Functional Role for Cas Cytoplasmic Adaptor Proteins During Cortical Axon Pathfinding

  1. Jason A. Estep
  2. Alyssa M. Treptow
  3. Payton A. Rao
  4. Patrick Williamson
  5. Wenny Wong
  6. Martin M. Riccomagno

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Abstract

Proper neural circuit organization requires individual neurons to project to their targets with high specificity. While several guidance molecules have been shown to mediate axonal fasciculation and pathfinding, less is understood about how neurons intracellularly interpret and integrate these cues. Here we provide genetic evidence that the Crk-Associated Substrate (Cas) family of intracellular adaptor proteins is required for proper fasciculation and guidance of two cortical white matter tracts: the Anterior Commissure (AC) and thalamocortical axons (TCAs). Using a Cas Triple Conditional Knock Out ( Cas TcKO ) mouse model, we show that Cas proteins are required for proper TCA projection by a non-neuronal cortical cell population. We also demonstrate a requirement of the β1-integrin receptor for TCA projection, similarly in a population of non-neuronal cortical cells. Additional analysis of Cas TcKO mutants reveals a role for Cas proteins in AC fasciculation, here within the neurons themselves. This AC fasciculation requirement is not phenocopied in β1-integrin deficient mutants, suggesting that Cas proteins might signal downstream of a different receptor during this axon pathfinding event. These findings implicate Cas proteins as key mediators of cortical axon tract fasciculation and guidance.

Author Summary

In the developing nervous system, neurons extend axons—long projections that relay information to their targets—to establish neural circuits. Axons follow specific pathways directed by extracellular guidance cues, much like street signs direct traffic. While these guidance cues are well studied, how neurons internally interpret and respond to these signals remains unclear. Here, we examine the role of the Crk-Associated Substrate (Cas) family of intracellular adaptor proteins in axon guidance within cortical axon tracts. Using genetic techniques to selectively remove Cas gene function from specific cell types, we demonstrate that Cas proteins are required for proper fasciculation (bundling) of anterior commissure axons, acting directly within the projecting axons themselves. Additionally, Cas proteins are required for proper guidance of thalamocortical projections—axons connecting the thalamus with the cortex. However, in this case, Cas proteins do not act within projecting axons but instead direct target neurons to their final positions. We further show that the β1-integrin receptor is similarly required for thalamocortical axon projection. These findings provide genetic evidence for a critical role of Cas adaptor proteins in both fasciculation and guidance of cortical axon tracts.

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    Reply to the reviewers

    Response to Reviewers and Revision Plan

    We thank all three reviewers for their thoughtful and constructive comments. We are pleased that the reviewers found our work to be "very interesting," "well written," with "high quality" data that is "convincing" and will be "of broad interest for the community of axon guidance, circuit formation and brain development." We particularly appreciate the recognition that our study provides "novel functions for Cas family genes in forebrain axon organization" and uses "state-of-the art mouse genetics" with "quantitative and statistical rigor." Below are our detailed responses to each reviewer's comments, including extensive additional experiments and analyses that we will perform to significantly strengthen the manuscript.

    Reviewer #1

    We thank this reviewer for recognizing that our experiments are "carefully done and quantified" with "clear and striking" phenotypes that "support most of the conclusions in the manuscript." We appreciate their acknowledgment that this work will be "of interest to developmental neurobiologists and the axon guidance and adhesion fields."

    Major Comments:

    __ Authors clearly show that misplaced TCA axons are coordinate with cortical layer defects, with misplaced tbr1+ neurons, in EMX-Cre cas and integrin knockouts, suggesting these axons are following misplaced cells. These results are described as 100% coordinate, but since there is no figure of quantification, authors need to clarify how many embryos were examined for each genotype, as this was not described in results or legends.__ We apologize for this oversight and will provide detailed quantification of this important finding. We examined a total of 11 Emx1Cre;TcKO embryos with 13 controls, and 14 Emx1Cre;Itgb1 embryos with 13 littermate controls at two developmental stages (E16.5 and P0) to quantify the coordination between misplaced Tbr1+ neurons and cortical bundle formation. This quantification will be presented in the main text and figure legend.

    Here's a more detailed breakdown of those numbers: For Emx1Cre;TcKO knockouts, we examined 7 controls and 5 mutants at P0, and 6 controls and 6 mutant embryos at E16.5. For the Emx1Cre;Itgb1 knockouts, we examined 5 controls and 6 mutant neonates at P0, and 8 controls and 8 mutant embryos at E16.5.

