Restoring Shank3 in a rostral sensorimotor brainstem nucleus rescues reduced light-evoked behaviors in shank3ab-/- zebrafish.


 People with Phelan-McDermid Syndrome, caused by mutations in the SHANK3 gene, commonly present with symptoms of sensory hyporeactivity. To investigate how shank3 mutations impact brain circuits and contribute to sensory hyporeactivity, we generated two shank3 zebrafish mutant models. These shank3 mutant models both exhibit hyporeactivity to visual stimuli. Using whole-brain activity mapping, we show that light receptive brain nuclei show normal levels of activity while sensorimotor integration and motor regions are less active in shank3-/- mutants. Specifically rescuing Shank3 in a sensorimotor nucleus of the rostral brainstem is sufficient to rescue shank3-/- mutant hyporeactivity. In summary, reduced sensory responsiveness in shank3-/- mutant is associated with reduced activity across the brain and can be rescued by restoring Shank3 function in the rostral brainstem.

the rostral brainstem is su cient to rescue shank3-/-mutant hyporeactivity. In summary, reduced sensory responsiveness in shank3-/-mutant is associated with reduced activity across the brain and can be rescued by restoring Shank3 function in the rostral brainstem.

Main Text
Altered sensory processing is a pervasive but poorly understood symptom in individuals with autism spectrum disorders (ASD) 1 . Sensory symptoms manifest as muted or excessive responses to light, sound, and/or touch. Because of variability in both the presence and presentation of sensory symptoms, gaining a mechanistic understanding of these sensory processing de cits remains a challenge. In contrast to ASD as a whole, genetically de ned forms of ASD share similar sensory de cits. For instance, individuals Here we identify the neurobiological basis of sensory hyporeactivity in shank3 loss-of-function zebra sh models of PMS.
Several animal models of PMS recapitulate muted responses to diverse sensory stimuli: pain in Shank3 mutant mice 5 , sound in Shank3 mutant rats 6 , and both touch and light in shank3ab mutant zebra sh 7,8 ; nonetheless, a brain-wide understanding of these muted responses is lacking. Hyporeactivity in PMS could re ect functional changes that either span the entire brain or are localized to speci c brain regions and/or muscle 9 . Zebra sh allow unique experimental approaches to identifying underlying mechanisms because, within the rst week of life, larvae have fully functional sensory-motor circuits and produce robust, stereotyped responses to calibrated sensory stimuli 10 These larval zebra sh have transparent vertebrate brains composed of only ~100,000 neurons, allowing unbiased functional approaches to map brain-wide neuronal activity. Moreover, embryonic transplantation can be used to make wildtype-mutant chimeras to test for brain-region-speci c functional rescue 11,12 . Here, we use brain-wide activity mapping and transplants to identify and functionally validate brain regions that underlie sensory hyporeactivity to changes in light in zebra sh shank3 mutant models.
In contrast to the single SHANK3 gene in people, the shank3 gene is duplicated in zebra sh; therefore, to generate zebra sh models of PMS we used CRISPR/Cas9 to mutate both the shank3a and shank3b (shank3ab) gene paralogs. Shank3 proteins are large, ~200 kD, with multiple isoforms that can be differentially impacted by mutations in different parts of the gene 13 . To capture this complexity, we generated two zebra sh PMS models, shank3abDN with mutations truncating both the Shank3 a and b proteins in the ankyrin repeat domains and shank3abDC with mutations truncating both the Shank3 a and b proteins near the proline-rich domain 13 (Fig. 1a; Supplementary Fig. S1, Supplementary Tables 1&2). These models mimic the most common types of SHANK3 mutations found in people with PMS and, by having two models, we control for genetic background. In mice and humans, Shank3 protein is expressed in glutamatergic granule cells of the cerebellum, colocalizing with the scaffolding protein PSD-95. Likewise, in wildtype zebra sh, we show that Shank3 protein colocalizes with PSD-95 in the cerebellum and along ventral neural tracts of the brainstem ( Fig. 1b; Supplementary Fig. S2). In contrast, in both shank3abDN-/and shank3abDC-/-PMS models Shank3 staining is lacking despite intact PSD-95 synaptic puncta (Fig. 1b). These data indicate that the four alleles that underlie the two shank3abDN-/and shank3abDC-/models are loss-of-function mutations. Hereafter, we refer to shank3abDN-/and shank3abDC-/models are as shank3ab-/mutant models except in cases that the results differ between the models.
The sensory reactivity of zebra sh shank3ab-/models was measured by quantifying behavioral changes to a light-based stimulus using the well-established visual motor response (VMR; 14 ). The VMR is characterized by dramatic increases in movement in response to sudden transitions from light to darkness (Fig. 1c). Both shank3ab-/mutant models exhibited reduced VMR responses as quanti ed by comparing the distance traveled in the thirty seconds before and after the transition from lights-on to lights-off conditions ( Fig. 1c & d; Supplementary Tables 3-8). Muted VMR responses were more pronounced in homozygous shank3ab-/larvae (p<0.001) than in heterozygous shank3ab+/larvae (p<0.05). We used the pronounced VMR de cits shank3ab-/mutants as the basis of all subsequent experiments to determine the mechanistic underpinnings of these altered sensorimotor integration phenotypes.
