DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation
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Summary: This study is a welcome follow-up to your earlier demonstration that midline zipper glia (MZG) migrate along the interhemispheric fissure (IHF) and intercalate across the hemispheres, and in doing so, remodel the meningeal basement membrane to provide a substrate for callosal axon growth. The authors identify DCC and its ligand Netrin1 to be important for this process, by acting on the distribution and morphology of MZG, in addition to their service as axon guidance signals for callosal axons to be attracted to and across the midline.
Co-submission with https://www.biorxiv.org/content/10.1101/2020.07.29.227827v1
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Abstract
The forebrain hemispheres are predominantly separated during embryogenesis by the interhemispheric fissure (IHF). Radial astroglia remodel the IHF to form a continuous substrate between the hemispheres for midline crossing of the corpus callosum (CC) and hippocampal commissure (HC). Deleted in colorectal carcinoma (DCC) and netrin 1 (NTN1) are molecules that have an evolutionarily conserved function in commissural axon guidance. The CC and HC are absent in Dcc and Ntn1 knockout mice, while other commissures are only partially affected, suggesting an additional aetiology in forebrain commissure formation. Here, we find that these molecules play a critical role in regulating astroglial development and IHF remodelling during CC and HC formation. Human subjects with DCC mutations display disrupted IHF remodelling associated with CC and HC malformations. Thus, axon guidance molecules such as DCC and NTN1 first regulate the formation of a midline substrate for dorsal commissures prior to their role in regulating axonal growth and guidance across it.
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Reviewer #3:
This is a very thorough study giving new insight into a non-cell autonomous mechanism for DCC in axon guidance in midline fusion important for corpus callosum axon guidance.
I have no substantive concerns.
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Reviewer #2:
This paper is the second in a series of landmark studies from the Richards lab that re-assess the molecular and cellular mechanisms that permit the corpus callosum (CC) to cross the interhemispheric midline in the telencephalon. The Richards lab previously showed key role for a specialized population of fetal astrocytes, the midline zipper glia (MZG), establishing this substrate when the MZG migrate into the interhemispheric fissure (IHF), intercalate with one another and degrade the intervening leptomeninges. In this manuscript, the authors now assess the requirement for the Ntn1/Dcc pathway in remodeling the IHF. In an elegant series of experiments, they show that Ntn1/Dcc regulate the migration pathway of the MZG, potentially by directly controlling cytoskeletal dynamics. This mechanism is conserved between humans and …
Reviewer #2:
This paper is the second in a series of landmark studies from the Richards lab that re-assess the molecular and cellular mechanisms that permit the corpus callosum (CC) to cross the interhemispheric midline in the telencephalon. The Richards lab previously showed key role for a specialized population of fetal astrocytes, the midline zipper glia (MZG), establishing this substrate when the MZG migrate into the interhemispheric fissure (IHF), intercalate with one another and degrade the intervening leptomeninges. In this manuscript, the authors now assess the requirement for the Ntn1/Dcc pathway in remodeling the IHF. In an elegant series of experiments, they show that Ntn1/Dcc regulate the migration pathway of the MZG, potentially by directly controlling cytoskeletal dynamics. This mechanism is conserved between humans and rodents; the authors show that Dcc mutations that cause CC dysgenesis in humans, cause striking changes in the morphology of astroglial-like cells, consistent with the regulation of MZG migration. Thus, Dcc appears to have two roles first, remodeling the MGZ and then guiding CC axons towards the telencephalic midline. Together, these studies continue the overgoing re-evaluation of the role of netrin1/Dcc in establishing neural circuitry, and shed further understanding on a fascinating and beautiful piece of biology.
This is a very beautiful manuscript, the authors are to be congratulated for the very high quality of their images, and detailed quantifications. Would that all studies were so thorough! These studies will be of great interest to the developmental neuroscience research and clinical communities.
Major comments
The authors should be congratulated by including what was clearly a difficult conditional analysis to assess whether Dcc is required in the callosal axons, or in the MZG radial fibers. This analysis was confounded a) by the low efficiency of the shRNA to knock down Dcc and b) the mosaic nature of Emx::cre line, which appears to be variably expressing cre in both callosal neurons and MZG, given that TDT/Dcc are present in both axons (Fig 5B), and the MZG (Fig 5O) in the less severely affected animals.
As currently presented, however, the analysis (sadly) does not greatly add to the paper, since technical issues beyond the authors' control, have made it difficult to assess specifically where Dcc is required with much confidence. Would the authors could consider removing the shRNA approach from the manuscript, and re-focusing the cKO data on a description of a Dcc phenotypic series? This analysis might fit better with the initial description of lack of interhemispheric remodeling observed in Dcc/Ntn mutant mice, and how they relate to (variable?) phenotypes observed in patients.
Minor Points:
Fig 3C, D. The failure of the MZG radial fibers to extend along the IHF in Dcc mutant at E15 is very striking, and well described in the text. However, there appears to be an additional more punctate Glast/Nestin signal immediately above the radial fibers in IHF in the E15 mutants, what is that?
Fig 4E. Could the increased numbers of migrating MGZ cells seen on the surface of the IHF in E16 Dcc mutants be because there is no "stop" signal created when the IHF is remodeled?
