The ciliary kinesin KIF7 controls the development of the cerebral cortex by acting differentially on SHH-signaling in dorsal and ventral forebrain

  1. María Pedraza
  2. Valentina Grampa
  3. Sophie Scotto-Lomassese
  4. Julien Puech
  5. Aude Muzerelle
  6. Azka Mohammad
  7. Sophie Lebon
  8. Nicolas Renier
  9. Christine Métin
  10. Justine Masson

  • Curated by eLife

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    This important study provides convincing evidence that the Kinesin protein family member KIF7 regulates the development of the cerebral cortex and its connectivity and the specificity of Sonic Hedgehog signaling by controlling the details of Gli repressor vs activator functions. This study provides important new insights into general aspects of cortical development.

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Abstract

Mutations of KIF7 , a key ciliary component of Sonic hedgehog (SHH) pathway, are associated in humans with cerebral cortex malformations and clinical features suggestive of cortical dysfunction. KIF7 regulates the processing of GLI-A and GLI3-R transcription factors in a SHH-dependent manner both in humans and mice. Here, we examine the embryonic cortex development of a mouse model that lacks the expression of KIF7 ( Kif7 -/-) . The cortex is composed of principal neurons generated locally in the dorsal telencephalon where SHH expression is low and inhibitory interneurons (cIN) generated in the ventral telencephalon where SHH expression is high. We observe a strong impact of Kif7 deletion on the dorsal cortex development whose abnormalities resemble those of GLI3-R mutants: subplate cells are absent, the intermediate progenitor layer and cortical plate do not segregate properly, and corticofugal axons do not develop timely, leading to a delayed colonization of the telencephalon by thalamo-cortical axons. These structural defects alter the cortical distribution of cIN, which moreover exhibit intrinsic migration defects and cortical trajectories resembling those of cyclopamine-treated cIN. Our results show that Kif7 deletion impairs the cortex development in multiple ways, exhibiting opposite effects on SHH pathway activity in the developing principal neurons and inhibitory interneurons.

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  1. eLife

    eLife Assessment

    This important study provides convincing evidence that the Kinesin protein family member KIF7 regulates the development of the cerebral cortex and its connectivity and the specificity of Sonic Hedgehog signaling by controlling the details of Gli repressor vs activator functions. This study provides important new insights into general aspects of cortical development.

  2. eLife

    Reviewer #1 (Public review):

    Summary:

    This is an interesting follow-up to a paper published in Human Molecular Genetics reporting novel roles in corticogenesis of the Kif7 motor protein that can regulate the activator as well as the repressor functions of the Gli transcription factors in Shh signalling. This new work investigates how a null mutation in the Kif7 gene affects the formation of corticofugal and thalamocortical axon tracts and the migration of cortical interneurons. It demonstrates that Kif7 null mutant embryos present with ventriculomegaly and heterotopias as observed in patients carrying KIF7 mutations. The Kif7 mutation also disrupts the connectivity between cortex and thalamus and leads to an abnormal projection of thalamocortical axons. Moreover, cortical interneurons show migratory defects that are mirrored in cortical slices treated with the Shh inhibitor cyclopamine suggesting that the Kif7 mutation results in a down-regulation of Shh signalling. Interestingly, these defects are much less severe at later stages of corticogenesis.

    Strengths/weaknesses:

    The findings of this manuscript are clearly presented and are based on detailed analyses. Using a compelling set of experiments, especially the live imaging to monitor interneuron migration, the authors convincingly investigate Kif7's roles and their results support their major claims. The migratory defects in interneurons and the potential role of Shh signalling present novel findings and provide some mechanistic insights but rescue experiments would further support Kif7's role in interneuron migration. Similarly, the mechanism underlying the misprojection which has previously been reported in other cilia mutants remains unexplored. Taken together, this manuscript makes novel contributions to our understanding of the role of primary cilia in forebrain development and to the aetiology of the neural symptons in ciliopathy patients.

