Role of Nrp1 in controlling cortical inter-hemispheric circuits
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Evaluation Summary:
Martín-Fernández et al. show that Nrp1 acts within the primary somatosensory cortex to control the refinement of axons and the topography of contralateral targeting, particularly with regards to homotopic "matching" of gradients. While this action of Nrp1 has already been discovered between cortical areas, this work elucidates its role within a cortical area, with further insight into the developmental dynamics of projection and refinement also reported. This is impactful to the field of cortical axon guidance and corpus callosum development. The data analysis is rigorous and most conclusions are justified by the data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Axons of the corpus callosum (CC) mediate the interhemispheric communication required for complex perception in mammals. In the somatosensory (SS) cortex, the CC exchanges inputs processed by the primary (S1) and secondary (S2) areas, which receive tactile and pain stimuli. During early postnatal life, a multistep process involving axonal navigation, growth, and refinement, leads to precise CC connectivity. This process is often affected in neurodevelopmental disorders such as autism and epilepsy. We herein show that in mice, expression of the axonal signaling receptor Neuropilin 1 (Nrp1) in SS layer (L) 2/3 is temporary and follows patterns that determine CC connectivity. At postnatal day 4, Nrp1 expression is absent in the SS cortex while abundant in the motor area, creating a sharp border. During the following 3 weeks, Nrp1 is transiently upregulated in subpopulations of SS L2/3 neurons, earlier and more abundantly in S2 than in S1. In vivo knock-down and overexpression experiments demonstrate that transient expression of Nrp1 does not affect the initial development of callosal projections in S1 but is required for subsequent S2 innervation. Moreover, knocking-down Nrp1 reduces the number of S2L2/3 callosal neurons due to excessive postnatal refinement. Thus, an exquisite temporal and spatial regulation of Nrp1 expression determines SS interhemispheric maps.
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Author Response:
Reviewer #3:
Weaknesses:
In utero electroporation as well as other in vivo gene manipulation techniques do not allow fine manipulations of expression gradients. Therefore some conclusions of the paper are not fully supported. Although the data presented in the paper clearly show that Nuropilin1 expression level is important for establishment of homotopic connections, it does not show directly that the gradient of expression indeed is in play, as suggested by the authors. Another week point is that there is no direct evidence that the Neuropilin1 protein level follows the mRNA expression gradient.
Therefore it remains an open question, whether it is a gradient of expression or a sharp border of cellular response to higher-lower levels of Neuropilin1 that controls area specific connections within somatosensory …
Author Response:
Reviewer #3:
Weaknesses:
In utero electroporation as well as other in vivo gene manipulation techniques do not allow fine manipulations of expression gradients. Therefore some conclusions of the paper are not fully supported. Although the data presented in the paper clearly show that Nuropilin1 expression level is important for establishment of homotopic connections, it does not show directly that the gradient of expression indeed is in play, as suggested by the authors. Another week point is that there is no direct evidence that the Neuropilin1 protein level follows the mRNA expression gradient.
Therefore it remains an open question, whether it is a gradient of expression or a sharp border of cellular response to higher-lower levels of Neuropilin1 that controls area specific connections within somatosensory cortex.
Another weak point is that the paper relies on in utero electroporation solely. This technique with all its advantages, has some disadvantages too. One of them is high variability of individual experiments. On the other hand, it targets only subsets of cells, and therefore is not the best to address cell extrinsic mechanisms, especially those that involve expression gradients.
The reviewer raised interesting comments. We would like to clarify that we never attempted to disrupt the gradient per se but to alter Nrp1 expression in individual cells. This evaluates how their projections are affected by the Nrp1 expression imposed by their location and this contributes to understand how the gradient contributes to connectivity. Nevertheless, the reviewer raised a fair point in that we realized it was important to revise the expression of Nrp1 to sharpen our interpretations. In this revised version we have performed in situ hybridization to investigate if we were dealing with gradients or sharp borders. Surprisingly we found unexpected patterns of expression. This is now shown in Figure 1. Interestingly, the expression pattern of Nrp1 in the postnatal brain is highly dynamic. At early stages of CC development in the cortex shows a discontinuity in L2/3 neurons of the SS, rather than a gradient. Nrp1 expression is upregulated after P7 in a manner that suggests a gradual activation from lateral to medial SS cortex. At P16, few cells are positive but they are equally distributed throughout the S1 and S2 cortex. Therefore, we have modified the text and avoided referring to the gradient. This gradient was described at embryonic stages and P0 (Zhou et al., 2013). The new version of the manuscript also adds results showing that changes in Nrp1 expression do not detectably modify contralateral innervation at P10 and that the S2 column is not formed at this stage. The quantification method suggested by reviewer #2 allows us to conclude that the reduction in the S2 columns in the shNrp1 condition, although is statistically significant. Together, the new data provides a better understanding of our phenotypes and explains why the phenotypes of CAG-Nrp1 and shNrp1 are so similar and both block innervation in S2, since they both disrupt the normal transient expression.
