Aberrant cortical development is driven by impaired cell cycle and translational control in a DDX3X syndrome model
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Evaluation Summary:
Hoye et al. analyzed conditional inactivation of Ddx3x gene in mouse dorsal forebrain, which leads to decreased brain size and widespread apoptosis in females but not males. Interestingly, the authors showed that Ddx3y was transcriptionally upregulated in cKO males, and suggested that Ddx3y compensated for the loss of Ddx3x. These results are attributed to prolonged cell cycle, impaired cell cycle exit, leading to increased progenitor populations. Ribo-Seq analysis showed differentially translated genes, providing potential new insights into Ddx3x function and pathogenic mechanisms. Overall, this study is of great importance and provides novel insights into the pathogenesis of DDX3X syndrome and the crucial role of DDX3X during cortical development.
(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
Mutations in the RNA helicase, DDX3X , are a leading cause of Intellectual Disability and present as DDX3X syndrome, a neurodevelopmental disorder associated with cortical malformations and autism. Yet, the cellular and molecular mechanisms by which DDX3X controls cortical development are largely unknown. Here, using a mouse model of Ddx3x loss-of-function we demonstrate that DDX3X directs translational and cell cycle control of neural progenitors, which underlies precise corticogenesis. First, we show brain development is sensitive to Ddx3x dosage; complete Ddx3x loss from neural progenitors causes microcephaly in females, whereas hemizygous males and heterozygous females show reduced neurogenesis without marked microcephaly. In addition, Ddx3x loss is sexually dimorphic, as its paralog, Ddx3y , compensates for Ddx3x in the developing male neocortex. Using live imaging of progenitors, we show that DDX3X promotes neuronal generation by regulating both cell cycle duration and neurogenic divisions. Finally, we use ribosome profiling in vivo to discover the repertoire of translated transcripts in neural progenitors, including those which are DDX3X-dependent and essential for neurogenesis. Our study reveals invaluable new insights into the etiology of DDX3X syndrome, implicating dysregulated progenitor cell cycle dynamics and translation as pathogenic mechanisms.
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Author Response
Reviewer #1 (Public Review):
In this work, Hoye et al. analyzed a conditional mouse model for DDX3X syndrome, an important cause of neurodevelopmental disorders in humans, and provide critical insights into the pathomechanisms of this disorder. They show that homozygous loss of DDX3X in females in neural progenitors leads to microcephaly and massive apoptosis due to impaired neural progenitor cells. Furthermore, they show that conditional DDX3X KO mice are sexually dimorphic. In males, it seems that the paralog DDX3Y on the Y chromosome is also required for neurogenesis and may be partially able to compensate DDX3X function leading to comparable phenotypes of cKO males comparable to cHET females. The authors therefore mostly focus their analysis on cHET females and cKO males. The number of progenitors is increased in …
Author Response
Reviewer #1 (Public Review):
In this work, Hoye et al. analyzed a conditional mouse model for DDX3X syndrome, an important cause of neurodevelopmental disorders in humans, and provide critical insights into the pathomechanisms of this disorder. They show that homozygous loss of DDX3X in females in neural progenitors leads to microcephaly and massive apoptosis due to impaired neural progenitor cells. Furthermore, they show that conditional DDX3X KO mice are sexually dimorphic. In males, it seems that the paralog DDX3Y on the Y chromosome is also required for neurogenesis and may be partially able to compensate DDX3X function leading to comparable phenotypes of cKO males comparable to cHET females. The authors therefore mostly focus their analysis on cHET females and cKO males. The number of progenitors is increased in cHET females and cKO males. Additionally, they show that DDX3X dosage is important for proper neuron numbers. They could link the abrogated number of neuronal cell types to a globally prolonged cell cycle and identify altered cell cycle exit of radial glial cells (RGCs) as a possible explanation for fewer neurons. Finally, the authors shed light on the molecular mechanisms by which DDX3X impairs neurogenesis using ribosomal profiling (RIBO-Seq). Large-scale studies of translational profiling are rare and this dataset provides a novel and valuable resource of translational profiles in early mouse brain development to the community. In addition, their RIBO-Seq data on DDX3X deficient cells reveal an essential requirement of DDX3X for the translation of cortical progenitor-specific mRNAs. Several targets are critical for cortical development, as investigated in more detail for Setd3 in this manuscript. Overall, this study is of great importance and provides novel insights into the pathogenesis of DDX3X syndrome and the crucial role of DDX3X during cortical development.
The manuscript is well written and the data, in general, support the conclusions drawn, but some aspects need to be clarified or modified:
We thank the reviewer for carefully reading our manuscript and for their helpful suggestions.
