Notochord induction of zebrafish slow muscle mediated by Sonic hedgehog

Abstract

The patterning of vertebrate somitic muscle is regulated by signals from neighboring tissues. We examined the generation of slow and fast muscle in zebrafish embryos and show that Sonic hedgehog (Shh) secreted from the notochord can induce slow muscle from medial cells of the somite. Slow muscle derives from medial adaxial myoblasts that differentiate early, whereas fast muscle arises later from a separate myoblast pool. Mutant fish lacking shh expression fail to form slow muscle but do form fast muscle. Ectopic expression of shh, either in wild-type or mutant embryos, leads to ectopic slow muscle at the expense of fast. We suggest that Shh acts to induce myoblasts committed to slow muscle differentiation from uncommitted presomitic mesoderm.

Figure 1

Slow and fast MyHC isoform-specific antibodies recognize discrete muscle fiber populations in developing embryonic 24-hr zebrafish. Slow BA-D5-reactive MyHC (green; B,C) is present in the outermost layer of A4.1025-reactive cells containing skeletal muscle MyHC (red; A,C) in a transverse cryosection through the trunk (A–C). A tail section (D–F) shows that fast EB165-reactive MyHC (red; D,F) was detected in medial fibers that do not express slow MyHC (green; E,F) so that muscle fibers at this stage can be divided into two subpopulations of slow lateral and fast medial cells (C,F). Western analysis (G) using protein extracted from 24-hr zebrafish embryos shows that A4.1025 and BA-D5 recognize major bands that migrate at the same rate as purified bovine cardiac muscle MyHC (∼220 kD). EB165 detects two bands ∼170 kD that could either reflect degradation of the fast MyHC or an unusual sized MyHC in zebrafish fast muscle. The lower ∼50-kD band is nonspecific because it is also detected with antibody F1.652 to mouse embryonic MyHC that does not react with fish sections. The ∼75-kD band in the A4.1025 lane reflects some degradation. (n) Notochord; (nt) neural tube.

Figure 2

Slow muscle differentiates before fast in zebrafish embryos. (A–C) Serial transverse cryosections of a methanol-fixed 15-somite embryo show that muscle first differentiates next to the notochord (A), and expresses slow MyHC (B). Fast MyHC (C) is undetectable at any level of the embryo. (D–L) At the 21-somite stage, a developmental progression exists along the anteroposterior axis. Posteriorly, slow muscle cells are still medial (D,E) and fast MyHC is undetectable (F). However, more anterior sections of the same embryo show loss of slow MyHC⁺ cells medially and their appearance laterally (H,K; arrows). Somite cells medial to slow MyHC⁺ cells initiate differentiation (G,J) and fast MyHC expression (L; arrowheads). Therefore, medial fast muscle differentiates slightly before lateral in each individual somite. (M–O) By 24 hr, the slow muscle cells are aligned in a layer one cell thick at the lateral margin (N), and the remaining medial muscle expresses fast MyHC (O).

Figure 3

Severe midline defects correlate with loss of slow muscle formation. (A–C) Serial transverse sections of severely affected 24-hr boz embryos, which have no axial mesoderm from gastrulation, have no detectable slow MyHC (B). A single fused somite (A) expressing fast MyHC (C) extends under the neural tube (cf. Fig. 2, M–O). (D–L) Successive transverse sections of a 24-hr ntl^b160 embryo. Anterior tail shows normal distribution of muscle fiber types (D–F), except for the lack of muscle pioneers at the dorsoventral midline (E). More posteriorly (G–I), slow cells are missing or migrate abnormally (arrows in H,K). Fast MyHC appeared unaffected (F,I,L). (M–Q) Whole-mount in situ mRNA hybridization for shh at 15 somites (M–O,Q) or 24 hr (P). Notochord and floorplate shh expression is completely absent in severe boz embryos (N,Q), compared with wild type (M,O). In contrast, only the most posterior shh expression is lacking in ntl mutants (P). (M,N) Dorsal views of the tail; (O–Q) lateral views of the tail.

Figure 4

Ectopic overexpression of shh induces extra slow muscle cells in the myotome of wild-type and mutant embryos. (A–C) shh mRNA was microinjected into the zebrafish blastulae at the 2- or 4-cell stage and 24-hr embryos examined in transverse serial cryosections. All differentiated cells in both myotomes contained slow MyHC (A,B) and did not express fast MyHC (C). Some injected embryos showed ectopic slow MyHC and fast MyHC suppression on only one side, probably caused by mosaic expression of injected mRNA (data not shown). (D–H) Embryos injected with shh mRNA were analyzed at the 15-somite stage by whole-mount or cryosection immunohistochemistry. Compared with wild type (G and Fig. 2A), injected embryos showed bilateral (H) or unilateral (D, arrow) expansion of muscle differentiation. Ectopic muscle contained slow (E, arrow), but not fast, MyHC (F). Note that although ectopic lateral muscle is present in the anterior portion of shh-injected embryos (H, left), premature differentiation is not observed in presomitic mesoderm (H, right). (I,J) Embryos from boz mutant crosses were injected with shh mRNA and severely affected boz embryos (lacking eyes and notochord), were examined by serial cryosection immunohistochemistry. Widespread slow MyHC rescue (I) and supression of fast MyHC (J) was observed.

Figure 5

Slow muscle forms beneath the floorplate in flh embryos. (A–C) Differentiated muscle visualized by MyHC-staining in whole-mount 15-somite wild-type (A) or flh (B,C) embryos observed after flat-mounting. Dual labeling for shh mRNA (red; C) and MyHC (green; C) confirmed that the most posterior differentiated muscle was at the midline (arrow, C). (D–I) Transverse cryosections show that differentiated muscle in flh midline mesoderm expresses slow but not fast MyHC at 15 somites (D–F). By 24 hr, flh embryos have a single fused myotome (G), with lateral slow and medial fast muscle (H,I). Despite the ectopic location of formation of slow muscle cells in the midline the slow muscle cells appear to migrate normally so that the only detectable residual slow muscle defect is lack of muscle pioneers. (J,K) Dual labeling for MyHC (green) and ptc1 mRNA (red) in the two most posterior MyHC-containing serial sections of a 15-somite flh embryo. Newly differentiating muscle cells close to the midline do not always express detectable ptc1 (arrows), even though ptc1 is expressed abundantly in the mesoderm underlying residual floor plate (K).

Figure 6

Three phases of zebrafish muscle development. A model in which presomitic mesoderm gains competence to respond to Shh early in development in wild-type zebrafish. The result of exposure to notochord-derived Shh at this stage is a slow myoblast phenotype involving myoD expression and up-regulation of ptc1 (orange). However, a second event, either intrinsically timed within the slow myoblast, or in the form of a signal from outside of the somite (indicated by ? on the diagram, but unlikely to be either Shh or notochord-derived), is required for the slow myoblast to differentiate into a fiber (red). Simultaneously, the MyoD-positive myoblast state in lateral somitic cells arises independent of notochord-derived signals (yellow). Later, adaxial slow fiber migration and concurrent differentiation of lateral myoblasts into fast muscle (green) is timed by a third event(s). Fast fiber differentiation is independent of slow fiber migration, although the converse may not be true.