DRAXIN regulates interhemispheric fissure remodelling to influence the extent of corpus callosum formation
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Summary: Your manuscript is an excellent account of the cellular and genetic mechanisms involved in the diversity of corpus callosum dysgenesis (CCD) phenotypes in humans and in a mouse model. Your work over the years has revealed that interhemispheric fissure (IHF) fusion is critical for proper formation of the callosum and its failure is the main cause of complete CCD. Here you nicely show that the extent of aberrant interhemispheric fissure (IHF) remodeling does in fact correlate with commissure dysgenesis severity, in inbred and outcrossed BTBR mouse strains, as well as in humans with partial CCD. The phenotypes in the mouse are very similar to what is found in humans, and also variable, perhaps related to stochasticity on the mechanisms involved, or to the dependency on other allelic variants.
You also identify an eight base pair deletion in Draxin and misregulated astroglial and leptomeningeal proliferation as genetic and cellular factors for variable IHF remodelling and CCD in BTBR acallosal strains. The Draxin mutations interrupt the normal remodeling (closing) of interhemispheric fissure necessary for callosal axons to cross. Your study thus places the focus on midline cellular populations and away from axonal navigation as the main source of corpus callosum dysgenesis. The findings are important to understand what mutations cause CCD in humans and how, mechanistically, it occurs.
This manuscript was co-submitted with https://www.biorxiv.org/content/10.1101/2020.08.03.233593v1
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Abstract
Corpus callosum dysgenesis (CCD) is a congenital disorder that incorporates either partial or complete absence of the largest cerebral commissure. Remodelling of the interhemispheric fissure (IHF) provides a substrate for callosal axons to cross between hemispheres, and its failure is the main cause of complete CCD. However, it is unclear whether defects in this process could give rise to the heterogeneity of expressivity and phenotypes seen in human cases of CCD. We identify incomplete IHF remodelling as the key structural correlate for the range of callosal abnormalities in inbred and outcrossed BTBR mouse strains, as well as in humans with partial CCD. We identify an eight base pair deletion in Draxin and misregulated astroglial and leptomeningeal proliferation as genetic and cellular factors for variable IHF remodelling and CCD in BTBR acallosal strains. These findings support a model where genetic events determine corpus callosum structure by influencing leptomeningeal-astroglial interactions at the IHF.
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Reviewer #3:
The manuscript by Morcom et al., describes mechanisms of Corpus callosum Diysgenesis in mice and how they relate to humans. It will be of interest to the field. It explains the spectrums of disorders of the corpus callosum in humans. It is an important study that sets the focus on midline populations and away from axonal navigation as the main source of corpus callosum dysgenesis.
The authors found that a mutation in Draxin carried by certain mouse strains is responsible for the heterogenicity of corpus callosum phenotypes found in these mice. Draxin mutations interrupt the normal remodeling (closing) of interhemispheric fissure necessary for callosal axons to cross. The phenotypes in the mouse are very similar to what is found in humans, and also variable, perhaps related to stochasticity on the mechanisms involved, or to …
Reviewer #3:
The manuscript by Morcom et al., describes mechanisms of Corpus callosum Diysgenesis in mice and how they relate to humans. It will be of interest to the field. It explains the spectrums of disorders of the corpus callosum in humans. It is an important study that sets the focus on midline populations and away from axonal navigation as the main source of corpus callosum dysgenesis.
The authors found that a mutation in Draxin carried by certain mouse strains is responsible for the heterogenicity of corpus callosum phenotypes found in these mice. Draxin mutations interrupt the normal remodeling (closing) of interhemispheric fissure necessary for callosal axons to cross. The phenotypes in the mouse are very similar to what is found in humans, and also variable, perhaps related to stochasticity on the mechanisms involved, or to the dependency on other allelic variants. The findings are important to understand what mutations cause CCD in humans and how, mechanistically, it occurs. The authors found that Draxin mutation misregulates astroglial and leptomeningeal proliferation. Mechanistically, how this more precisely affects interhemispheric remodeling is still unclear. This is a point that may reinforce the work.