    Comments on revisions:

    The authors addressed most of the points I raised in my original review. However, I am not convinced by the figures the authors present on Shh protein expression. The "bright tiny dots" of Shh protein in the cortex are not visible on the images in Figure 7. I wonder whether the authors could present higher magnification and/or black and white images with increased contrast.

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    This study investigates the role of KIF7, a ciliary kinesin involved in the Sonic Hedgehog (SHH) signaling pathway, in cortical development using Kif7 knockout mice. The researchers examined embryonic cortex development (mainly at E14.5), focusing on structural changes and neuronal migration abnormalities.

    Strengths:

    (1) The phenotype observed is interesting, and the findings provide neurodevelopmental insight into some of the symptoms and malformations seen in patients with KIF7 mutations.

    (2) The authors assess several features of cortical development, including structural changes in layers of the developing cortex, connectivity of the cortex with thalamus, as well as migration of cINs from CGE and MGE to cortex.

    Weaknesses:

    (1) The Kif7 null does have phenotype differences from individual mutations seen in patients. It would be interesting to add more thoughts about how the null differs from these mutants in ciliary structure and SHH signaling via the cilium.

    (2) The description of altered cortex development at E14.5 is perhaps rather descriptive. It would be useful to assess more closely the changes occurring in different cell types and stages. For this it seems very important to have a time course of cortical development and how the structural organization changes over time. This would be easy to assess with the addition of serial sections from the same mice. It might also be interesting to see how SHH signaling is altered in different cortical cell types over time with a SHH signaling reporter mouse.

    (3) Abnormal neurodevelopmental phenotypes have been widely reported in the absence of other key genes affecting primary cilia function (Willaredt et al., J Neurosci 2008; Guo et al., Nat Commun 2015). It would be interesting to have more discussion of how the Kif7 null phenotype compares to some of these other mutants.

    (4) The authors see alterations in cIN migration to the cortex and observe distinct differences in the pattern of expression of Cxcl12 as well as suggest cell intrinsic differences within cIN in their ability to migrate. The slice culture experiments though make it a little difficult to interpret the cell intrinsic effects on cIN of loss of Kif7, as the differences in Cxcl12 patterns still exist presumably in the slice cultures. It would be useful to assess their motility in an assay where they were isolated, as well as assess transcriptional changes in cINs in vivo lacking KIF7 for expression patterns that may affect motility or other aspects of migration.

    Comments on revisions:

    The authors have made significant and thoughtful responses as well as experimental additions to the authors comments. Their efforts are appreciated and the manuscript is much improved.

  4. eLife

    Author response:

    The following is the authors’ response to the original reviews

    Reviewer #1 (Public review):

    Summary:

    This is an interesting follow-up to a paper published in Human Molecular Genetics reporting novel roles in corticogenesis of the Kif7 motor protein that can regulate the activator as well as the repressor functions of the Gli transcription factors in Shh signalling. This new work investigates how a null mutation in the Kif7 gene affects the formation of corticofugal and thalamocortical axon tracts and the migration of cortical interneurons. It demonstrates that the Kif7 null mutant embryos present with ventriculomegaly and heterotopias as observed in patients carrying KIF7 mutations. The Kif7 mutation also disrupts the connectivity between the cortex and thalamus and leads to an abnormal projection of thalamocortical axons. Moreover, cortical interneurons show migratory defects that are mirrored in cortical slices treated with the Shh inhibitor cyclopamine suggesting that the Kif7 mutation results in a down-regulation of Shh signalling. Interestingly, these defects are much less severe at later stages of corticogenesis.

    Strengths/weaknesses:

    The findings of this manuscript are clearly presented and are based on detailed analyses. Using a compelling set of experiments, especially the live imaging to monitor interneuron migration, the authors convincingly investigate Kif7's roles and their results support their major claims. The migratory defects in interneurons and the potential role of Shh signalling present novel findings and provide some mechanistic insights but rescue experiments would further support Kif7's role in interneuron migration. Similarly, the mechanism underlying the misprojection which has previously been reported in other cilia mutants remains unexplored. Taken together, this manuscript makes novel contributions to our understanding of the role of primary cilia in forebrain development and to the aetiology of neural symptoms in ciliopathy patients.