    __ Are the neurons not misplaced in Nex cre cas or integrin knockouts? One would think presumably not, but then what are the tbr1+ cell migration defect caused by? I struggle with the semantics of non-neuronal autonomous role of cas in cortex, since tbr1+ neurons are misplaced, and this is what the axons are mistargeting too. So yes, potentially cas or b1 is not needed in those neurons, but those misplaced neurons are presumably driving the phenotype.__

    We agree that this important point requires better explanation. You are absolutely correct that Tbr1+ neurons are not misplaced in NexCre;TcKO mutants (Wong et al., 2023), which is precisely why these animals do not exhibit cortical bundle formation. In addition to our previously published data showing normal location of Tbr1+ neurons in those mutants, we can also provide similar analysis at E16.5 and P0 as a supplemental figure. The model we propose is that Cas genes are required in radial glial cells for proper positioning of deep layer cortical neurons. These correctly positioned neurons, in turn, provide appropriate guidance cues for TCA projections. Hence, our model is that while the role of Cas genes is non-neuronal-autonomous (acting in radial glia rather than in the neurons themselves), the mispositioned Tbr1+ neurons in Emx1Cre;TcKO mutants drive the TCA misprojection phenotype. We will clarify this mechanism in the discussion and provide a new graphical model as a supplemental figure to facilitate conceptualization of our conclusions.

    __ Authors need to clarify in the discussion that they can't rule out the cas not also needed in tca neurons, Since neither emx or nex cre would hit those cells.__

    We will add the following clarification to the discussion: The analysis of cortical bundle formation in Emx1Cre;TcKOrevealed a comparable phenotype to that observed in NestinCre;TcKO, strongly suggesting a cortical-autonomous role for *Cas *genes in CB formation. "However, we cannot formally exclude a thalamus-autonomous role for *Itgb1 *or Cas genes in TCA pathfinding, as we did not ablate these genes exclusively in the thalamus. Future studies using thalamus-specific Cre drivers would be needed to definitively address this question."

    __ Could authors add boxes in zoomed out brain images to denote zoom regions. And potentially a schematic demonstrating placement of DiI for lipophilic tracing experiments.__

    We will add boxes to denote zoom regions where possible throughout the manuscript. For some high magnification panels, we selected the best representative images, which don't necessarily correspond to specific regions of the lower magnification panels, but we will note this in the figure legends. We will also add a schematic diagram to a supplemental figure illustrating DiI placement for all lipophilic tracing experiments.

    Reviewer #2

    We thank this reviewer for describing our study as "very interesting," "well written," with data that are "of high quality" and findings that are "convincing." We appreciate their recognition that we used "state-of-the art mouse genetics" and that our work will be "of broad interest for the community of axon guidance, circuit formation and brain development."

    Major Comments:

    __ Immunofluorescence labeling for other β-integrin family members to examine expression in AC axons may provide insights into why β1-integrin deficiency does not replicate the Cas TcKO phenotype.__ This is an excellent suggestion that we will address experimentally. We will perform RNAscope analysis for integrin β5, β6, and β8 in developing piriform and S1 cortex at E14.5, E16.5, and E18.5, as these are the only other β-integrins expressed during cortical development. We anticipate that this analysis may reveal expression of alternative β-integrins in the neurons that extend axons along the developing anterior commissure, which would provide a potential explanation for why β1-integrin deficiency does not replicate the AC phenotype observed in Cas TcKO animals. These new data will be presented as part of a new figure.

    __ Is there any evidence that β1-integrin in developing cortical axons is colocalized with Cas proteins (in vivo or in vitro)?__

    We have tested multiple antibodies for p130Cas and CasL without success in cortical tissue. However, we will test two new integrin β1 antibodies and a new p130Cas antibody. While direct colocalization may be challenging due to species restrictions and tissue-specific antibody performance, we will attempt to show regional co-expression in consecutive sections. If the integrin antibodies work, we will present data as a supplemental figure demonstrating that p130Cas (using our BAC-EGFP reporter) and β1-integrin show overlapping expression patterns in developing cortical white matter tracts and neurons, supporting their potential functional interaction. In the end, while we will try to address this critique, we will be limited by the reagents that are available to us.

    Minor Comments:

    __ How long do the Cas TcKO with the various cre driver survive?__

    We have not systematically quantified survival beyond 6 months, but surprisingly, survival up to 6 months of age appears normal for all genotypes examined. This information will be included in the Methods section.

    Reviewer #3

    We thank this reviewer for acknowledging that our "main claims and conclusions are solidly supported by the data" with "good overall data quality" and "high quantitative and statistical rigor." We appreciate their recognition that we "uncover novel functions for Cas family genes in forebrain axon organization" and that our "overall reporting and discussion of findings is data-driven and refrains from excessive speculation."

