A tissue-targeted auxin-inducible degradation toolkit for C. elegans embryogenesis
Varshney, N.; Green, R. A.; Desai, A. O.; Moghareh, S.; Green, D. J.; Desai, A.; Oegema, K.
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Many proteins required for embryogenesis are expressed in multiple tissues and across developmental time, often with tissue- and stage-specific roles. To resolve where and when proteins function, we developed a tissue-targeted auxin-inducible degradation toolkit for C. elegans embryogenesis. We generated a plasmid set compatible with single-copy insertion or high-copy extrachromosomal array generation that employs tissue-specific promoters (active in the germline, intestine, muscle, pharynx, neuroblasts, neurons, sensory neurons, epidermis, and pan soma) to drive an operon expressing an optimized auxin-dependent plant F-box factor TIR1 and, in most cases, a fluorescent histone. Using a ubiquitous nuclear envelope marker as a test substrate, we identified single-copy insertions and arrays that exhibit efficient auxin-dependent, tissue-specific target degradation. We also establish a workflow that ensures all functionally important isoforms of a target are tagged and that degradation reproduces the loss-of-function phenotype prior to tissue-specific analysis. Applying the toolkit to the broadly expressed Rho kinase LET-502, we find that degradation in the epidermis, but not in other embryonic tissues, disrupts elongation. Unexpectedly, temporal analysis revealed that LET-502 is required for late as well as early elongation.
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