    We again thank Reviewer 1 for her/his positive assessment of our article. We have addressed several weaknesses identified by the reviewer, supplementing the initial results with new data, and correcting or clarifying the text where necessary. Our detailed responses to the reviewer’s recommendations appear at the end of each comment.

    Reviewer #1 (Recommendations for the authors):

    (1) The authors report remarkable phenotypic changes in E14.5 embryos in the projection patterns of corticofugal/thalamocortical axons and in interneuron migration, but some of those phenotypes appear much less severe at E16.5. This might be indicative of a delay in development. Does the migration of interneurons to more dorsal regions correspond to an extended Cxcl12 expression? Do interneuorons still show migratory defects at E16.5? To address a potential delay, the authors could, if feasible, repeat Tbr2/Tomato and L1 or neurofilament stainings in E18.5 embryos?

    The question of a possible developmental delay in Kif7 -/- embryos is important. To document this topic, we have extended our study initially focused on embryonic stage E14.5 to earlier (E12.5) and later (E16.5, E18.5/P0) developmental stages. We added new data on E12.5 (Fig. 1, Fig. 3, Fig. S4) and E18.5 (Fig. 3, Fig. 4) embryos in the main figures, and considerably extended the data on E16.5 embryos (Fig. 1, Fig. 3). The legends of figures and the text of the result section (p5-p6) have been modified accordingly. We now describe developmental defects in Kif7 -/- embryos, which are not simple developmental delays. The sequences of thalamic axon development and cIN migration are representative of this complexity.

    Thalamic axons: the pioneer projection is misrouted to the amygdala at E14.5 (Fig. 4B) whereas most Kif7 -/- thalamic axons extend to the cortex at E16.5, with a slight delay compared to WT axons (Fig. 4D). At E18.5, the Kif7 -/- thalamo-cortical projection appears rather normal in the rostral forebrain but is drastically reduced in the median and caudal forebrain (Fig. 4E). This strong decrease is confirmed by neurofilament staining performed at E18.5 which identifies a major loss of corticofugal and thalamo-cortical projections in Kif7 -/- brains (Fig. 4F).

    Migrating cIN: During normal development, CXCL12 maintains cIN in their tangential pathways as they start to colonize the cortical wall (E13.5/E14.5). Then CXCL12 drops in the SVZ (Tiveron et al., 2006; Caronia-Brown and Grove, 2011) allowing wild type cIN to invade the cortical plate (Stumm et al., 2003; Li et al., 2008; Atkins et al., 2023). In Kif7 -/- embryos, CXCL12 is never expressed in the SVZ of the dorsal cortex. Therefore Kif7 -/- cIN migrate radially in the dorsal cortex instead of tangentially. We have improved our text in the result section to clarify this transient defect (p8-9).

    (2) Figure 1D: The overview of the Gsh2 and Tbr2 stainings does not allow us to see details of the PSPB. The lines indicating the position of the PSPB are not helpful either. Higher magnifications are required to see whether there are subtle differences at these boundaries as observed for other cilia mutants.

    We thank the reviewer for her/his question that allowed us to identify a mild default of patterning at the PSB, illustrated by high magnification pictures in the Fig. 1D and described in the result section (p5). This subtle defect of PSB patterning is consistent with previous observations in Kif7 -/- embryos (Putoux et al, 2019) and appears milder than the PSB defect in hypomorphic Gli3 Pdn mutants (PSB shifted dorsally and less well defined as illustrated in Kuschel et al, 2003 and Magnani et al., 2010).

