Complimentary vertebrate Wac models exhibit phenotypes relevant to DeSanto-Shinawi Syndrome
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eLife Assessment
This important study establishes the first vertebrate models of DeSanto-Shinawi Syndrome, revealing conserved craniofacial and social and behavioral phenotypes across mouse and zebrafish that mirror key clinical features. The solid evidence is supported by behavioral, anatomical, and molecular analyses of Wac animal mutants that broadly support the authors' claims, though additional mechanistic investigation would strengthen the conclusions. This study sets a baseline for future mechanistic studies and reports a platform to test approaches to reverse phenotypes.
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Abstract
Monogenic syndromes are associated with neurodevelopmental changes that result in cognitive impairments, neurobehavioral phenotypes including autism and seizures. Limited studies and resources are available to make meaningful headway into the underlying molecular mechanisms that result in these symptoms. One such example is DeSanto-Shinawi Syndrome (DESSH), a rare disorder caused by pathogenic variants in the WAC gene. Individuals with DESSH syndrome exhibit a recognizable craniofacial gestalt, developmental delay/intellectual disability, neurobehavioral symptoms that include autism, ADHD, behavioral difficulties and seizures. However, no thorough studies from a vertebrate model exist to understand how these changes occur. To overcome this, we developed both murine and zebrafish Wac/wac deletion mutants and studied whether their phenotypes recapitulate those described in individuals with DESSH syndrome. We first show that the two Wac models exhibit craniofacial and behavioral changes, reminiscent of abnormalities found in DESSH syndrome. In addition, each model revealed impacts to GABAergic neurons and further studies showed that the mouse mutants are susceptible to seizures, changes in brain volumes that are different between sexes and relevant behaviors. Finally, we uncovered transcriptional impacts of Wac loss of function in mice that will pave the way for future molecular studies into DESSH. These studies present two new animals that begin to uncover some biological underpinnings of DESSH syndrome and elucidate the biology of Wac .
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eLife Assessment
This important study establishes the first vertebrate models of DeSanto-Shinawi Syndrome, revealing conserved craniofacial and social and behavioral phenotypes across mouse and zebrafish that mirror key clinical features. The solid evidence is supported by behavioral, anatomical, and molecular analyses of Wac animal mutants that broadly support the authors' claims, though additional mechanistic investigation would strengthen the conclusions. This study sets a baseline for future mechanistic studies and reports a platform to test approaches to reverse phenotypes.
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Reviewer #1 (Public review):
Summary:
The authors generated mouse and zebrafish models for DeSanto-Shinawi Syndrome, caused by loss-of-function variants in the WAC gene. Using these vertebrate systems, they demonstrate conserved craniofacial and social-behavioral phenotypes that parallel human clinical features, along with deficits in GABAergic markers. They observe increased seizure susceptibility and male-biased brain volumetric changes in Wac mutant mice. Together, these findings begin to define the biological consequences of Wac haploinsufficiency and provide valuable resources for future mechanistic studies.
Strengths:
WAC is a high-confidence neurodevelopmental disorder gene and one of the genes identified by large-scale exome sequencing efforts, including the Satterstrom et al. (2020) autism spectrum disorder cohort. This study …
Reviewer #1 (Public review):
Summary:
The authors generated mouse and zebrafish models for DeSanto-Shinawi Syndrome, caused by loss-of-function variants in the WAC gene. Using these vertebrate systems, they demonstrate conserved craniofacial and social-behavioral phenotypes that parallel human clinical features, along with deficits in GABAergic markers. They observe increased seizure susceptibility and male-biased brain volumetric changes in Wac mutant mice. Together, these findings begin to define the biological consequences of Wac haploinsufficiency and provide valuable resources for future mechanistic studies.
Strengths:
WAC is a high-confidence neurodevelopmental disorder gene and one of the genes identified by large-scale exome sequencing efforts, including the Satterstrom et al. (2020) autism spectrum disorder cohort. This study establishes the first vertebrate Wac models, addressing a major gap in the understanding of DeSanto-Shinawi Syndrome, and provides a framework for studying other syndromic forms of autism. The models generated will be impactful and useful to the community to study and understand DeSanto-Shinawi Syndrome.
