Figure 4. TRIM promotes a CNC progenitor cell fate and disrupts chondrogenic differentiation.

(A-E) CNC marker analysis at 24 hpf demonstrate that TRIM treatment expanded the number of crestin⁺ CNC progenitors (A), inhibited dlx2a⁺ migratory cells (B, arrows) and the sox9a⁺ pre-chondrogenic cells (C, yellow dotted) in all PA. However, the mitf ⁺ melanocytes (D) and foxd3⁺ neuronal/glial cells (E) were not seriously affected by TRIM. Notably, sox9a expression was localized to an aberrant lateralized location (C, green brackets) compared to the ventral-lateral distribution pattern in DMSO control. Conversely, leflunomide exerted a more general and profound depletion effect on all CNC cells and their derivatives (A-E, bottom panel).

(F) At 48 and 72 hpf, TRIM treatment caused loss of dlx2a⁺ or sox10⁺ migratory cells in the anterior PA (yellow dotted), whereas aberrant dlx2a⁺ or sox10⁺ CNC cells were sequestered in the posterior domain and retained in a progenitor state (green dotted, arrowheads).

(G) At 48 and 72 hpf, chondrogenic differentiation of sox9a⁺ CNC cells or terminal col2a⁺ chondrocytes in anterior PA was significantly inhibited by TRIM (yellow dotted). Instead, ectopic posterior expression of sox9a and col2a was trapped in the lateralized ceratobranchial (green dotted, arrowheads).

Scalebar: 100 μm.