    (3) Figure 3: The authors report an interesting mis-projection of thalamocortical axons towards the amygdala. A very similar pattern has been described in Gli3 hypomorphic Pdn mutants (Magnani et al., 2010), in Rfx3, and in Inpp5e null mutant embryos (Magnani et al., 2015). These papers lend further support that this Kif7 phenotype is Gli3 dependent and should be cited in the manuscript. Moreover, the mechanism(s) underlying this mis-projection remain unexplored. Is this phenotype rescued in the previously reported Kif7/ Gli3D699 double mutants? Is there an abnormal expression of axon guidance molecules?

    We deeply thank the reviewer for drawing our attention to the abnormal projection of thalamic axons to the amygdala described in the Gli3 Pdn mutant and in two ciliary mutants, Rfx3 -/- and Lnpp5e -/-. We cite these two papers (Magnani et al., 2010, 2015) in the revised manuscript (p7). In the Gli3 Pdn mutant, transplantation experiments show that a patterning defect of the ventral telencephalon (VT) underlies the mis-projection of the thalamus to the amygdala (Magnani et al, 2010). In the Rfx3 ciliary mutant, two possible mechanisms are proposed: pre-thalamus patterning defect and ectopic Netrin and Slit1 expression in the VT (Magnani et al, 2015). We do agree that understanding the mechanism of the thalamic misprojection in the Kif7 mutant would be of great interest. However, given the complexity of the putative mechanisms described in the Gli3 Pdn and Rfx3 mutants, we believe that this question deserves further investigation in a future study. Finally, the possibility that the thalamic projection defect observed in Kif7 -/- embryos could be rescued in Kif7/Gli3699 (double mutants in which Gli3R is overexpressed in the dorsal and ventral forebrain) is very unlikely. Our two main arguments are:

    (1) Magnani et al (2015) did not rescue the TCA pathfinding defect in the Rfx3 -/- ciliary mutant when they overexpressed GLI3-R (see TCA description in the Rfx3/ Gli3699 double mutant, last paragraph of the result section). The authors concluded “This finding could be explained by a requirement for Gli activator and not Gli repressor function in VT {ventral telencephalon} patterning and indeed, Gli3 western blots showed that the levels of Gli3R are not altered in the VT of Rfx3 -/- embryos”.

    (2) The GLI3-R/Gli3-FL ratio is decreased in the cortex of the Kif7 -/- embryos (dorsal telencephalon) as expected, whereas it is very low in the MGE of WT embryos (ventral telencephalon) and remains unaltered in the Kif7 -/- embryos (Fig. 2B).

    Similarly, the analysis of Kif7 -/- cIN migratory defects leads us to conclude that Kif7 ablation impairs Gli activation function rather than Gli repressor function in the VT where cIN are generated.

    (4) Figure 4: The authors should discuss the difference between Tbr2 and Cxcl12 expression which does not extend into the dorsal-most cortical SVZ.

    We observed that the transient CXCL12 expression is lacking in the SVZ of the dorsal cortex of Kif7 -/- embryos at E14.5, in a region where TBR2 cells abnormally reach the cortical surface and intermingle with post-mitotic cells. A sentence in our previous version (lines 233-234) could suggest a link between the abnormal location of TBR2 expressing cells and the lack of CXCL12 expression. Having found no data in the literature to explain the absence of CXCL12 expression in the brain by an abnormal cellular environment or by a defect in transcription factor expression, we do not want to further elaborate on differences and similarities between TBR2 and CXCL12 expression patterns in the Kif7 -/- brain. We have modified our text accordingly in the result section of the revised manuscript (p8-9).

    (5) Figure 5: The authors convincingly describe migratory defects of interneurons. The treatment with Shh agonist and antagonist provides some mechanistic insights but genetic or pharmacological rescue experiments would lend further support. For example, they could treat Kif7 mutant sections with Shh agonists or analyse Kif7/Gli3D699 double mutants.

