Complementary vertebrate Wac models exhibit phenotypes relevant to DeSanto-Shinawi Syndrome

This study establishes murine and zebrafish *Wac* deletion mutants as complementary vertebrate models that recapitulate key craniofacial, neurobehavioral, and molecular features of DeSanto-Shinawi Syndrome, thereby providing a foundation for understanding the disorder's underlying biological mechanisms.

The Gist

Imagine the human body as a massive, complex construction site. Every building needs a foreman to ensure the blueprints are followed, the materials are mixed correctly, and the workers (cells) stay on task. In the world of genetics, the WAC gene acts like one of these crucial foremen.

When this foreman is missing or broken, the construction site gets messy. This specific mess is called DeSanto-Shinawi Syndrome (DESSH). People with this condition often face developmental delays, learning difficulties, seizures, and unique facial features. But for a long time, scientists didn't have a good way to study how this happens because they couldn't easily test these ideas in living animals that look and act like humans.

This paper is like a "field test" where scientists built two different construction sites to see what happens when they remove the WAC foreman: one site is a mouse (very similar to us), and the other is a zebrafish (a tiny, fast-growing fish).

Here is the story of what they found, broken down into simple parts:

1. The Two Test Sites: Mice and Fish

The scientists created "mutant" mice and fish that were missing the WAC gene.

• The Mouse Site: They found that if they removed the gene completely, the mouse embryos didn't survive (the construction site collapsed too early). So, they created mice with half the foreman (one working copy, one broken). These mice survived but showed clear signs of trouble.
• The Fish Site: Fish have two copies of the gene (like having two foremen). The scientists had to knock out both copies of the main gene to see the effects. When they did, the baby fish looked mostly normal in size, but their jaws were too short, and their heads looked a bit different.

The Takeaway: Both animals showed craniofacial changes (changes to the face and skull), which matches what doctors see in human patients with DESSH. It's like if you removed the foreman, the blueprint for the front door and the roof got a little crooked.

2. The "Brain Wiring" Problem

The most interesting part of the story is what happened inside the brain.

• The GABA Problem: Think of your brain as a busy city with traffic lights. Some lights tell cars to stop (inhibitory neurons), and others tell them to go (excitatory neurons). The "stop" lights are made of a chemical called GABA.
• The Finding: In both the mice and the fish, the scientists found that the "stop lights" (GABAergic neurons) were dimmer or fewer than usual.
• The Consequence: In the mice, this dimming of the stop lights made them super sensitive to seizures. When given a mild chemical trigger that usually wouldn't bother a normal mouse, the mutant mice had seizures. This explains why people with DESSH often have epilepsy. Interestingly, the fish didn't get seizures, showing that different animals handle this "wiring error" in different ways.

3. Social Behavior: The "Party" Test

• The Mice: When put in a room with other mice, the mutant mice were okay, but they had trouble with memory tasks (like remembering which path they took in a maze). They were a bit forgetful.
• The Fish: The fish were much more dramatic. Normal fish like to swim in a tight group (a school). The mutant fish, however, swam all over the place, completely scattered. They lost their "social cohesion." This is a great model for the autism and social difficulties seen in humans with DESSH.

4. The "Boy vs. Girl" Mystery

One of the coolest discoveries was that gender matters.

• Brain Size: When they scanned the mouse brains with an MRI, they found that male mutant mice had slightly larger brains than normal males. The females didn't show this change.
• Gene Activity: When they looked at the genetic "to-do lists" (RNA) inside the cells, the males showed much bigger changes than the females. It seems the male brain is more sensitive to the loss of the WAC foreman. This might explain why some symptoms of the syndrome vary between boys and girls in humans.

5. The "Paperwork" Glitch

Finally, the scientists looked at the actual instructions (the RNA) the cells were reading.