Major concerns:
- The authors have done an excellent job identifying the mutation and characterizing and comparing in detail the phenotypes in mice and humans. They also provide very interesting hints about how Draxin regulates the remodeling of the interhemispheric fissure. But mechanistically, their findings only offer an incomplete view. In my opinion, the findings would be reinforced by a deeper digging into how, cellularly or molecularly, Draxin makes glial and leptomeningeal cells remodel the interhemispheric fissure. Proliferation by itself does not seem to explain the phenotypes. It is not fully clear the model that they are proposing. Does it affect cell-cell adhesion, cell-cell signaling, membrane processes, metalloproteinase activity? Perhaps they could characterize some more the morphology and junctions of the affected cells or perform some studies in acute models or in vitro.
Minor comments:
Fig 4C-the expression patterns of mRNA Draxin in C57 or BTBR does not seem so similar as it is mentioned in the description of the results.
Fig 4D-The full versión of western-blots shown in supplementary showing all forms is more informative than the cuts shown in principal Figure. Please indicate molecular weights.
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Reviewer #2:
This is an interesting study that provides convincing evidence that a Draxin mutation underpins forebrain commissure phenotypes in BTBR mice and crosses.
The use of BTBR x C57 N2 crosses where commissure phenotype is correlated with the Draxin mutation (Figure 5) is a nice illustration of unpicking variable penetrance. The phenocopy of the BTBR/c57 phenotype to Draxin mutants is a nice confirmatory experiment.
Further, analysis of midline fusion shows that problems in MZG proliferation and hemisphere fusion are prevalent in BTBR mice supporting the hypothesis that Draxin is needed for midline fusion.
MRI scans of human subjects with a spectrum of CC abnormalities show that commissure abnormalities correlate with midline fusion defects.
Major comments.
As a central contention of this study is that variable penetrance of the …
Reviewer #2:
This is an interesting study that provides convincing evidence that a Draxin mutation underpins forebrain commissure phenotypes in BTBR mice and crosses.
The use of BTBR x C57 N2 crosses where commissure phenotype is correlated with the Draxin mutation (Figure 5) is a nice illustration of unpicking variable penetrance. The phenocopy of the BTBR/c57 phenotype to Draxin mutants is a nice confirmatory experiment.
Further, analysis of midline fusion shows that problems in MZG proliferation and hemisphere fusion are prevalent in BTBR mice supporting the hypothesis that Draxin is needed for midline fusion.
MRI scans of human subjects with a spectrum of CC abnormalities show that commissure abnormalities correlate with midline fusion defects.
Major comments.
As a central contention of this study is that variable penetrance of the commissure phenotypes in the BTBR x C57 mice stems from an earlier midline fusion phenotype is would have been useful to see if the (embryonic) midline fusion phenotype also showed the same partial penetrance in BTBR x C57 mice, perhaps also correlated with the WT/MUT Draxin alleles (as in Figure 5). This would be a testable prediction of the hypothesis that midline fusion (and not something else) mediates the Draxin phenotype.
I am not sure the human data adds substantially to the paper as it is not related to Draxin mutations. It is already well known that corpus callosum phenotypes are variable in humans (and mice).
Minor comments:
Some of the data are not normally distributed (particularly clear for pink data points in Fig 5a,e,i,m) so it is not appropriate to show standard errors (the SEM bars could simply be removed), a non-parametic Kruskal-Wallis ANOVA has been used which is appropriate.
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Reviewer #1:
This is an interesting translational and comprehensive study which examines cellular and genetic mechanisms involved in the diversity of corpus callosum dysgenesis (CCD) phenotypes. Using mouse models and human cohorts with a spectrum CCD, it is found that the extent of aberrant interhemispheric fissure (IHF) remodeling predicts commissure dysgenesis severity. Elegant neuroanatomical experiments show that abnormal proliferation/migration of midline zipper glia (MZG) progenitors underlies aberrant IHF remodeling. Thus, in addition to genetic perturbations linked to aberrant callosal axon guidance in humans and mice (i.e. variants in DCC guidance cue receptor gene), disruption to IHF remodeling also causes CCD. Indeed, an 8-base pair deletion in the DCC receptor ligand, Draxin, which is expressed in MZG, associates with CC …
Reviewer #1:
This is an interesting translational and comprehensive study which examines cellular and genetic mechanisms involved in the diversity of corpus callosum dysgenesis (CCD) phenotypes. Using mouse models and human cohorts with a spectrum CCD, it is found that the extent of aberrant interhemispheric fissure (IHF) remodeling predicts commissure dysgenesis severity. Elegant neuroanatomical experiments show that abnormal proliferation/migration of midline zipper glia (MZG) progenitors underlies aberrant IHF remodeling. Thus, in addition to genetic perturbations linked to aberrant callosal axon guidance in humans and mice (i.e. variants in DCC guidance cue receptor gene), disruption to IHF remodeling also causes CCD. Indeed, an 8-base pair deletion in the DCC receptor ligand, Draxin, which is expressed in MZG, associates with CC malformations in mice. The findings are novel and important to both basic and clinical scientists.