- I have one concern regarding the mouse model, which the authors need to better explain. It is unclear to me why the expression levels in the male cKO are reduced to levels comparable to the cHET females at D11.5 (Figure 1D, E), and not comparable to cKO females as one might expect. This to me raises the question if this is indeed a good model for male cKO of DDX3X. Do the authors have an explanation for this? Could it be that the probes/primers used here are indeed specific for DDX3X or also detecting DDX3Y, or is there is another explanation?
We apologize for the lack of clarity regarding this point. We quantified that WT females had ~25% higher levels of Ddx3x mRNA expression than WT males (Figure 1G). Thus, we originally plotted males and females separately to illustrate the reduction of Ddx3x in conditional mice relative to their sex-matched controls (original Figures 1D, E). Because females have higher levels than males at baseline, the relative reduction in Ddx3x levels in cHet females and cKO males is similar, particularly at E11. In this revision, we now plot all of the sexes together which makes it easier to compare levels across all genotypes (new Figures 1E and 1F). Our probes are specific for Ddx3x, as evidenced in Figure 2C (in which we knockdown Ddx3y but observe no change in Ddx3x).
Importantly, males also express Ddx3y, which acts redundantly with Ddx3x. While there are no available DDX3Y specific antibodies, Ddx3y is upregulated at the RNA level in cKO males (Figure 2A). Thus we posit that the overall levels of DDX3 protein in males and females is relatively similar.
- While the increase in progenitor cells in cHET females and cKO males is convincing, the reduction in neurons is only supported by weak evidence and trends. Significance levels used to draw these conclusions are somewhat inconsistent (Figure 3 - figure supplement 2). For instance, in Figure 3 - figure supplement 2E results with a p-value of 0.2 are communicated as a trend, whereas in Figure 3 - figure supplement 2F a p-value of 0.15 is marked as no difference. Overall, the findings of reduced number of neurons during development are not well supported by the data in this manuscript, which should be improved or toned down.
We apologize for this lack of consistency and thank the reviewer for pointing it out. We have modified the text throughout the manuscript to ensure we are consistent in what we call significant. We agree that the reduction in neurons is modest, especially at E14.5 (Figure 3-figure supplement 1D). However, three additional pieces of data support the conclusion that excitatory neurons are reduced. First, the lamination at P0 clearly show significant reductions in several cortical excitatory neurons (layers V, VI, Figure 3D-G). Second, our cell cycle exit data (% EdU+Sox2-Tbr2-) supports the observation of fewer neurons (Figure 4E). Third, the live imaging reflects reduced production of neurons (Figure 5E). We thus observed significant differences at multiple timepoints (E14.5 and P0) and with multiple markers (Tbr2-Sox2-, Ctip2, Tbr1). However, we agree with the reviewer that the reduction in neurons is modest and have modified the results (p. 9 and 11) and discussion (p. 18) to reflect this modest reduction.
- Do the authors have an explanation for why the increased cell cycle duration and reduced neuron numbers may not lead to microcephaly?
We cannot rule out subtle brain size defects with our current analysis at P0. However, in human DDX3X syndrome, the microcephaly is mild or absent in patients with nonsense mutations (microcephaly is primarily associated with missense mutations). Thus, our findings are consistent with disease pathology. We have added this point to the discussion (p. 19).
- For their ribosomal profiling experiments, the authors focused on cKO females and males, while in the rest of the paper they argue that cKO males are actually comparable to cHET females. And then for polysome fractionation, they go back to cHET females. Those inconsistencies are not well justified in the manuscript. I am worried that those data are then not really comparable, and differences in RNA abundance that they attribute to different developmental time points in RIBO seq vs. polysome fractionation (E11.5 vs. E14.5) may actually be due to different DDX3X levels.
We thank the reviewers for this suggestion. While we did use cKO females and males for the ribosome profiling experiment, this was done for several reasons.
- Ribo-seq is more technically challenging than a standard RNAseq experiment, so we aimed to maximize the effect of Ddx3x knockout by using the cKO females. Because of the profound apoptosis that begins at E12.5 in cKO females, we opted to do these experiments at E11.5 to avoid potential complications due to cell death and composition changes.
- The bulk of our paper focuses on the cortical development phenotypes of the cHet females and cKO males (to best model DDX3X syndrome), with significant phenotypes at E14.5. Thus, we initially performed the polysome fractionation of these genotypes at E14.5 to determine whether any of the DDX3X-dependent translation changes might be contributing to phenotypes at this stage. We did not include cKO females in this assay because at E14.5, most of the cells in the cortex are apoptotic.
In response to reviewer concerns, in this revised manuscript we include new polysome fractionation analyses at E11.5 using cKO females and cKO males-this provides validation of Ribo-seq of the same genotypes. These data show significant enrichment in monosome fractions for 2 targets (Rcor2 and Topbp1) and trends for a 3rd (Setd3, p=0.10). This also validates the same transcripts which are altered in polysomes at E14.5 in cHet females and cKO males. We include these new data in Figures 6H, J and Figure 6- figure supplement 2A,B.