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Evaluation Summary:
Martín-Fernández et al. show that Nrp1 acts within the primary somatosensory cortex to control the refinement of axons and the topography of contralateral targeting, particularly with regards to homotopic "matching" of gradients. While this action of Nrp1 has already been discovered between cortical areas, this work elucidates its role within a cortical area, with further insight into the developmental dynamics of projection and refinement also reported. This is impactful to the field of cortical axon guidance and corpus callosum development. The data analysis is rigorous and most conclusions are justified by the data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers …
Evaluation Summary:
Martín-Fernández et al. show that Nrp1 acts within the primary somatosensory cortex to control the refinement of axons and the topography of contralateral targeting, particularly with regards to homotopic "matching" of gradients. While this action of Nrp1 has already been discovered between cortical areas, this work elucidates its role within a cortical area, with further insight into the developmental dynamics of projection and refinement also reported. This is impactful to the field of cortical axon guidance and corpus callosum development. The data analysis is rigorous and most conclusions are justified by the data.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Martín-Fernández et al. present an important advance in the understanding of how Nrp1 controls formation of the corpus callosum. It had previously been shown that the neocortical Nrp1 gradient (high medial, low lateral) is important for controlling the separation of axon bundles from two different cortical areas (S1 and M1) within the corpus callosum midline, and therefore also controlling the homotopic pattern of contralateral targeting (Zhou et al., 2013). The major advance of the work presented here is showing that the Nrp1 gradient within S1 acts in a similar fashion as between S1 and M1, as well as elucidating some further details about the mode of action. The conclusions regarding these further details are generally supported by the results, although select further controls to ensure comparability …
Reviewer #1 (Public Review):
Martín-Fernández et al. present an important advance in the understanding of how Nrp1 controls formation of the corpus callosum. It had previously been shown that the neocortical Nrp1 gradient (high medial, low lateral) is important for controlling the separation of axon bundles from two different cortical areas (S1 and M1) within the corpus callosum midline, and therefore also controlling the homotopic pattern of contralateral targeting (Zhou et al., 2013). The major advance of the work presented here is showing that the Nrp1 gradient within S1 acts in a similar fashion as between S1 and M1, as well as elucidating some further details about the mode of action. The conclusions regarding these further details are generally supported by the results, although select further controls to ensure comparability between conditions are recommended. This is an important addition to the field of axon guidance and callosal development, and presents intriguing insights into the complex mechanics of exuberance, refinement and competition occurring in the corpus callosum that, while not completely elucidated here, do provide candidate mechanistic processes for future work to follow.