    We thank the reviewer for her/his positive assessment of our analysis of the cIN migration. Unfortunately, the rescue experiments proposed by the reviewer should not help to further support our conclusions. First, Kif7 ablation in cIN prevents the processing of any SHH signal in the transcriptional pathway. Second, increasing GLI3R by crossing Kif7 -/- animals with Gli3D699 mice could possibly rescue the alterations of layering in the dorsal cortex where the GLI3R/GLI-FL ratio is strongly decreased and the SHH pathway activated. Such a rescue had been previously described for corpus callosum defects (Putoux et al., 2019). However, because cIN are generated in the ventral forebrain where SHH signaling predominantly activates the formation of GLI-A and where Kif7 ablation does not alter the GLI3 ratio, GLI3R re-introduction in the basal forebrain should rather increase the migratory defects of Kif7 -/- cIN instead of producing a rescue. To further support our conclusion, we analyzed the migratory behavior of Kif7 -/- cIN in a WT cortical environment. The results illustrated in the Fig. 6A and described in page 9 of the result section confirm that the migration defects of Kif7 -/- cIN are reminiscent of an inhibition but not an activation of the transcriptional SHH pathway (same phenotype as in Kif3a ciliary mutants described in Baudoin et al, 2012).

    (6) Figure 6: The authors describe the Shh mRNA and protein expression with relevance to interneuron migration. In contrast to the in situ hybridisation, the immunofluorescence analysis is not very convincing and requires further controls. The authors should at least show a no primary antibody control and, if available, could include a staining on Shh mutants. These additional controls are important as Shh protein expression in the developing cortex is highly controversial and a recent paper describes a different pattern (Manuel et al., 2022: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001563#). Moreover, it remains unclear whether the Shh protein expression is uniform within the cortex or follows lateral to medial or ventricular to pial gradients. A more thorough description and corresponding figures would be helpful.

    Manuel et al. (2022) used the SHH KO (generated by Chiang et al., 1996) that develops a long proboscis to validate the rabbit anti-SHH antibodies (from Genetech) used in their study. They show a lack of SHH signal in the SHH KO. However, it is difficult to identify the cortex in this mouse line and the authors did not specify which part of the SHH protein was used to generate antibodies. We wished to use the SHH KO generated by Chiang and backcrossed on a C57B/6 line (Rash and Grove, 2007) that develops a layered neocortex at E17.5. However,

    (1) the SHH KO was obtained by replacing exon2 with a PGK-neo cassette and could express a 101 aa truncated protein comprising the N-ter part of the protein, and

    (2) the antibody we used, is a polyclonal N-ter antibody that targets the active SHH protein (Cys25-Gly198 part of SHH protein used as immunogen to produce the antibody). We thus thought that this labeling experiment will not give information on the specificity of the antibody, some epitopes being able to recognize the truncated protein produced in the SHH KO.

    To overcome the lack of a good mutant mice to validate the SHH N-ter antibodies, we analyzed the SHH immunostaining pattern at E12.5 and compared the expression profile with previously published SHH mRNA expression patterns. The border of the third ventricle and the ZLI were strongly immunostained by SHH-Nter antibodies and these regions were shown to express SHH mRNA at E12.5-E13.5 (Kicker et al. 2004, Loulier et al., 2005, Sahara et al., 2007 and Fig. 7B1). In brain sections at E14.5, only the choroid plexus was strongly labeled and some structures showed diffused labeling. We analyzed the distribution of SHH mRNAs in the cortex using a highly sensitive technique (RNAscop) at E14.5 and showed that very few cortical cells expressed SHH mRNA and at very low level. Anti-SHH-Nter antibodies immunostained numerous bright dots throughout the cortical neuropile, which is not surprising for a diffusible factor like SHH. However, the labeling was not homogeneous and showed a ventricle to pial gradient at E12.5 and aligned distributions in the different cortical layers at E14.5. We have described the expression pattern in more detail and modified the Fig. S4 by adding an image of immunostaining performed without SHH N-ter antibody.

