FIGURE 8

snai2 regulates jag1b expression via its double zinc‐finger domain. (A and B) Summary of mt mRNA(A) and jag1b mRNA (B) measured in sox10 ⁺ cells isolated from control embryos treated with zinc and/or injected with snai2 mRNA. (C) Schematic diagram depicting the wild‐type (WT) Snai2 protein and three mutant Snai2 constructs lacking the indicated zinc‐finger (ZF) domains. (D and E) Summary of mt mRNA (D) and jag1b mRNA (E) measured in sox10 ⁺ cells isolated from control embryos co‐injected with snai2 CRISPR/Cas9 ribonucleoprotein complexes together with either WT snai2 mRNA or the indicated mutant snai2 mRNAs; where indicated, the embryos were treated 1 mM zinc. (F) Representative images of cartilage staining in the heads of control and mutant embryos co‐injected with snai2 CRISPR/Cas9 ribonucleoprotein complexes together with either WT snai2 mRNA or the indicated mutant snai2 mRNAs. (G) Summary of Mt1, Mt2, Snai2, Jag1, and Cdh1 mRNA measured in mouse primary mesenchymal stem cells (MSCs) cultured in the absence or presence of 10 μM zinc. (H) Schematic illustration of the Jag1 promoter region, showing the approximate locations and sequences of the five E2 boxes. P1 through P4 indicate the primer pairs used for chromatin immunoprecipitation (ChIP) analysis, and the transcription start site (TSS) is indicated. (I) ChIP assay of mouse primary MSCs using the SNAI2 antibody for pull‐down followed by quantitative polymerase chain reaction (qPCR) using the indicated primer pairs to amplify the Jag1 promotor region. Where indicated, the cells were cultured in the absence or presence of 10 μM zinc. The Cdh1 promoter was used as a positive control. (J) Under normal conditions in wild‐type embryos (top left), slc30a1 is expressed in the ventral region to balance zinc homeostasis, jag1b is expressed in the dorsal region, and snai2 is expressed in the ventral pharyngeal arch (PA) more robustly than in the dorsal PA. Ventral snai2 expression suggests a stemness state in this region (bottom left). In the absence of Slc30a1 proteins (top right), zinc accumulates both as free ions and bound to metallothionein (MT) proteins. The expression of snai2 is increased in the ventral region, thereby expanding the pattern jag1b expression. In neural crest progenitor cells (bottom right), the expression of jag1b is upregulated via the double zinc‐finger in the Snai2 protein in a zinc‐dependent manner. The resulting increase in Jag1b activates downstream Notch signaling, arresting chondrocyte differentiation and resulting in “dorsalized” neural crest progenitor cells. *p < 0.05, **p < 0.005, ***p < 0.001, and n.s., not significant