    Addressing Concerns About Novelty and Impact:

    We respectfully disagree with the characterization of our findings as "somewhat incremental." While we acknowledge that similar axonal defects have been described in other lamination mutants, our study makes several novel and significant contributions:

    First demonstration of Cas family requirement in forebrain axon tract development: This is the first study to establish roles for Cas proteins in axon guidance, representing a completely new function for these well-studied signaling molecules. Novel β1-integrin-independent role for Cas proteins: Our finding that AC defects occur in Cas mutants but not β1-integrin mutants reveals a previously unknown signaling pathway and challenges the assumption that Cas proteins always function downstream of β1-integrin. Mechanistic insights into cortical-TCA interactions: While the general principle that cortical lamination affects TCA projections has been established, our work provides the first demonstration of how specific adhesion signaling molecules (Cas proteins) control this process through radial glial function. Cell-type specific requirements: Our systematic analysis using multiple Cre drivers provides unprecedented detail about where and when Cas proteins function during brain development, revealing both neuronal-autonomous (AC) and non-neuronal autonomous (TCA) roles.

    As Reviewer #2 noted, "The main advancement is a more nuanced understanding of where and when these molecules function during brain development and insights into the origin of the defects observed." This represents significant mechanistic progress in understanding forebrain circuit assembly.

    Specific Comments:

    Suggestion about cell autonomy testing: We appreciate the optional suggestion to test strict cell autonomy using sparse deletion approaches. While this would indeed be interesting, it would represent a substantial undertaking beyond the scope of the current study. However, we believe our current data using NexCre (which hits early postmitotic neurons) versus NestinCre (CNS-wide deletion) and Emx1Cre (cortical progenitors) provides supportive evidence for neuronal autonomy of the AC phenotype, as mentioned by the reviewer.

    In vitro axon guidance assays: This is an excellent suggestion for future molecular studies. In the discussion we identify specific candidate guidance molecules (e.g. Ephrins) that would be prime targets for such experiments.

    Cross-Reviewer Comments:

    We appreciate Reviewer #3's agreement with the other reviewers' suggestions and will address the quantification of neuronal mispositioning/axon bundle correlation as requested by Reviewer #1.

    Additional Improvements:

    Beyond addressing the specific reviewer comments, we will make several additional improvements to strengthen the manuscript:

    Enhanced statistical analysis: All quantifications will include appropriate statistical tests with clearly stated n values and multiple litters represented. Expanded discussion: We will better contextualize our findings within the broader axon guidance literature and discuss future directions (e.g. TCAs). New data: Additional controls, expression analysis, and quantifications will strengthen our conclusions.

    We believe these revisions, particularly the new experimental data addressing integrin family expression and the detailed quantification of phenotype coordination, will significantly strengthen our conclusions and demonstrate the novelty and impact of our findings. We hope the reviewers will find these improvements satisfactory and agree that our work makes important contributions to understanding axon guidance mechanisms in the developing forebrain.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript by Estep et al., the authors use conditional in vivo mouse genetics to study roles for Cas family intracellular adaptor proteins in forebrain axon tract development. They report two phenotypes after simultaneous nervous system-wide deletion of three Cas family genes - (1) defasciculation and misprojection of anterior commissure axons and (2) ectopic formation of thalamocortical axon bundles that penetrate the cortex. Further investigation using specific Cre lines and other conditional knockout alleles demonstrates that the anterior commissure defect results from a requirement for Cas genes in cortical projection neurons, whereas thalamocortical axons are misguided due to Cas functional requirements in cortical lamination, as ectopic axon bundles are confined to sites of disrupted cortical layer formation. Overall, this study uncovers novel functions for Cas family genes in forebrain axon organization, one of which likely reflects a direct role in axon guidance and/or fasciculation, while another one is indirect and based in the previously established role of Integrin-Cas signaling in radial glia organization and cortical neuron migration.

    The main claims and conclusions of the paper are solidly supported by the data. The study is fairly descriptive in nature, being limited to in vivo analyses of Cas expression patterns and characterization of the knockout phenotypes, and does not uncover novel molecular mechanisms for axon guidance, but it also does not attempt to make any claims to that effect. The overall reporting and discussion of findings is data-driven and refrains from excessive speculation, which is commendable. The overall data quality is good, and data organization and presentation are clear. Quantitative and statistical rigor are high.

    The characterization of Itgb1 knockout animals and various conditional Cas knockouts provides strong evidence that the thalamocortical axon phenotypes are simply a secondary consequence of cortical disorganization, as they strictly segregate with defects in cortical lamination.