    (7) Figure S1: The Gli3 Western blot needs to be quantified. As the authors only show one control and one mutant sample, it remains unclear how representative this blot is. In addition to Gli3R and Gli3FL, the authors should also determine the ratio of both isoforms. Are there also differences in the MGE?

    We now produce results of Gli3 western blots in the cortex and MGE of several E14.5 Kif7 KO (n=4) and WT (n=4) embryos. The GLI3R/GLI3FL ratio has been determined in the cortex and in the MGE of WT and mutant embryos. Results are illustrated in the Fig. 2.

    Minor points:

    The authors should carefully amend the literature on Gli genes and forebrain development. For example:

    (1) Line 85: Add Hasenpusch-Theil et al., 2018.

    We added this reference.

    (2) Line 141: Remove Magnani et al., 2010 (they characterized hypomorphic Gli3 Pdn mutants) and replace with Kuschel et al., 2003.

    Since our revised figure 2 illustrates GLI3 western blots and compare GLI3R/GLI3FL ratios in the cortex and MGE of WT and Kif7-/- embryos, we no longer cite these papers in the result section.

    (3) Line 380: Replace reference with Theil, 2005.

    We have replaced Magnani et al, 2014 by Theil 2005 in the sentence.

    (4) Line 414: Rallu et al is not an appropriate reference for this as this manuscript does not investigate the expression of a single cortical marker in Shh/Gli3 double mutants.

    We removed the reference Rallu et al. in the sentence.

    (5) Reference in line 355: do not use Vancouver style.

    We apologize for the mistake that was corrected.

    (6) Spelling: Line 447 it should read "choroid plexus"

    We again apologize for the mistake that has been corrected.

    Reviewer #2 (Public review):

    Summary:

    This study investigates the role of KIF7, a ciliary kinesin involved in the Sonic Hedgehog (SHH) signaling pathway, in cortical development using Kif7 knockout mice. The researchers examined embryonic cortex development (mainly at E14.5), focusing on structural changes and neuronal migration abnormalities.

    Strengths:

    (1) The phenotype observed is interesting, and the findings provide neurodevelopmental insight into some of the symptoms and malformations seen in patients with KIF7 mutations.
    (2) The authors assess several features of cortical development, including structural changes in layers of the developing cortex, connectivity of the cortex with the thalamus, as well as migration of cINs from CGE and MGE to the cortex.

    We greatly thank Reviewer 2 for her/his positive assessment of our work that characterize the neurodevelopmental defects induced by KIF7 ablation. We have deeply reorganized and implemented data in the figures to show changes occurring in different cortical cell types and at different stages. We have moreover corrected and clarified the text where necessary. Our detailed responses to the reviewer’s recommendations appear at the end of each comment.

    Weaknesses:

    (1) The Kif7 null does have phenotype differences from individual mutations seen in patients. It would be interesting to add more thoughts about how the null differs from these mutants in ciliary structure and SHH signaling via the cilium.

    We are grateful to the Reviewer for recalling that Kif7 ablation alters SHH signaling within primary cilium and has a strong effect on ciliary structure. In the revised version of the manuscript, we discuss data from the literature that describe these alterations in human (Putoux et al, 2011) and in murine KIF7 depleted cells (He et al, 2015; Cheung et al., 2009; Lai et al., 2021) (discussion p13).

    (2) The description of altered cortex development at E14.5 is perhaps rather descriptive. It would be useful to assess more closely the changes occurring in different cell types and stages. For this it seems very important to have a time course of cortical development and how the structural organization changes over time. This would be easy to assess with the addition of serial sections from the same. It might also be interesting to see how SHH signaling is altered in different cortical cell types over time with a SHH signaling reporter mouse.

