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Rapid Assessment of the Temporal Function and Phenotypic Reversibility of Neurodevelopmental Disorder Risk Genes in C. elegans

Hundreds of genes have been implicated in neurodevelopmental disorders. Previous studies have indicated that some phenotypes caused by decreased developmental function of select risk genes can be reversed by restoring gene function in adulthood. However, very few risk genes have been assessed for adult reversibility. We developed a strategy to rapidly assess the temporal requirements and phenotypic reversibility of neurodevelopmental disorder risk gene orthologs using a conditional protein degradation system and machine vision phenotypic profiling in Caenorhabditis elegans. Using this approach, we measured the effects of degrading and re- expressing orthologs of 3 neurodevelopmental risk genes EBF3, BRN3A, and DYNC1H1 across 30 morphological, locomotor, sensory, and learning phenotypes at multiple timepoints throughout development. We found some degree of phenotypic reversibility was possible for each gene studied. However, the temporal requirements of gene function and degree of phenotypic reversibility varied by gene and phenotype. The data reflects the dynamic nature of gene function and the importance of using multiple time windows of degradation and re-expression to understand the many roles a gene can play over developmental time. This work also demonstrates a strategy of using a high-throughput model system to investigate temporal requirements of gene function across a large number of phenotypes to rapidly prioritize neurodevelopmental disorder genes for re-expression studies in other organisms. SUMMARY STATEMENT We developed a strategy that combines a conditional and reversible protein degradation system with our high-throughput machine vision tracking system to assess the temporal windows of gene function and reversibility of phenotypic disruptions associated with neurodevelopmental disorder risk gene orthologs using C. elegans. Using this approach, we assessed 3 genes (unc- 3, unc-86, and dhc-1) and found that post-embryonic rescue was possible for each gene and each phenotypic feature class assessed. Re-activation of certain genes was able to reverse multiple phenotypic disruptions late into development without inducing novel phenotypes, prioritizing them for further study.


INTRODUCTION
7 Auxin Inducible degradation (AID) and high-throughput machine vision phenotyping (Fig. 1). We 155 took advantage of the genetic tractability and rapid, stereotyped development of C. elegans to 156 precisely investigate the effects of degrading and re-expressing the proteins of interest at multiple

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For example, reversal duration seems to be mediated by a mechanism that acts earlier in 273 development, with reversibility only possible with early re-expression ( Fig. 3G-I

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3C) and exposing animals to Auxin from L4 onwards did not induce the hyporesponsive 312 phenotype seen with continuous degradation (Fig. 4B)

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We next investigated the phenotypic consequences of degrading and re-expressing UNC-

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Instead, only select components of the mechanosensory reversal response were affected while 370 others were left intact. Response probability was unaffected, as the proportion of worms that 371 responded to each mechanosensory stimulus was similar to the no Auxin control (Fig. 5B).

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However, degrading dynein at 72 hrs post-hatch caused animals to display deeper habituation of 373 response duration (Fig. 5C) and a slower response speed compared to animals not exposed to 374 Auxin (Fig. 5D). Interestingly, the absolute speed trace across the entire experiment shows 375 animals have the ability to respond as fast as control animals but decrement their response speed 376 faster with repeated stimulation (Fig. 5E). These findings suggest that, in adulthood, dynein may 377 function to promote normal habituation of response duration and speed, but not response 378 probability. Taken together, these results support the hypothesis that different components of  the rab-3 promoter (Fig. 6A). Continuous pan-neuronal degradation of dynein did not cause     habituation of response probability (animals were hyperresponsive and did not learn to decrease 428 their likelihood of responding to repeated stimuli) (Fig. 6B). Adult pan-neuronal degradation of 429 dynein (beginning Auxin exposure at L4) did not alter initial response probability but did decrease 430 habituation of response probability (Fig. 6B). For response duration, adult re-expression of dynein 431 was sufficient to fully rescue the impairment seen with continuous degradation, but adult 432 degradation did not induce the response duration impairment (Fig. 6C). Consistent with our 433 findings for ubiquitous dynein degradation in 72 hr old adult animals, continuous degradation of 434 pan neuronal dynein also caused animals to exhibit slower reversal speed. The speed impairment 435 could be rescued with dynein re-expression in early adulthood and was also induced by adult 436 degradation (Fig. 6D) suggesting that dynein is continuously required in neurons to mediate 437 response speed.

Figure 7. Pan-neuronally degrading and re-expressing dynein reveals distinct temporal
24 development by re-expression of one of the genes, but not others (Fig. 8A-C). Based on our 486 results, EBF3•unc-3 is a promising candidate for further research as many impaired phenotypes 487 could be reversed even when gene function was restored later stages of development (Fig. 8A),

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and re-expression did not induce novel phenotypic disruptions (Fig. 2 & 3). Further, identifying 489 which phenotypes are most commonly affected across risk genes may provide insight into points 490 of convergence in the mechanisms that are altered in neurodevelopmental disorders (Fig. 8B).

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We found that reversal probability metrics appeared to be both the most affected and reversible 492 phenotypes across all genes assessed (Fig. 8B & C). This finding supports previous work from  neurodevelopmental disorder risk gene orthologs across a suite of morphological, locomotor, 512 sensory, and learning phenotypes in thousands of freely behaving animals using our high-513 throughput machine vision tracking system. Taking advantage of the CRISPR-Cas9 AID system 514 allowed us to test whether restoring protein levels through re-expression from the endogenous 515 locus was sufficient for phenotypic rescue at multiple time points throughout development. We

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In addition, we found that time windows for when a gene is required and when re-573 expression can reverse impairments did not always align. While EBF3•unc-3 showed reciprocal 574 functional and reversibility windows for many affected phenotypes, we found other genes (e.g.

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were selected for analysis. Note that throughout the manuscript the "•" symbol is used to denote being placed on the MWT. After acclimation, we collected data for an additional 5 min period to 644 assess baseline locomotion and morphology features (Fig. 1D). Following this baseline period, 645 thirty mechanosensory stimuli were administered to the side of the Petri plate using an automated 646 push-solenoid at a 10 second inter-stimulus interval (Fig. 1D). These non-localized