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Reviewer #2 (Public Review):
While midline crossing of callosal axons has been extensively studied, how axons make precise connections with contralateral target areas is less understood. Neuropilin 1 (Nrp1), a well-known guidance receptor, mediates the crossing of callosal axons at the midline through its interaction with the Semaphorin (Sema) 3C and the segregation of motor from somatosensory (SS) callosal axons within the corpus callosum (CC) through its interaction with Sema3A. Here, the authors address the role of this guidance cue in the invasion of SS callosal axons to homotopic or heterotopic SS cortical areas. Using in utero electroporation of an shRNA against Nrp1 (shNrp1) or a CAG-Nrp1 construct to respectively down- or up- regulate Nrp1 expression in SS layer 2/3 neurons combined with stereotaxic injection of CTB to label …
Reviewer #2 (Public Review):
While midline crossing of callosal axons has been extensively studied, how axons make precise connections with contralateral target areas is less understood. Neuropilin 1 (Nrp1), a well-known guidance receptor, mediates the crossing of callosal axons at the midline through its interaction with the Semaphorin (Sema) 3C and the segregation of motor from somatosensory (SS) callosal axons within the corpus callosum (CC) through its interaction with Sema3A. Here, the authors address the role of this guidance cue in the invasion of SS callosal axons to homotopic or heterotopic SS cortical areas. Using in utero electroporation of an shRNA against Nrp1 (shNrp1) or a CAG-Nrp1 construct to respectively down- or up- regulate Nrp1 expression in SS layer 2/3 neurons combined with stereotaxic injection of CTB to label callosally projecting neurons, the authors study the importance of Nrp1 expression levels in establishing primary (S1) and secondary (S2) somatosensory interhemispheric homotopic and heterotopic connections. They conclude that neurons overexpressing Nrp1 project contralaterally in the S1/S2 but not the S2 column. Neurons with lower levels of Nrp1 can project contralaterally both in the S1/S2 and the S2 columns. This study suggests that precise controls of guidance cue expression levels are necessary for the proper establishment of cortical connections.
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Reviewer #3 (Public Review):
In this paper Martin-Fernandes et al. investigated establishment of topographically organized connections between cortical hemispheres. Specifically, they studied homotopic connectivity, when cortical neurons project to the same topographic region in the opposite hemisphere versus heterotopic projections, when neocortical neurons project to other regions. They focused on primary and secondary somatosensory areas of the neocortex (S1 and S2). They hypothesized that a well known axon guidance molecule, Neuropilin1, that has a gradient of expression in the neocortex, is a potential controller of homotopic connectivity. They show that manipulation of Neuropilin1 levels in the neocortical neurons would change their target areas in the opposite hemisphere. Using in utero electroporation (IUE) of both Neuropilin1 …
Reviewer #3 (Public Review):
In this paper Martin-Fernandes et al. investigated establishment of topographically organized connections between cortical hemispheres. Specifically, they studied homotopic connectivity, when cortical neurons project to the same topographic region in the opposite hemisphere versus heterotopic projections, when neocortical neurons project to other regions. They focused on primary and secondary somatosensory areas of the neocortex (S1 and S2). They hypothesized that a well known axon guidance molecule, Neuropilin1, that has a gradient of expression in the neocortex, is a potential controller of homotopic connectivity. They show that manipulation of Neuropilin1 levels in the neocortical neurons would change their target areas in the opposite hemisphere. Using in utero electroporation (IUE) of both Neuropilin1 "gain-of-function" and "loss-of-function" genetic constructs into developing brain, the authors find that neurons that downregulate Neuropilin1 become less selective in projecting to homotopic area S1 and target S2 area as well. On the other hand, neurons in the areas S1 and S2 with artificially elevated levels of Neuropilin1 can only project to S1 area of the opposite hemisphere. Interestingly, the data also indicate that this selectivity is achieved mostly at postnatal stages due to selective elimination of axons that project to heterotopic areas. These data support a model of cortical area map establishment that is based on gradients of expression of certain signaling molecules.
Strengths:
The authors used technically challenging combination of in utero electroporation of precise cortical regions with axonal labelling. This approach allowed genetic manipulation of local neurons in restricted neocortical regions. The conclusions of this paper are mostly well supported by data.
Weaknesses:
In utero electroporation as well as other in vivo gene manipulation techniques do not allow fine manipulations of expression gradients. Therefore some conclusions of the paper are not fully supported. Although the data presented in the paper clearly show that Nuropilin1 expression level is important for establishment of homotopic connections, it does not show directly that the gradient of expression indeed is in play, as suggested by the authors. Another week point is that there is no direct evidence that the Neuropilin1 protein level follows the mRNA expression gradient.
Therefore it remains an open question, whether it is a gradient of expression or a sharp border of cellular response to higher-lower levels of Neuropilin1 that controls area specific connections within somatosensory cortex.
Another week point is that the paper relies on in utero electroporation solely. This technique with all its advantages, has some disadvantages too. One of them is high variability of individual experiments. On the other hand, it targets only subsets of cells, and therefore is not the best to address cell extrinsic mechanisms, especially those that involve expression gradients.
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