|
Fig. 5
AP regionalization of vitamin D signaling controlled by Hand2. (A) cyp24a1, which encodes a vitamin D-inactivating enzyme, is more highly expressed in posterior regions of uninjured pectoral fins. (B) Posterior enrichment of cyp24a1 is maintained during regeneration at 4 dpa. (C) hand2 induction daily during regeneration elevates anterior cyp24a1 expression <7.5-fold at 4 dpa relative to heat-shocked wild-type clutchmate controls. (D) hand2 morpholino injection into pectoral fin regenerates reduces posterior cyp24a1 expression by <28% relative to vehicle-injected clutchmate controls. (A-D) Expression is normalized to actb1. *P<0.05, **P<0.005, Student’s t-test. n=3; mean ± s.e.m. (E) Appearance of 7-dpa fins from hsp70l:hand2 and wild-type clutchmates given a daily heat shock and either daily vehicle or vitamin D injections. Vitamin D injection had little effect on wild-type regeneration, but partially suppressed the effects on bone patterning caused by hand2 overexpression. Representative regenerative growth of hsp70l:hand2 vehicle-injected rays 3 and 8 is denoted by dotted lines. Arrowheads indicate amputation plane. (F) Quantification of the effects of daily vitamin D injection on ray lengths, indicating improvement in hsp70l:hand2 animals. (G) Daily vitamin D injection increases ray widths in regenerating hsp70l:hand2 fins. (F,G) n=20-28; bar indicates mean. **P<0.05, Student’s t-test. Scale bars: 0.6 mm.