To identify the neural circuits underlying hyporeactivity in shank3ab mutant models, we used an unbiased brain-wide Mitogen Activated Protein (MAP)-mapping 12 approach, based on phosphorylation of extracellular signal-regulated kinase (pERK). Because ERK phosphorylation increases when calcium is elevated during action potentials, staining for pERK provides a proxy for neuronal activity ( Fig. 2a & b).
Brain regions differentially active between light-on and lights-off conditions were identi ed by statistically comparing relative ERK signals (pERK/total ERK) in two groups of 15-21 larvae per group (p<10 5 ; Fig. 2c & d, Supplementary Figs. S3 & S6). In response to the lights-on stimulus, wild type (WT) and shank3ab-/models showed similarly elevated pERK staining in the optic tectum (green) that receives input from retinal ganglion cells. In response to the lights-off stimulus, WT showed elevated pERK staining in the pineal, the telencephalic pallium and subpallium, the torus semicircularis of the midbrain, brainstem, and spinal cord (magenta). While shank3ab-/mutant models showed similarly elevated pERK staining in the pineal, they showed little or no elevated pERK other brain regions. These VMR brain activity maps in shank3ab-/models show that sensory brain regions including the pineal, retina, and optic tectum detect changes in light normally, but that downstream brain regions fail to integrate and respond to dark transitions consistent with muted lights-off behavioral responses.
Next we explored whether restoring Shank3 function would be su cient to rescue hyporeactivity in both shank3abDN-/and DC-/mutant models. We generated genetically mosaic larvae by transplanting WT cells into otherwise shank3ab mutant embryos at the late gastrula shield stage, ~ six hours postfertilization (Fig. 3a, Supplementary Fig. S5). WT donor cells were deposited in the region of the shank3ab-/embryo fated to become brainstem. To track the fate of transplanted cells, WT donor Zebrabow embryos expressing dTomato under a ubiquition promoter 15 were used as the source of WT cells, referred to as ZbT for Zebrabow transplants (Fig. 3). Remarkably, when tested as six-day-old larvae, transplanted ZbT cells were su cient to rescue shank3ab-/mutant lights-off reactivity in the VMR assay ( Fig. 3 b-d, Supplementary Fig. S5; Supplementary Tables S9-16). To determine ZbT brain regions in common among behaviorally rescued shank3abD:ZbT larvae, we registered shank3abD:ZbT larvae to the Z-brain atlas. We found that the majority of rescued shank3abD:ZbT larvae had integrated ZbT cells in a rostral dorsal glutamatergic brainstem nucleus known in zebra sh brain atlases as vGluT cluster 2 (90.5%; n=19/21; Supplementary Fig. S6; Supplementary Table S17). Previous studies using whole-brain gCaMP have identi ed this brainstem nucleus as important in transforming sensory inputs to behavioral responses 16,17 . To control for non-speci c transplantation effects, we performed within genotype transplants. WT donor to WT recipient chimeras and shank3abDN-/donor to shank3abDN-/recipient chimeras had no effects on VMR behaviors compared to unmanipulated larvae of the corresponding genotype. shank3abDC-/donor to shank3abDC-/recipient chimeras had more severe hyporeactivity compared to unmanipulated larvae of the same genotype (Supplementary Figs. S7, Supplementary Tables S18-23). Consistent with the MAP-mapping experiments, these results indicate Shank3ab function in rostral brainstem is su cient for WT levels of light-evoked activity.
Previous work applying whole brain imaging in zebra sh larvae has highlighted the roles of the cerebellum and rostral brainstem as regions that receive inputs from sensory centers to coordinate the appropriate motor output [16][17][18] . Brainstem de cits in shank3abDN-/and DC-/mutants could be due to synaptic de cits and/or altered development. In support of a synaptic role, loss of Shank3 protein in mammalian models is known to decrease glutamate receptor expression, disrupt post-synaptic density composition, and reduce synaptic transmission 19 . Weaker excitatory synaptic responses could therefore explain the failure of sensory brain regions to evoke responses at the levels of both other brain regions and motor behaviors in shank3ab mutant PMS models. Functional de cits could also be due to altered development that could disrupt functional connectivity. Supporting this possibility, global developmental delay has previously been reported in shank3ab zebra sh models 7,8 . Moreover, altered brainstem development has been suggested as the likely basis for multisensory integration and sensory-motor de cits more generally in ASD 20, 21 . Such developmental de cits in brainstem regions could help explain the e cacy of rostral sensorimotor brainstem transplants in rescuing VMR behaviors in shank3ab-/-mutants in this study. With the recent inclusion of sensory de cits, more clinical research is needed to determine links between changes in the brainstem function and sensory de cits in individuals with autism.