    The requirement for Cas genes in anterior commissure axon organization is accurately reported as "neuronal-autonomous", but not as cell-autonomous. It would be interesting, yet not essential (i.e. this suggestion is optional), to test for strict cell autonomy by sparsely deleting Cas family genes in a subset of the neurons that project axons through the anterior commissure and analyzing the projection patterns of Cas mutant and control neurons in such a genetic mosaic side by side.

    In the discussion, the authors highlight a few of the axon guidance signaling pathways that would be strong candidates for requiring Cas in the context of the anterior commissure. If the authors wanted to develop this idea further, they should consider using in vitro axon guidance assays to study the requirement for Cas function in the axonal response to these candidate guidance molecules.

    Referee Cross-commenting

    I generally agree to the comments by reviewers 2 and 3. I especially like reviewer 1's suggestion to provide quantitative support for the correlation between sites of neuronal mispositioning and sites of ectopic axon bundle emergence in the cortex. I also agree with that reviewer's idea to box regions in micrographs that are shown in high-magnification panels.

    Significance

    The strengths of the study lie in its simplicity and limited scope, yet so do its weaknesses. The authors uncover requirements for Cas genes in axon tract organization, but mechanistic insights are extremely limited. On the plus side, the authors refrain from excessive speculation and stay very close to the data in the interpretation and discussion of their findings.

    The reported findings are novel, at least to some extent. The same group had previously established the Itgb1-Cas signaling axis as an important regulator of cortical architecture, and results presented here document a thalamocortical axon guidance phenotype that results from defective cortical lamination. Similar axonal defects have been described in other mouse models with lamination phenotypes, and these studies are cited in the manuscript at hand. So while the study is not first to show this interplay between cortical neuronal positioning and thalamocortical axon organization, it does add to the growing body of evidence for this phenomenon. As for the anterior commissure defect, the study is first to establish a role for Cas family genes in development of this axon tract, but beyond evidence that this might be a neuronal-autonomous requirement (but see earlier comment), it does not provide any mechanistic insights into this Cas function. Had the authors identified an actual signaling pathway for axon guidance or bundling that is mediated by Cas proteins and explains their requirement for anterior commissure formation, this study would be a lot more impactful. In its current form, however, the limited genetic and functional insights from this manuscript will largely be of interest to a specialized audience. The overall advance provided by this work is somewhat incremental.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary: In this very interesting study, Estep and colleagues investigate the role of Cas family members (p130Cas, CasL/Nedd9 and Sin/Efs) by generating triple conditional mutant (Cas TcKO) mice to investigate to role in the developing brain (E14.5 - P0), focusing on thalamocortical axons (TCA) and the anterior commissure (AC). For visualization of p130Cas expressing neurons, the p130Cas-EGFP-BAC allele was used. This revealed EGFP (p130Cas) expression in all major cortical tracts and overlap with L1 distribution. Conditional ablation using Nestin-cre (Nes-cre;TcKO) revealed defects in the AC and the external capsule (EC). In addition, these mice show pathfinding defects resulting cortical bundles (CBs) in white matter within the cortical plate. Evidence is provided that these CBs originate from TCA afferents. To assess the cell autonomy of these phenotypes, Emx1-cre;TcKO and Nex-Cre;TcKO mice were generated. Analysis of these mice revealed that Cas genes function in cortical neurons is required for proper TCA development. Nex-cre;TcKO mice only replicated the AC phenotypes observed in Nestin-cre;TcKO mice. Moreover, evidence is provided that proper development of TCA afferents requires non-neuronal functions of Cas genes. Because Cas proteins function downstream of integrins, including beta1-integrin, Itgb1 cKO mice (using Emx-cre or Nex-cre) to examine similarities to Cas TcKO mice. Indeed, Emx-cre;Itgb1 cKO mice phenocopy CB defects observed in the Emx-cre; CasTcKO, while Nex-cre;Itgb1 mutants do not, and neither of the Itgb1 mutants phenocopied the Cas TcKO defects in the AC. Correlative evidence is provided that CBs observed in Cas TcKO mutants originate from disorganization of the subplate.

    Overall, this manuscript is well written, and most of the data presented are of high quality. It is also clear that a great deal of effort was put into the experiments, however some issues were identified, and the authors should address them to further clarify and strengthen the work.