The cross-species analysis is important and well executed, and reveals both conserved and divergent phenotypes. The behavioral and anatomical assays are rigorously executed and well-controlled, and the inclusion of RNA-sequencing analyses adds valuable insights into the mechanisms underlying brain function in Wac mutants. Notably, the RNA-seq data reveal upregulation of several clustered protocadherins, genes central to neuronal identity and cell-cell interactions, which are known to be regulated by dynamic developmental regulation of chromatin architecture. This observation provides an intriguing hint that could link Wac function to higher-order chromatin organization and neuronal connectivity.
Weaknesses:
The evidence is solid, but the study remains incomplete in its mechanistic depth and molecular interpretation. The authors compellingly describe behavioral, anatomical, and transcriptomic phenotypes associated with WAC loss, yet do not explore how WAC mechanistically regulates chromatin or transcription. Given prior evidence that WAC interacts with the RNF20/40 ubiquitin ligase complex and promotes histone H2B ubiquitination and transcriptional elongation, the paper would benefit from a discussion of these functions as a potential link between Wac haploinsufficiency and the observed changes in neuronal gene expression. Similarly, the authors mention WAC's WW and coiled-coil domains but do not consider how these domains could mediate nuclear interactions or recruitment of transcriptional cofactors that shape gene regulation and chromatin organization in neurons.
The transcriptomic analysis is rich but largely descriptive. Although the upregulation of clustered protocadherins is particularly intriguing, these findings are not validated or localized to specific neuronal populations. The study would be strengthened by independently validating the most significant RNA-seq changes, such as protocadherin gamma genes, using in situ hybridization methods to confirm the spatial and cellular specificity of expression changes.
Finally, while the behavioral and MRI results add valuable breadth, their interpretation would be improved by clearer reporting of sample sizes, statistical corrections, and effect sizes to support claims of sex-specific and regional brain volume differences.
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Reviewer #2 (Public review):
The authors describe the first deep neurological characterization of WAC mutation in two vertebrate species (zebrafish and mouse). They examine these at various levels, guided by the work in humans that has associated a heterozygous WAC mutation with DeSantos Shinawi Syndrome (DESSH). Therefore, they investigate the animals for a variety of phenotypes, following a template for what is seen when characterizing a new mouse/fish model of a developmental disability gene. Investigations include analysis of skull and jaw for abnormalities(both species), MRI of brain structure(in mice), electrophysiology(mice), assessment of signaling pathways (by Western blot, in mice), cell counts (both, more in mice), transcriptomics (mice), and behavior (both).
Generally, this describes an important first characterization of …
Reviewer #2 (Public review):
The authors describe the first deep neurological characterization of WAC mutation in two vertebrate species (zebrafish and mouse). They examine these at various levels, guided by the work in humans that has associated a heterozygous WAC mutation with DeSantos Shinawi Syndrome (DESSH). Therefore, they investigate the animals for a variety of phenotypes, following a template for what is seen when characterizing a new mouse/fish model of a developmental disability gene. Investigations include analysis of skull and jaw for abnormalities(both species), MRI of brain structure(in mice), electrophysiology(mice), assessment of signaling pathways (by Western blot, in mice), cell counts (both, more in mice), transcriptomics (mice), and behavior (both).
Generally, this describes an important first characterization of the consequences of the mutation. Most of the studies appear well-conducted and reasonably powered, thus solid or convincing. However, there are a few places where the data presentation could be improved for clarity, and a few concerns about some choices in analytical approach for a couple of the experiments, where improved statistical approaches could improve their sensitivity and/or better rule out false positives, and thus the support of some of these claims is currently incomplete. There is also some lack of clarity about the rationale for some decisions regarding the fish genetics. Nonetheless, this is an important and useful first characterization of many phenotypes of these lines. Such experiments form a baseline for future mechanistic studies in the same lines and a platform to test approaches to reverse phenotypes.