Conclusion
Brain-wide activity mapping and transplant rescue experiments provide robust evidence that hyporeactivity to light-based stimuli in zebra sh shank3ab mutants is due to functional de cits downstream of sensory reception that can be rescued by restoring wild type Shank3 in the rostral brainstem. Boxes denote the median, 1st and 3rd quartile, while whiskers represent the minimum and maximum values. Groups were statistically compared using a Kruskal-Wallis ANOVA, and when statistically signi cant, were followed by a Dunn's multiple comparison test. P value asterisks represent; p<0.05 -*, p<0.01 -**, p<0.001 -***, p<0.0001-****.

Figure 2
Brain-wide neural activity mapping reveals shank3abΔ-/-mutant models sense light normally but fail to activate downstream brain regions underlying sensorimotor integration. Brain-wide activity maps were generated by using phosphorylated-ERK (pERK) antibody staining as a proxy for neuronal activity.  Hyporeactivity is rescued in both ΔN and ΔC shank3 mutant models by restoring wild-type shank3ab positive neurons in dorsal/rostral glutamatergic brainstem nuclei. (a) A cartoon above shows how cells from wild type donor embryos marked by a ubiquitously expressed dTomato uorescent protein (ubi:zebrabow) are transplanted into the presumptive hindbrain of shank3ab-/-mutant recipient embryos at mid-gastrulation stages. (b) Chimeric embryos at 1 day post-fertilization (dpf), with donor cells expressing the uorescent protein (false-colored in cyan) in recipient shank3abΔN-/-or shank3abΔC-/embryos. Chimeric six-day-old larvae (shank3ab-/-:Zb-T) were imaged to determine the fate of the transplanted cells. (c) Confocal images of chimeric larvae at 6 dpf following behavioral screening, demonstrating transplanted cells in rescued larvae populate the dorsal/rostral brainstem nuclei.
Individual larvae are numbered 1-3, with the three averaged in the right most stack. Scale bars = 50 µm. (d) VMR line graphs with ample sizes are indicated below the paired dot plots and apply to plots in d, e, and f. and (e) paired-plots show lights-off behavioral phenotypes are rescued in both shank3abΔ-/mutant models with wild-type-derived brainstems (shank3abΔ-/-:Zb-T). Within shank3 model comparisons were conducted using Dunn-Bonferroni p-value corrected t-tests. (f) Box plots displaying median swimming distances for individuals following the rst 30-seconds following lights-off. Individual values are medians representing all four lights-off transitions for individual larvae. Boxes denote the median, 1st and 3rd quartile, while whiskers represent the minimum and maximum values. Groups were statistically compared using Kruskal-Wallis one-way ANOVA, and when statistically signi cant, followed by Dunn's multiple-comparisons. p<0.05 -*, p<0.01 -**, p<0.001 -***, p<0.0001-****.

Supplementary Files
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