Cereblon influences the timing of muscle differentiation in Ciona tadpoles

Thalidomide has a dark history as a teratogen, but in recent years its derivates have been shown to function as a chemotherapeutic agent. These drugs bind cereblon (CRBN), the substrate receptor of an E3 ubiquitin ligase complex and modify its degradation targets. Despite these insights, remarkably little is known about the normal function of cereblon in development. Here we employ Ciona, an invertebrate chordate, to identify endogenous Crbn targets. In Ciona, Crbn is specifically expressed in developing muscles during tail elongation before they acquire contractile activity. Crbn expression is activated by Mrf, the ortholog of MYOD1, a transcription factor important for muscle differentiation. CRISPR/Cas9-mediated mutations of Crbn lead to precocious onset of muscle contractions. By contrast, overexpression of Crbn delays contractions and is associated with decreased expression of contractile protein genes such as troponin. This reduction is possibly due to reduced Mrf protein levels without altering Mrf mRNA levels. Our findings suggest that Myod and Crbn form a negative feedback loop to control the precision of muscle differentiation during tail elongation. Single-cell transcriptomes from mid tailbud embryos expressing either membrane GFP in the tail muscle (control condition) or both membrane GFP and Cereblon (Crbn overexpression condition) were analyzed using Chromium Single Cell Gene Expression (10X Genomics©). 9854 cells for the control condition and 8540 cells for the Crbn overexpression condition, which include 212 and 174 muscle cells, respectively, were sequenced.