On Day 11, cells were passaged at a 1:2 ratio onto LN-521-coated wells using ReLeSR. After centrifugation (260 × g, 3 minutes), cells were resuspended in CMM containing 10 µM Y27632 and plated in 800-2000 µL volumes per well. From Days 12-17, CMM was supplemented with FGF2 (1:5000) to promote progenitor proliferation, with medium changes every other day. A second passage was performed on Day 17, after which cells entered the maturation phase (Days 18-34) and were maintained in CMM with medium refreshed every other day.
 
CHL1 presentation and treatment regimen
 
To assess the effects of soluble and adherent CHL1 on cortical differentiation, two treatment conditions were employed. For soluble CHL1, recombinant human CHL1 protein (R and D Systems, Cat. No. 2126-CH) was added to the culture medium (10 µg/mL) during the seeding step and maintained throughout the culture period with each medium change. For membrane-bound CHL1, LN-521-coated wells were first incubated overnight at 4oC with anti-His-tag antibody (Fc control), followed by PBS washes, blocking with 1% BSA (1 hour at 37oC) and incubation with recombinant CHL1 protein (overnight, 4oC). After washing, cells were seeded and cultured under standard differentiation conditions. Assessments were conducted at Day 28, Day 35 and Day 42. All treatments were performed with n= 3 independent biological replicates per condition.
 
Quantitative real-time PCR
 
Total RNA was extracted from differentiated cortical neurons using Trizol reagent (Sigma-Aldrich) following the manufacturer’s instructions. RNA quality and quantity were assessed using spectrophotometry and 1 µg of RNA was reverse transcribed into cDNA using SuperScript IV First-Strand Synthesis System (Invitrogen, Cat. No. 18091050). qRT-PCR was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Cat. No. A25742) on a QuantStudio 3 Real-time PCR System (Thermo Fisher Scientific). Primers targeting cortical development markers were used, including Tbr1, Ctip2, Satb2, CHL1, Tbr2, Brn2, Cux1 and Cux2. GAPDH was used as a housekeeping gene for normalization (Table 1). Reactions were run in triplicate and gene expression was analyzed using the 2-ΔΔCt method (Alsanie et al., 2017).

Table 1: List of primer sequences of the genes analyzed by qRT-PCR.


 
Ethical approval
 
This study, along with all experimental procedures, was conducted according to the principles outlined by the ethics committee of the University of Taif (Approval No. TUZ-18-T-277).
 
Statistical analysis
 
All data are presented as mean ± standard error of the mean (SEM) from three independent biological replicates (n=3). Statistical analysis was conducted using GraphPad Prism (v9, GraphPad Software, San Diego, CA, USA). Data normality was assessed using the Shapiro-Wilk test. For comparisons between two groups, unpaired two-tailed Student’s t-test was used. For multiple group comparisons, one-way ANOVA followed by Tukey’s post hoc test was performed. All qRT-PCR reactions were run in technical triplicates. Statistical significance was set at p<0.05. For multiple comparisons within the same dataset, p-values were adjusted using the Benjamini-Hochberg method to control false discovery rate. Effect sizes were calculated using Cohen’s d for t-tests for ANOVA.
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.

Fig 1: Temporal expression of CHL1 during cortical differentiation.


 
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.

Fig 2: CHL1 suppresses Tbr1 expression in both orientations.



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.

Fig 3: CHL1 does not affect early neurogenic markers.


 
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.

Fig 4: Orientation-specific regulation of Tbr1 and Ctip2.


 
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.

Fig 5: CHL1 modulates cortical markers in orientation-dependent manner.


 
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.

Fig 6: Selective modulation of cortical markers by CHL1.


 
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.

Fig 7: CHL1 does not affect neurite morphology.


       
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.
In conclusion, CHL1 acts as an orientation-dependent molecular switch that precisely regulates cortical neuron fate without affecting morphology. The opposing effects of CHL(S) and CHL(B) suppressing versus promoting differentiation markers-combined with stage-specific regulation, reveal CHL1 as a critical determinant of cortical layer identity. These findings establish a new paradigm for understanding how spatial presentation of adhesion molecules controls neurodevelopment and offer therapeutic targets for CHL1-related.
 
Author contributions
 
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Ashraf Albrakati, Majid Alhomrani and Walaa F. Alsanie. The first draft of the manuscript was written by Majid Alhomrani and all authors commented on previous versions of the manuscript. All authors read and approved of the final manuscript.
 
Data availability statement
 
All the data supportive stated results exist in the manuscript.
 
Funding
 
This work was funded by the Deanship of Graduate Studies and Scientific Research, Taif University (project number 1-441-18).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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