Fig. 5

Cited3 regulates expression of mef2c and myogenin and it acts cell-autonomously.

(A–D) Expression of myoD and myf5 was not perturbed in embryos injected with the cited3 MO at 19 hpf. (E,F) Myogenin transcripts were retained in mature somites in the cited3 morphants at 22 hpf (F) where it was usually downregulated in the control MO-injected embryos (E). Transcription of mef2ca was reduced in cited3 morphants (H) compared to control MO-injected embryos (G) and it was restored to normal levels when coinjected with cited3 RNA (I) at 19 hpf. (J–L) Mef2d expression was not affected in embryos injected with the cited3 MO or cited3 MO + cited3 RNA at 19 hpf. (M–O) Immunostaining with the general anti-Mef2 antibody (recognizes all Mef2 variants) revealed that cumulative Mef2 protein levels were reduced in the cited3 morphants (N compared to M) and were restored to normal levels when cited3 RNA was coinjected (O) at 22 hpf (images spanning somites 16^th–18^th are shown) . Similarly, Mef2 protein levels were reduced in the heart of cited3 morphants (N′) compared to the control MO-injected embryo (M′) and rescued in the cited3 RNA coinjected embryos (O′) at 30 hpf. (P–U′) Slow muscle fibers labeled with rhodamine dextran dye were transplanted from wild-type donor embryos into control MO-injected (P–R′) or cited3 MO-injected host embryos (S–U′). Anti-Mef2 immunostaining revealed that Mef2 protein levels in wild-type transplanted myofibers (arrowhead) were not different from those in the control MO injected host myofibers (Q), but were brighter compared to those in the cited3 MO containing host slow myofibers (T). (V–X2) Mef2 staining in the transplanted cited3 morphant myofibers is fainter than their wild-type host counterparts. (Y,Z) The normalized intensity of Mef2 protein level is plotted for different transplantation conditions. The number of quantified transplanted slow myofibers ranged from 23 to 141. P-values are 10⁷ in Y and 10¹² in Z. Scale bars: 100μm in A and 25μm in M,P. Images in (P–U′) are from trunk somites, while those in (V–X′) are from anterior somites.