    We thank the Reviewer for her/his request that helped us to improve our description of developmental defaults in the Kif7 -/- cortex. In the revised manuscript, we have expanded our study initially focused on embryonic stage E14.5 to earlier (E12.5) and later (E16.5, E18.5 /P0) developmental stages. Instead of focusing on median forebrain sections, we have expanded our observations to rostral and caudal sections. Altogether, these new observations allow us to describe more precisely the complex developmental defects in the Kif7 -/- cortex over time, in specific cortical regions (dorsal versus lateral cortex, and rostral versus caudal levels). Figures 1, 3, 4, and S4 have been deeply edited to present new data on E12.5 (Fig. 1, Fig. 3, Fig. S4), E16.5 (Fig. 1, Fig. 3) and E18.5 (Fig. 3, Fig. 4) embryos. We have modified the legends and text in the result section (p5-6) accordingly. We agree with the Reviewer that deciphering how SHH signaling is altered in the different cortical cells over time should be highly interesting and relevant. Nevertheless, we anticipate complex analyses and consider that they should be retained for future studies.

    (3) Abnormal neurodevelopmental phenotypes have been widely reported in the absence of other key genes affecting primary cilia function (Willaredt et al., J Neurosci 2008; Guo et al., Nat Commun 2015). It would be interesting to have more discussion of how the Kif7 null phenotype compares to some of these other mutants.

    We agree with this Reviewer concern. In the revised manuscript, we discuss our results with regard to previous observations in other ciliary mutants. The murine cobblestone mutant described in Willaredt et al. (2008) indeed shows defects similar to those we describe in the Kif7 -/- mouse. We thank again the Reviewer for her/his helpful comment that allowed us to strengthen and better interpret our results. Guo et al (2015) did not conduct a study of ciliary mutants. Nevertheless, their characterization of cortical developmental defects following invalidation of genes involved in human ciliopathies identified cell autonomous defects in cortical progenitors and in differentiating cortical neurons, which corroborate our observations (p.15)

    (4) The authors see alterations in cIN migration to the cortex and observe distinct differences in the pattern of expression of Cxcl12 as well as suggest cell-intrinsic differences within cIN in their ability to migrate. The slice culture experiments though make it a little difficult to interpret the cell intrinsic effects on cIN of loss of Kif7, as the differences in Cxcl12 patterns still exist presumably in the slice cultures. It would be useful to assess their motility in an assay where they were isolated, as well as assess transcriptional changes in cINs in vivo lacking KIF7 for expression patterns that may affect motility or other aspects of migration.

    To circumvent the difference in the expression profile of CXCL12 in the dorsal cortex of WT and Kif7 -/- embryos on the migratory behavior of cIN, we compared the trajectories and dynamics of WT and Kif7 -/- cIN imaged in the lateral cortex where CXCL12 expression appears similar in WT and Kif7 -/- brains.

    We moreover followed the reviewer recommendation and analyzed the migratory behavior of Kif7 -/- cIN that migrate as isolated cells on a dissociated substrate of WT cortical cells. We sincerely thank the reviewer for her/his suggestion as the results revealed an interesting and relevant ciliary phenotype in migrating Kif7 -/- cIN. This additional experiment confirms that Kif7 -/- cIN exhibit the same migratory defects as those initially characterized in the Kif3a -/- ciliary mutant. The new results are illustrated in the Fig. 6A and described in the result section (p9). We agree with the reviewer that the analysis of transcriptional changes that could affect Kif7 -/- cIN motility and migration would be very interesting to study, but this study is beyond the scope of the present article.

  5. Version published to 10.1101/2024.03.21.586159v4 on bioRxiv
  6. Version published to 10.1101/2024.03.21.586159v3 on bioRxiv
  7. eLife

    eLife Assessment

    This study provides convincing evidence that the Kinesin protein family member KIF7 regulates the development of the cerebral cortex and its connectivity and the specificity of Sonic Hedgehog signaling by controlling the details of Gli repressor vs activator functions. This study provides important new insights into general aspects of cortical development.