Fig 5B. The failure of the GFAP cells to move away from the third ventricle in Dcc mutants seems profound in both the figures and the quantification. Can the authors elaborate more on why the 0-400 um measurement doesn't rise to being significant in the Dcckanga mutants? Perhaps spell out (p=0.0?) where the trend lies on Fig 5B. ?
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Reviewer #1:
In this manuscript, the authors revisit DCC and NTN1 mutants in order to better define the basis for midline crossing defects. This group recently demonstrated that midline zipper glia (MZG) must migrate along the interhemispheric fissure (IHF) and intercalate across the midline while remodeling the meningeal basement membrane to provide a substrate for callosal axons to cross the midline. In this study, they show that DCC and its ligand NTN1 are required for proper midline zipper glia (MZG) distribution/morphology along the IHF, proper remodeling of the basement membrane, and subsequent corpus callosum (CC) formation. The data in figures 2 and 3 generally do a nice job of supporting the model that DCC and NTN1 are expressed in MZG and that the morphology and distribution of MZG are affected in DCC/NTN1 mutants. There …
Reviewer #1:
In this manuscript, the authors revisit DCC and NTN1 mutants in order to better define the basis for midline crossing defects. This group recently demonstrated that midline zipper glia (MZG) must migrate along the interhemispheric fissure (IHF) and intercalate across the midline while remodeling the meningeal basement membrane to provide a substrate for callosal axons to cross the midline. In this study, they show that DCC and its ligand NTN1 are required for proper midline zipper glia (MZG) distribution/morphology along the IHF, proper remodeling of the basement membrane, and subsequent corpus callosum (CC) formation. The data in figures 2 and 3 generally do a nice job of supporting the model that DCC and NTN1 are expressed in MZG and that the morphology and distribution of MZG are affected in DCC/NTN1 mutants. There appear to be some defects in MZG migration that may account for this (Figure 4). Due to technical limitations, the author's attempt to use a conditional knockout of DCC to genetically dissect whether CC formation defects are due to defects in MZG or callosal axons are a bit inconclusive (Figure 6). Finally, the paper ends with experiments showing that mutations in DCC identified in acallosal patients are loss-of-function using an in vitro cell morphology assay (Figure 7 and 8).
The authors are commended for the quality of their imaging data and for being as quantitative as possible when measuring their in vivo phenotypes, which is not often done with these types of studies. There are few issues that need to be addressed.
Major points:
In Figure 4, in addition to the migration defects of Sox9+ MZG, there seems to be a rather large increase in the total number of Sox9+ cells along the IHF by E16 (more than 2 fold, Figure 4G). The authors show there is no change in cell cycle or apoptosis of these cells in the supplemental data (Figure S4), so what accounts for this increase? Is this also seen with NFIA/B staining at E16?
Regarding the attempt to distinguish between DCC in MZG versus callosal axons (Figure 6), the incomplete deletion/loss of DCC protein (Figures 6C, I, J) is a bit concerning. It's not clear to me why this would happen, but it confounds the interpretation of the results. While the authors state "The severity of callosal agenesis was associated with the extent to which the IHF had been remodeled" (pg 15), they don't actually quantify this. It might be informative to generate scatterplots of IHF length vs. CC/HC length to determine if there is a significant correlation between the two. This might lend more evidence to a causal relationship between IHF remodeling and CC/HC formation.
At the end of the result section, the authors state: "mutations that affect the ability for DCC to regulate cell shape (Figure 8F), are likely to cause callosal agenesis through perturbed MZG migration and IHF remodelling." (pg. 19). While the authors nicely show that patient mutations in DCC affect the morphology of cells in cell lines (Figure 7-8), it is not clear why simply transfecting WT DCC into cell lines results in such a dramatic change in morphology, or why addition of NTN1 doesn't increase this. The authors mention that the cell lines could express NTN1 or that NTN1 is not required for the effect. This seems an important distinction. Did the authors check this? Could they use a function blocking antibody or a soluble fragment of the NTN1 binding domain of DCC to block NTN1:DCC interactions? DCC has been shown to function as a "dependence receptor" that can induce apoptosis in the absence of ligand; are the authors certain that the morphology changes they are seeing in DCC transfected cells aren't cytoskeletal changes resulting from caspase activation?
Minor points:
The authors should mention recent work showing Netrin localization to basement membranes during axon guidance (Varadarjan et al, Neuron 2017). The data in Figure 2 are very much in agreement with this previous work, and it should be mentioned in this context.
Figure S5A: Representative images from each genotype don't look comparable, even though there's no difference in quantification.
Did the authors check whether the cell lines they used in Figure 7-8 express DCC?
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Summary: This study is a welcome follow-up to your earlier demonstration that midline zipper glia (MZG) migrate along the interhemispheric fissure (IHF) and intercalate across the hemispheres, and in doing so, remodel the meningeal basement membrane to provide a substrate for callosal axon growth. The authors identify DCC and its ligand Netrin1 to be important for this process, by acting on the distribution and morphology of MZG, in addition to their service as axon guidance signals for callosal axons to be attracted to and across the midline.
Co-submission with https://www.biorxiv.org/content/10.1101/2020.07.29.227827v1
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