Pelvic fin muscle formation in bony fish.

Pelvic fin muscle formation in P. spathula (A–J), D. rerio (K–T), and N. forsteri (U–DD). Larvae at stages when pelvic fin muscles form (A, K, and U). Developing pelvic fin bud (B, L, and V). Muscle fibres in developing pelvic fin are separate and distinct from the muscle of the somite (C, M, and W). Immediately before pelvic fin formation epithelial buds (mb) head the myotomal extension (me) (D, E, F, N, O, X, Y). The pelvic fin muscles (pfm) have formed within the pelvic fin and are separate from the myotomal extension (me) (G, H, I, P, Q, Z, AA, BB). myoD is restricted to individual, post-migratory, differentiating pelvic fin muscles (R). lbx1 positive cells (purple) at the position of the forming pelvic fin muscles (pfm) in lungfish (DD). Lbx1 expression in 14 dph pelvic fin of P. spathula, inset is a cross-section of the pelvic fin at the same stage, revealing lbx expression in the muscle masses (J). Lbx1 positive (blue) precursors in the tip of the extension position of the future pelvic fin muscles in D. rerio at 8 mm TL (S) and 9 mm TL (T). Lbx1-positive precursors in stage 50 pelvic fin bud of N. forsteri (DD). (ep, epithelial bud; me, myotome extension; pf, pelvic fin; pfm, pelvic fin muscle).

Transgenic somite transplantation in D. rerio reveals pelvic fin muscles derive from myotomal extension.

(A) Donor embryos are generated by crossing Tg(acta1:mCherry)^pc4 with Tg(acta1:GFP)^zf13 [33]. (B) Donor somites are surgically removed and transplanted into the host. (C–I) Myotomal extensions derived from somites 10 and 11 generate the pelvic fin muscles (Section through s10 (blue line in F) of host (5 wk post-operation) (G, H, and I)). (Comprehensive methods are provided in supplementary data, however surgery involving transplantation of two consecutive somites lead to a greater probability of transplanting the entire somite, including the ventral aspect required for pelvic fin muscle formation). (J–L) Contribution to the pelvic fin muscle requires the most ventral tip of the somite to be transplanted. Full transplantation of somite 9 contributes to ventral muscle anterior to pelvic fin. but a partial transplant of somite 10 does not result in contribution to the pelvic fin muscle. (M, N) Section through s10 (blue line in M) of the transplanted host (5 wk post-operation). (N) If the donor somite is not included in the most ventral tip of the extension, contribution to the fin does not occur . (O–T) The most ventral group of pelvic fin muscles are derived from somite 11 (s11+s12) section through s10 (S) (blue line in R) and s11 (T) (blue line in R) of host (5 wk post-operation). Pf, pelvic fin; rfp, red fluorescent protein; pfm, pelvic fin muscle; me, myotomal extension; s9,s10,s11,s12, somite numbered from anterior to posterior.

Donor somite transplants contribute only muscle to the host. (A) Fish transgenic for a ubiquitously expressed promoter (beta actin2), expressing mCherry (Tg(bact2:mCherry)^pc3), are crossed with fish transgenic for GFP driven by the alpha actin skeletal muscle specific promoter (Tg(acta1:GFP)^zf13). (B) The recipient host embryos are transgenic for BFP also expressed via the alpha actin skeletal muscle specific promoter (Tg(acta1:BFP)^pc5). (C) The donor somites from embryos doubly transgenic for Tg(bact2:mCherry^pc3) and Tg(acta1:GFP^zf13) were transplanted into a Tg(acta1:BFP^pc5) host. In each of 12 transplants performed in this way, co-expression of both green and red fluorescent protein was only ever observed, indicating that the donor somite somitic tissue only ever generated donor-derived muscle in the host. (D–G) Transplant of somite 10 using the method outlined in (C).

Myotomal extensions derived from somite 8 generate the individual muscle adjacent to the ventral tip of the extending myotomes and anterior to pelvic fin. Pf, pelvic fin; pfm, pelvic fin muscle; rfp, red fluorescent protein; s8, the 8th somite numbered from anterior to posterior.