Individual claims and their strength & weaknesses:
(1) The authors developed mouse and zebrafish models of WAC deletion
They used the existing KOMP floxed WAC line to generate a null allele. For the mouse, there is a Western showing that it is indeed null for the protein. The fish data is less robustly validated - they don't confirm the allele in null at the protein or RNA level, and fish have two paralogs (waca and wacb), and this paper only characterizes one of these. So this evidence is less clear. The evaluated mice are heterozygous (Het), similar to patients, while the fish appear to be evaluated as homozygous mutants.
(2) The authors show that both species show altered craniofacial features
These data appear well powered, and the findings are robust.
(3) Each model altered GABAergic neurons
In mice, the authors stained with PV antibodies and saw a decrease in cells positive for this staining. A second marker, Lhx6, does not show a difference, suggesting this might be a change in PV expression rather than cell number. They could maybe look into the literature to see if this loss of just the protein also occurs in other models. Overall, the sample size here is a bit smaller than other parts of the paper (n=3), and the methods on the cell counts were less clear, so it is not as clear that this finding is as robust. The authors counted several other broad classes of cells, and those appear normal. Interestingly, there might also be some TBR1 mislocalization in layer 6 that might be significant with added power.
The fish data is based on an in situ hybridization for GAD. The measure shown is the width of the positive area in the forebrain. This measure is not one I have seen much before, and has potential to be driven by something unrelated to GABA (e.g., if the whole forebrain were simply a bit smaller). So this analysis could use a couple of other approaches (density of signal?) and/or a control probe for some other brain gene showing the measure is normal, and thus it is not just a size issue.
(4) Mice were more susceptible to the seizure-inducing agent PTZ
These data appear well powered, and the findings are robust. The authors also did a fair amount of useful electrophysiology that was all normal, but appeared to be well executed.
(5) Mice had changes in brain volume that interact with sex
The authors conducted an MRI on a good number of mice and reported a slight increase in global volume just in males. Sample size is fair, but the statistical approach here may be better if it puts males and females in the same model (to boost power and explicitly test for sex by genotype interaction that they report), and there is some chance that the brain region level differences that they report could include some false positives. They tested many regions, and it is not clear whether or not they corrected for the number of tests. Often, an FDR correction would be used in such imaging studies. It may be that only the most robust regional findings will survive those corrections. It is interesting data either way, but the analysis could be improved.
(6) Several behaviors are altered in the mice as well
These studies were fairly well-powered (n=15,16), and they found several positive and negative results, including alterations in memory and sociability in both species. There is a minor statistical flaw in the three-chamber analysis (they don't actually compare the Hets directly to the wildtypes in their statistical testing - a common mistake in neuroscience that should be addressed. But the data look like they will probably still be significant when correctly analyzed. In the supplement, the authors could do a bit more with the data they have to look at hyperactivity (i.e., show total motion in open field, not just time in center vs. periphery), and adding sex to their model might improve sensitivity for genotype effects.
(7) Some biochemical signaling pathways are altered in the brain
These are n=4 immunoblots, and show altered phospho ERK, but no changes in other signaling events predicted from prior WAC literature like H2B ubiquitination. They appear well done, and the authors share the full blots in the supplement.
(8) WAC deletion also alters gene expression in the brain
These studies were well-powered for RNAseq, with 10 and 14 samples, using neonates (P2), just the forebrain. The sequencing quality metrics all looked good, and the approach to analysis was okay. It would be stronger to again include sex in the model, rather than separate by sex. There were some typos in this part of the paper that made part of the conclusions unclear, but the RNAseq nicely confirmed the mutation of the mice, and discovered many differentially expressed genes, consistent with the role of this gene as a regulator of transcription. The presentation could be expanded to make more use of the data. Overall, though, this is a useful first characterization of the transcriptome in the line.
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