We agree that differences in RNA abundance could be due to different developmental timepoints and DDX3X levels. We have included this important possibility in the discussion and removed this point from the results.
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Evaluation Summary:
Hoye et al. analyzed conditional inactivation of Ddx3x gene in mouse dorsal forebrain, which leads to decreased brain size and widespread apoptosis in females but not males. Interestingly, the authors showed that Ddx3y was transcriptionally upregulated in cKO males, and suggested that Ddx3y compensated for the loss of Ddx3x. These results are attributed to prolonged cell cycle, impaired cell cycle exit, leading to increased progenitor populations. Ribo-Seq analysis showed differentially translated genes, providing potential new insights into Ddx3x function and pathogenic mechanisms. Overall, this study is of great importance and provides novel insights into the pathogenesis of DDX3X syndrome and the crucial role of DDX3X during cortical development.
(This preprint has been reviewed by eLife. We include the public …
Evaluation Summary:
Hoye et al. analyzed conditional inactivation of Ddx3x gene in mouse dorsal forebrain, which leads to decreased brain size and widespread apoptosis in females but not males. Interestingly, the authors showed that Ddx3y was transcriptionally upregulated in cKO males, and suggested that Ddx3y compensated for the loss of Ddx3x. These results are attributed to prolonged cell cycle, impaired cell cycle exit, leading to increased progenitor populations. Ribo-Seq analysis showed differentially translated genes, providing potential new insights into Ddx3x function and pathogenic mechanisms. Overall, this study is of great importance and provides novel insights into the pathogenesis of DDX3X syndrome and the crucial role of DDX3X during cortical development.
(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):
In this work, Hoye et al. analyzed a conditional mouse model for DDX3X syndrome, an important cause of neurodevelopmental disorders in humans, and provide critical insights into the pathomechanisms of this disorder. They show that homozygous loss of DDX3X in females in neural progenitors leads to microcephaly and massive apoptosis due to impaired neural progenitor cells. Furthermore, they show that conditional DDX3X KO mice are sexually dimorphic. In males, it seems that the paralog DDX3Y on the Y chromosome is also required for neurogenesis and may be partially able to compensate DDX3X function leading to comparable phenotypes of cKO males comparable to cHET females. The authors therefore mostly focus their analysis on cHET females and cKO males. The number of progenitors is increased in cHET females and …
Reviewer #1 (Public Review):
In this work, Hoye et al. analyzed a conditional mouse model for DDX3X syndrome, an important cause of neurodevelopmental disorders in humans, and provide critical insights into the pathomechanisms of this disorder. They show that homozygous loss of DDX3X in females in neural progenitors leads to microcephaly and massive apoptosis due to impaired neural progenitor cells. Furthermore, they show that conditional DDX3X KO mice are sexually dimorphic. In males, it seems that the paralog DDX3Y on the Y chromosome is also required for neurogenesis and may be partially able to compensate DDX3X function leading to comparable phenotypes of cKO males comparable to cHET females. The authors therefore mostly focus their analysis on cHET females and cKO males. The number of progenitors is increased in cHET females and cKO males. Additionally, they show that DDX3X dosage is important for proper neuron numbers. They could link the abrogated number of neuronal cell types to a globally prolonged cell cycle and identify altered cell cycle exit of radial glial cells (RGCs) as a possible explanation for fewer neurons. Finally, the authors shed light on the molecular mechanisms by which DDX3X impairs neurogenesis using ribosomal profiling (RIBO-Seq). Large-scale studies of translational profiling are rare and this dataset provides a novel and valuable resource of translational profiles in early mouse brain development to the community. In addition, their RIBO-Seq data on DDX3X deficient cells reveal an essential requirement of DDX3X for the translation of cortical progenitor-specific mRNAs. Several targets are critical for cortical development, as investigated in more detail for Setd3 in this manuscript. Overall, this study is of great importance and provides novel insights into the pathogenesis of DDX3X syndrome and the crucial role of DDX3X during cortical development.
The manuscript is well written and the data, in general, support the conclusions drawn, but some aspects need to be clarified or modified:
1. I have one concern regarding the mouse model, which the authors need to better explain. It is unclear to me why the expression levels in the male cKO are reduced to levels comparable to the cHET females at D11.5 (Figure 1D, E), and not comparable to cKO females as one might expect. This to me raises the question if this is indeed a good model for male cKO of DDX3X. Do the authors have an explanation for this? Could it be that the probes/primers used here are indeed specific for DDX3X or also detecting DDX3Y, or is there is another explanation?