Below are comments and suggestions that need to be addressed:
- Introduction:
-More detailed information about the BTBR mouse line and the rationale for using the BTBR x C57 mouse cross should be provided.
-The main question addressed in the study should be clearly stated.
- Methods:
The Statistical analysis section needs to provide a more detailed description of the statistical tests that were used and the reason why these tests were chosen.
- Results:
In general, the description of the statistical results lacks important details. For example:
-For figure 1, there is very little information about statistical analysis. For figure 1 C, it needs to be explained why a Welsh test was used instead of a one-way ANOVA. The errors on the bars do not seem to correspond to SEM, this needs to be clarified.
-For figures 3 G and H, if the data are presented in single graphs, it is not clear why unpaired t tests or Mann-Whitney tests were conducted (instead of ANOVAs). Why a non-parametric test was used is not explained.
-The description of the findings that prompted the authors to investigate the role of Draxin in CCD needs to be clearer.
-The references to the different panels of Figures 5 and 4 need to be revised in the Results section.
-It is not clear what is the impact of the Draxin deletion to IHF remodeling. There seems to be an effect shown in one of the supplementary figures (in BTRB mice), but there is no discussion in this regard. This is particularly important considering that Draxin is expressed by MZG.
-It seems that the Draxin deletion does not affect HC formation. However, at some point in the Results section it is stated "To investigate how DRAXIN regulates CC and HC formation...". This is confusing. It seems that the effect varies between BTRB mice and the BTRB x C57 cross, but this is not discussed clearly.
-Figure 7 should indicate the mouse genotype on the actual figure to avoid confusion.
-The study by Vosberg et al, 2019 in Annals in Neurology needs to be included when referring to studies linking DCC variance and CC dysgenesis in humans.
Minor Comments:
The organization of the manuscript could be improved to increase its clarity. The authors may want to consider moving the Draxin findings to the last part of the Results.
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Summary: Your manuscript is an excellent account of the cellular and genetic mechanisms involved in the diversity of corpus callosum dysgenesis (CCD) phenotypes in humans and in a mouse model. Your work over the years has revealed that interhemispheric fissure (IHF) fusion is critical for proper formation of the callosum and its failure is the main cause of complete CCD. Here you nicely show that the extent of aberrant interhemispheric fissure (IHF) remodeling does in fact correlate with commissure dysgenesis severity, in inbred and outcrossed BTBR mouse strains, as well as in humans with partial CCD. The phenotypes in the mouse are very similar to what is found in humans, and also variable, perhaps related to stochasticity on the mechanisms involved, or to the dependency on other allelic variants.
You also identify an eight base pair …
Summary: Your manuscript is an excellent account of the cellular and genetic mechanisms involved in the diversity of corpus callosum dysgenesis (CCD) phenotypes in humans and in a mouse model. Your work over the years has revealed that interhemispheric fissure (IHF) fusion is critical for proper formation of the callosum and its failure is the main cause of complete CCD. Here you nicely show that the extent of aberrant interhemispheric fissure (IHF) remodeling does in fact correlate with commissure dysgenesis severity, in inbred and outcrossed BTBR mouse strains, as well as in humans with partial CCD. The phenotypes in the mouse are very similar to what is found in humans, and also variable, perhaps related to stochasticity on the mechanisms involved, or to the dependency on other allelic variants.
You also identify an eight base pair deletion in Draxin and misregulated astroglial and leptomeningeal proliferation as genetic and cellular factors for variable IHF remodelling and CCD in BTBR acallosal strains. The Draxin mutations interrupt the normal remodeling (closing) of interhemispheric fissure necessary for callosal axons to cross. Your study thus places the focus on midline cellular populations and away from axonal navigation as the main source of corpus callosum dysgenesis. The findings are important to understand what mutations cause CCD in humans and how, mechanistically, it occurs.
This manuscript was co-submitted with https://www.biorxiv.org/content/10.1101/2020.08.03.233593v1
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