  8. eLife

    Reviewer #1 (Public review):

    Summary:

    This is an interesting follow-up to a paper published in Human Molecular Genetics reporting novel roles in corticogenesis of the Kif7 motor protein that can regulate the activator as well as the repressor functions of the Gli transcription factors in Shh signalling. This new work investigates how a null mutation in the Kif7 gene affects the formation of corticofugal and thalamocortical axon tracts and the migration of cortical interneurons. It demonstrates that the Kif7 null mutant embryos present with ventriculomegaly and heterotopias as observed in patients carrying KIF7 mutations. The Kif7 mutation also disrupts the connectivity between the cortex and thalamus and leads to an abnormal projection of thalamocortical axons. Moreover, cortical interneurons show migratory defects that are mirrored in cortical slices treated with the Shh inhibitor cyclopamine suggesting that the Kif7 mutation results in a down-regulation of Shh signalling. Interestingly, these defects are much less severe at later stages of corticogenesis.

    Strengths/weaknesses:

    The findings of this manuscript are clearly presented and are based on detailed analyses. Using a compelling set of experiments, especially the live imaging to monitor interneuron migration, the authors convincingly investigate Kif7's roles and their results support their major claims. The migratory defects in interneurons and the potential role of Shh signalling present novel findings and provide some mechanistic insights but rescue experiments would further support Kif7's role in interneuron migration. Similarly, the mechanism underlying the misprojection which has previously been reported in other cilia mutants remains unexplored. Taken together, this manuscript makes novel contributions to our understanding of the role of primary cilia in forebrain development and to the aetiology of neural symptoms in ciliopathy patients.

  9. eLife

    Reviewer #2 (Public review):

    Summary:

    This study investigates the role of KIF7, a ciliary kinesin involved in the Sonic Hedgehog (SHH) signaling pathway, in cortical development using Kif7 knockout mice. The researchers examined embryonic cortex development (mainly at E14.5), focusing on structural changes and neuronal migration abnormalities.

    Strengths:

    (1) The phenotype observed is interesting, and the findings provide neurodevelopmental insight into some of the symptoms and malformations seen in patients with KIF7 mutations.

    (2) The authors assess several features of cortical development, including structural changes in layers of the developing cortex, connectivity of the cortex with the thalamus, as well as migration of cINs from CGE and MGE to the cortex.

    Weaknesses:

    (1) The Kif7 null does have phenotype differences from individual mutations seen in patients. It would be interesting to add more thoughts about how the null differs from these mutants in ciliary structure and SHH signaling via the cilium.

    (2) The description of altered cortex development at E14.5 is perhaps rather descriptive. It would be useful to assess more closely the changes occurring in different cell types and stages. For this it seems very important to have a time course of cortical development and how the structural organization changes over time. This would be easy to assess with the addition of serial sections from the same mice. It might also be interesting to see how SHH signaling is altered in different cortical cell types over time with a SHH signaling reporter mouse.

    (3) Abnormal neurodevelopmental phenotypes have been widely reported in the absence of other key genes affecting primary cilia function (Willaredt et al., J Neurosci 2008; Guo et al., Nat Commun 2015). It would be interesting to have more discussion of how the Kif7 null phenotype compares to some of these other mutants.

    (4) The authors see alterations in cIN migration to the cortex and observe distinct differences in the pattern of expression of Cxcl12 as well as suggest cell-intrinsic differences within cIN in their ability to migrate. The slice culture experiments though make it a little difficult to interpret the cell intrinsic effects on cIN of loss of Kif7, as the differences in Cxcl12 patterns still exist presumably in the slice cultures. It would be useful to assess their motility in an assay where they were isolated, as well as assess transcriptional changes in cINs in vivo lacking KIF7 for expression patterns that may affect motility or other aspects of migration.

  10. Version published to 10.7554/elife.100328.1 on eLife
  11. Version published to 10.7554/elife.100328 on eLife
  12. Version published to 10.1101/2024.03.21.586159v2 on bioRxiv
  13. Version published to 10.1101/2024.03.21.586159v1 on bioRxiv