2. While the increase in progenitor cells in cHET females and cKO males is convincing, the reduction in neurons is only supported by weak evidence and trends. Significance levels used to draw these conclusions are somewhat inconsistent (Figure 3 - figure supplement 2). For instance, in Figure 3 - figure supplement 2E results with a p-value of 0.2 are communicated as a trend, whereas in Figure 3 - figure supplement 2F a p-value of 0.15 is marked as no difference. Overall, the findings of reduced number of neurons during development are not well supported by the data in this manuscript, which should be improved or toned down.
3. Do the authors have an explanation for why the increased cell cycle duration and reduced neuron numbers may not lead to microcephaly?
4. For their ribosomal profiling experiments, the authors focused on cKO females and males, while in the rest of the paper they argue that cKO males are actually comparable to cHET females. And then for polysome fractionation, they go back to cHET females. Those inconsistencies are not well justified in the manuscript. I am worried that those data are then not really comparable, and differences in RNA abundance that they attribute to different developmental time points in RIBO seq vs. polysome fractionation (E11.5 vs. E14.5) may actually be due to different DDX3X levels.
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Reviewer #2 (Public Review):
Hoye and colleagues use a mouse model of DDX3X syndrome, with Emx1-Cre-driven loss-of-function of Ddx3x, which encodes an RNA helicase. The authors first validate the conditional knockout of Ddx3x in neural progenitors and assess the impact on brain development via gross cortical anatomy and immunostaining for apoptosis. They also used in utero electroporation for CRISPR-based deletion to show that in males, DDX3Y can compensate for the loss of DDX3X during neurogenesis. These changes in neurogenesis were driven primarily by alterations in cell composition, with laminar distribution unaffected. The mechanism underlying increased progenitors and fewer mature neurons was a requirement for Ddx3x in progenitor cell cycle exit, primarily in radial glial cells, and semi-cumulative labeling demonstrated that loss …
Reviewer #2 (Public Review):
Hoye and colleagues use a mouse model of DDX3X syndrome, with Emx1-Cre-driven loss-of-function of Ddx3x, which encodes an RNA helicase. The authors first validate the conditional knockout of Ddx3x in neural progenitors and assess the impact on brain development via gross cortical anatomy and immunostaining for apoptosis. They also used in utero electroporation for CRISPR-based deletion to show that in males, DDX3Y can compensate for the loss of DDX3X during neurogenesis. These changes in neurogenesis were driven primarily by alterations in cell composition, with laminar distribution unaffected. The mechanism underlying increased progenitors and fewer mature neurons was a requirement for Ddx3x in progenitor cell cycle exit, primarily in radial glial cells, and semi-cumulative labeling demonstrated that loss of Ddx3x leads to longer cell cycles and fewer neurogenic divisions. Ribosome profiling, RNA-seq, and polysome fractionation were utilized to study DDX3X translation targets, resulting in the identification of several DDX3X-dependent targets. Overall, the study is rigorous, the manuscript is well-written, and the results are interesting and significant.
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Reviewer #3 (Public Review):
De novo DDX3X mutations are associated with intellectual disability and variable degrees of brain malformation such as hypoplastic corpus callosum - the authors reported over 100 mutations and their disease presentations in 2020. In the present study, Hoye et al. analyzed a floxed Ddx3x mouse model and showed that Emx1-Cre mediated deletion in the dorsal brain leads to decreased brain size and widespread apoptosis in females but not males. Interestingly, the authors showed that Ddx3y was transcriptionally upregulated in cKO males, and suggested that Ddx3y compensated for the loss of Ddx3x. The authors analyzed heterozygous Ddx3x cKO females and cKO males together and concluded that Ddx3x deletion prolonged cell cycle, impaired cell cycle exit, leading to increased progenitor populations as well as fewer Tbr1 …
Reviewer #3 (Public Review):
De novo DDX3X mutations are associated with intellectual disability and variable degrees of brain malformation such as hypoplastic corpus callosum - the authors reported over 100 mutations and their disease presentations in 2020. In the present study, Hoye et al. analyzed a floxed Ddx3x mouse model and showed that Emx1-Cre mediated deletion in the dorsal brain leads to decreased brain size and widespread apoptosis in females but not males. Interestingly, the authors showed that Ddx3y was transcriptionally upregulated in cKO males, and suggested that Ddx3y compensated for the loss of Ddx3x. The authors analyzed heterozygous Ddx3x cKO females and cKO males together and concluded that Ddx3x deletion prolonged cell cycle, impaired cell cycle exit, leading to increased progenitor populations as well as fewer Tbr1 and Ctip2 neurons. To gain molecular insights, the authors performed Ribo-Seq in E11.5 cortical tissues and reported target genes that showed Ddx3x-dependent translation efficiency. This is beautiful work backed by mouse genetics and high-quality molecular, cellular, and developmental studies. The Ribo-Seq targets provide potential new insights into Ddx3x function and pathogenic mechanisms.
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