• They found that without the WAC foreman, the cells were messing up their paperwork. Specifically, they were having trouble "splicing" (cutting and pasting) the genetic instructions correctly.
• This led to a buildup of certain proteins (like protocadherins) that are important for how brain cells talk to each other. It's like the construction crew started using the wrong type of glue, causing the walls to be built a bit too thick or in the wrong places.

The Big Picture

This paper is a major step forward because it gives scientists two new "living models" to study DeSanto-Shinawi Syndrome.

• The Zebrafish are great for seeing how the face and social behavior change quickly.
• The Mice are great for understanding seizures, brain size, and the complex molecular "glitches" that happen in a mammal brain.

In short: By removing the WAC foreman in these animals, the scientists confirmed that this gene is essential for building a normal face, keeping brain traffic lights working, and ensuring social behavior is on track. Most importantly, they found that boys might be more affected than girls, and that fixing the "stop lights" (GABA) in the brain could be a future way to treat the seizures and behavioral issues in people with this syndrome.

Technical Summary

1. Problem Statement

DeSanto-Shinawi Syndrome (DESSH) is a rare neurodevelopmental disorder caused by pathogenic variants in the WAC gene. Clinical features include developmental delay, intellectual disability, craniofacial dysmorphism, hypotonia, seizures, and neurobehavioral issues (autism, ADHD, anxiety).

• Knowledge Gap: While WAC is known to be involved in mTOR signaling, mitosis, and transcription in invertebrates and cell lines, its function in vertebrate neurodevelopment is poorly understood.
• Limitation: No comprehensive vertebrate models existed to study the molecular and phenotypic mechanisms underlying DESSH, hindering the development of therapeutic strategies.

2. Methodology

The authors generated and characterized two complementary vertebrate loss-of-function models: murine (mouse) and zebrafish.

• Mouse Model (Wac):
  • Utilized a conditional allele (Wac^tm2c) with loxP sites flanking exon 5.
  • Crossed with Beta-actin-Cre mice to generate constitutive heterozygous (Het) knockouts.
  • Lethality: Homozygous knockouts were embryonic lethal; only heterozygotes survived to adulthood.
  • Validation: Confirmed via Sanger sequencing (frameshift/premature stop) and Western blot (64% reduction in WAC protein in Hets).
• Zebrafish Model (wac):
  • Zebrafish possess two paralogs: waca and wacb.
  • Used CRISPR/Cas9 to generate single and double knockouts.
  • Selection: Focused on waca single knockouts (KOs) as wacb KOs showed no phenotype and double KOs exhibited high lethality (heart edema, swim bladder loss).
  • Validation: Confirmed via PCR genotyping and morphological assessment.
• Phenotypic Screening:
  • Craniofacial: Alizarin Red/Alcian Blue staining for bone and cartilage morphology.
  • Behavioral:
    • Mice: 3-chamber social test, Y-maze (working memory), elevated plus maze (anxiety), radial arm water maze (spatial memory).
    • Zebrafish: Social cohesion assay, novel tank assay (hyperactivity).
  • Seizure Susceptibility: Pentylenetetrazol (PTZ) challenge (subthreshold dose) in mice.
  • Cellular/Molecular: Immunofluorescence for interneuron markers (PV, SST, PROX1, LHX6), Western blotting for synaptic/signaling proteins (mTOR, PSD95, Gephyrin, pERK), and cell density counts (NeuN, OLIG2, S100β, IBA1).
  • Anatomical: Ex vivo MRI (9.4T) to measure brain and regional volumes.
  • Transcriptomics: Bulk RNA-seq of P2 forebrains (14 WT, 10 Het) with sex-stratified analysis. Differential expression (DE) and splicing analysis (MAJIQ) were performed.

3. Key Results

A. Craniofacial Dysmorphism (Recapitulated in both models)

• Mice: Wac Het mice exhibited widened anterior fontanels and sutures at birth (P0) and a persistent gap in the interfrontal suture at P30. Skull width across frontal bones was significantly increased.
• Zebrafish: waca KOs displayed a shortened lower jaw (mandible) and widened Meckel's cartilage.
• Significance: Both models successfully recapitulate the craniofacial features common in DESSH patients.