    Major comments:

    1. Immunofluorescence labeling for other b-integrin family members to examine expression in AC axons may provide insights into why b1-integrin deficiency does not replicate the Cas TcKO phenotype.
    2. Is there any evidence that b1-integrin in developing cortical axons is colocalized with Cas proteins (in vivo or in vitro)

    Minor comments:

    1. How long do the Cas TcKO with the various cre driver survive?

    Significance

    Elucidation of molecular mechanisms of axon pathfinding and brain wiring in vivo. Using state-of-the art mouse genetics; this includes genetic labeling of specific axon tracts, generation of compound mutants in a cell type specific manner and gene products that are thought to function in the same pathway. This was confirmed for some fiber systems, but not for others. The findings presented are convincing and the manuscript is well written. The guidance molecules investigated are not novel and have been analyzed previously, however not with the same rigor or the use of compound mutants. The main advancement is a more nuanced understanding of where and when these molecules function during brain development and insights into the origin of the defects observed. Of broad interest for the community of axon guidance, circuit formation and brain development. I have been studying molecules that regulate axon guidance, growth and regeneration for the past 20+ years.

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    Referee #1

    Evidence, reproducibility and clarity

    In the manuscript by Estep et al., the authors studied Cas proteins expressed during brain development, particularly during the formation of the anterior commissure (AC) and thalamocortical (TCA) projection, using conditional alleles in mice, immunohistochemistry, and a combination of lipophilic axon tracers or genetically encoded fluorophores that mark cells that have expressed Cre. They found that Cas proteins were required for proper guidance of TCA projections and fasciculation of posterior AC axons using broad deletion of Cas gene function by crossing Cas TcKO animals with Nestin-Cre mice- CNS-wide deletions in both neuronal and glial populations, results in axon misprojection and aberrant cortical bundles, of both AC and TCA. With a time course, they find these axon misguidance phenotypes appear at different developmental time points, with AC defasciulation not apparent until e18.5, whereas aberrent TCA Cortical bundles were detected already at e14.5, and increasing over developmental time.

    They then go on to use more specific Cre drivers, EMX cre (RGCs, excitatory neurons, mqcroglia in cortex), vs Nex Cre early postmitotic neurons in cortex. EMX cre, still shows TCA defects, even though cas not knocked out of tca axons, suggesting cortical autonomous expression of cas, affects these axons. Because Nex Cre mice didn't show this phenotype, this suggested that the TCA phenotype was cortical-autonomous but not neuronal-autonomous, with mis-projecting TCA processes (cortical bundles) closely associating with misplaced subplate and deep layer neurons. cortical- and neuronal autonomous role for Cas genes during AC fasciculation showed that defasciculating AC axons originated from the dorsolateral cortex. Defects in AC fasciculation were dissimilar to β1-integrin mutants, suggesting that Cas proteins can act independently of β1-integrin during AC formation.

    Overall, these data indicate a requirement for Cas family genes during cortical white matter tract formation. The experiments are carefully done and quantified, and the phenotypes are clear and striking, and support most of the conclusions in the manuscript. I only suggest a few points for clarification.

    Authors clearly show that misplaced TCA axons are coordinate with cortical layer defects, with misplaced tbr1 + neurons, in EMX-Cre cas and integrin knockouts, suggesting these axons are following misplaced cells. These results are described as 100% coordinate, but since there is no figure of quantification, authors need to clarify how many embryos were examined for each genotype, as this was not described in results or legends.

    Are the neurons not misplaced in Nex cre cas or integrin knockouts? One would think presumably not, but then what are the tbr1+ cell migration defect caused by? I struggle a with the semantics of non-neuronal autonomous role of cas in cortex, since tbr1+ neurons are misplaced, and this is what the axons are mistargeting too. So yes, potentially cas or b1 is not needed in those neurons, but those misplaced neurons are presumably driving the phenotype.

    Authors need to clarify in the discussion that they can't rule out the cas not also needed in tca neurons, Since neither emx or nex cre would hit those cells.

    Could authors add boxes in zoomed out brain images to to denote zoom regions. And potentially a schematic demonstrating placement of DiI for lipophilic tracing experiments.

    Significance

    The study demonstrates the different requirement for Cas proteins and b1 integrin in different cell populations for appropriate white matter tract formation, and further supports the that cortical layering helps direct TCA projections. It also provides evidence for a b1 integrin independent role for cas proteins. The authors nicely discuss this with several alternative upstream receptors that may be involved detailed, that set the stage for future work, but this would be quite a large endeavor. I would add that it is unclear if cas proteins are needed in the TCA neurons, as they did not use a cre driver that would clarify this. The final limitation I will mention is that EM would likely be required to demonstrate a role for fasiculation, but this also seems beyond this original manuscript. This study will be of interest to developmental neurobiologists and the axon guidance and adhesion fields.

  5. Version published to 10.1101/2025.04.08.647868 on bioRxiv