B. Behavioral and Neurological Phenotypes

• Mice:
  • Seizures: Wac Hets showed significantly elevated susceptibility to PTZ-induced seizures compared to WT, mirroring the ~40% seizure prevalence in DESSH patients.
  • Cognition: Significant deficits in working memory (Y-maze). Social discrimination trends were observed but not statistically significant.
  • Anatomy: MRI revealed increased whole brain volume in male Hets (but not females). Specific regional increases were noted in the retrosplenial cortex (males) and parietal cortex (females).
• Zebrafish:
  • Social Behavior: waca KOs showed profound deficits in social cohesion (dispersed grouping), with males showing greater deficits than females.
  • Hyperactivity: Male waca KOs exhibited increased locomotion in the novel tank assay.
  • Seizures: Unlike mice, zebrafish did not show increased PTZ susceptibility.

C. Cellular and Molecular Mechanisms

• GABAergic Deficits: Both models showed impacts on inhibitory neurons.
  • Mice: ~18% reduction in Parvalbumin (PV)+ interneuron density in the somatosensory cortex. However, LHX6+ cell counts were normal, suggesting a defect in PV expression rather than cell loss.
  • Zebrafish: Reduced expression area of the pan-GABAergic marker gad1b in the forebrain.
• Signaling Pathways:
  • No changes in mTOR activity or major synaptic markers (PSD95, Gephyrin).
  • MAPK Pathway: Significant reduction in phosphorylated ERK1 and ERK2 in mouse Hets.
  • Cell Types: No changes in total neuronal, oligodendrocyte, astrocyte, or microglial counts.
• Transcriptomics (RNA-seq):
  • Sex Bias: Male Hets showed a stronger transcriptional response (more DE genes) than females.
  • Pathways: Upregulated genes enriched for RNA processing/splicing; downregulated genes enriched for metabolic processes (purine/fatty acid metabolism).
  • ASD Relevance: Male-specific DE genes showed a trend toward enrichment for high-confidence Autism Spectrum Disorder (ASD) genes (e.g., Prr12, Chd2).
  • Protocadherins: Significant upregulation of Pcdhga family genes in Hets, confirmed at the protein level.
  • Splicing: 13 differential splicing events detected, including Snhg14 (linked to Angelman/Prader-Willi syndromes).

4. Key Contributions

1. First Vertebrate Models: Established the first Wac deletion models in mouse and zebrafish, providing essential tools for studying DESSH syndrome.
2. Phenotypic Validation: Demonstrated that Wac haploinsufficiency causes craniofacial defects, GABAergic dysfunction, and behavioral abnormalities in vertebrates, validating the relevance of these models to human disease.
3. Sex-Specific Effects: Uncovered a strong male bias in phenotypic severity (brain volume expansion, seizure susceptibility in mice, social deficits in zebrafish) and transcriptomic dysregulation, suggesting sex-specific mechanisms in DESSH.
4. Molecular Insights: Identified that Wac loss leads to reduced PV expression (potentially driving seizures) and altered RNA processing/splicing, moving beyond previous invertebrate-only hypotheses.
5. Divergent Model Strengths: Highlighted that while mice better model seizure susceptibility and brain volume changes, zebrafish better model social cohesion deficits, offering complementary insights.

5. Significance

This study provides a foundational framework for understanding the biological underpinnings of DeSanto-Shinawi Syndrome. By linking Wac loss to GABAergic dysfunction and sex-specific transcriptomic changes, the authors propose potential therapeutic targets (e.g., restoring PV expression or modulating inhibitory tone). The identification of male-biased phenotypes and ASD-linked gene dysregulation offers new hypotheses for why DESSH symptoms vary by sex and severity. These models will serve as critical platforms for future drug screening and mechanistic studies into neurodevelopmental disorders.