Pioneer and MFF precursors develop in close proximity to Laminin-rich extracellular matrices and Engrailed expression is absent from pioneers and MFFs in sly. (A,B) Transverse sections of 20 s stage wild-type (A) and sly embryos (B) at the level of early adaxial cell stacking. Dorsal is upwards. F59 (in green) labels adaxial cells and Laminin is in red; nuclei are counterstained with DAPI (in blue). (C-F) Transverse sections of 26 hpf wild-type (C,E) and sly (D,F) embryos at the level of the anterior tail (C,D) and the anterior trunk (E,F). (C,D) Laminin is in red. Owing to the somite chevron shape, vertical myosepta of the neighbour somite (blue arrows) are visible on these transverse sections. (C-F) 4D9 (in green) labels pioneers (arrowheads, C,E) and MFFs (arrows, E) in wild-type embryos and is absent in sly embryos (D,F). n, notochord; nt, neural tube. Scale bar: 50 μm.

eng2a expression in pioneers requires maternal and/or residual lamc1, while ptc1 expression in the caudal part of the embryo is not affected in lamc1-depleted embryos. (A-F) Lateral views of eng2a expression at the 15 (A-C) and 20 s stage (D-F) in wild-type (A,D), sly (B,E) and morpholino-injected sly embryos (C,F). The fraction of embryos displaying the corresponding phenotype is shown on each panel. In 20 s stage sly embryos, no (n=3/11) to weak and transient expression (n=8/11) is found in only two or three caudal somites (red arrow in E). (G-I) Dorsal views of Laminin labelling (in red, blue arrows) at the tail bud (Tb) stage in wild-type (G), sly (H) and lamc1 MO-injected sly embryos (I). Dorsal (J-L) and lateral views (M-O) of ptc1 expression at the 15 s stage (J-L) and 24 hpf (M-O) in wild-type (J,M), sly (K,N,O) and lamc1 MO-injected sly embryos (L). Scale bar: 100 μm.

pSmad expression is precisely regulated in pioneers and their precursors in wild-type, sly and cyclopamine-treated embryos. (A-E) Transverse sections of 18-20 s stage wild-type (A,B), sly (C,D) and cyclopamine-treated embryos (E) at the level of adaxial cell pre-stacking (A,C,E) and stacking (B,D). Dorsal is upwards. F59 (in red) labels adaxial cells and nuclear pSmad labelling is in green; nuclei are counterstained with DAPI (in blue). (F) Lateral view (projection of 11 confocal sections, 0,6 μm z-step) of a 18-20 s stage wild-type embryo, at the trunk level. Anterior is on the left, dorsal is upwards. 4D9 (in red) labels pioneers and nuclear pSmad labelling is in green. n, notochord; nt, neural tube. Scale bar: 50 μm.

pSmad expression in MFFs and their precursors is regulated in a similar way to that in pioneers. (A-F) Transverse sections of 26 hpf wild-type (A-C) and sly (D-F) embryos at the level of the posterior tail (A,D), the anterior tail (B,E) and the anterior trunk (C,F). Dorsal is upwards. Nuclear pSmad labelling is in green. Anti-pSmad antibody decorates myofibrils in differentiating slow and fast muscle fibres (C,F). (B,E) F59 (in red) labels migrating slow muscle fibres. (C,F) 4D9 (in red) labels pioneers (arrowhead) and MFFs (arrow). The horizontal white lines delimitate the pSmad-negative central domain. n, notochord; nt, neural tube. Scale bar: 50 μm.

Knockdown of Bmp signal rescues Engrailed expression in sly pioneers and MFFs and ectopically activates Engrailed expression in wild-type slow and fast lineages. (A,E,I,M) Lateral views of wild-type (A), sly (E), dorsomorphin-treated sly (I) and dorsomorphin-treated wild-type embryos (M) at 26 hpf. Anterior is leftwards, dorsal is upwards. (B-D,F-H,J-L,N-P) Magnification views (projection of confocal sections) of the regions around somite 10 (B-C,F-G,J-K,N-O) and somite 18 (D,H,L,P) for each type of embryo. (A-P) 4D9 (in green) labels pioneer (arrowheads) and MFF nuclei; Prox1 (in red) labels slow muscle fibre nuclei, including pioneers. Because of the projection, some nuclei artificially overlap. All pioneer nuclei are therefore identified by arrowheads. (A,C,G,I,K,O) 4D9 labelling only. The asterisk locates ectopic tails in dorsomorphin-treated embryos. Scale bar: 50 μm.

Heparan sulfate proteoglycans are absent in sly embryos and Heparinase III treatment affects Engrailed and pSmad expression in a manner similar to the sly mutant. (A-D) Transverse sections of wild-type (A,C) and sly (B,D) embryos at the 20 s stage (A,B, adaxial cell stacking level) and 26 hpf (C,D, anterior tail level). Dorsal is upwards. In addition to its extracellular signal, anti-HS antibody (in green) labels all nuclei. (E,I) Lateral views of vehicle solution-injected wild-type (E) and Heparinase III-injected wild-type (I) embryos at 26 hpf. Anterior is leftwards, dorsal is upwards. (F-H,J-L) Magnification views (projection of confocal sections) of the regions around somite 10 (F-G,J-K) and somite 18 (H,L) for each type of embryo. (E-L) 4D9 (in green) labels pioneer (arrowheads) and MFF nuclei; Prox1 (in red) labels slow muscle fibre nuclei, including pioneers. Because of the projection, some nuclei artificially overlap. All pioneer nuclei are therefore identified by arrowheads. (E,G,I,K) 4D9 labelling only. (M-P) Transverse sections of vehicle solution-injected wild-type (M,O) and Heparinase III-injected wild-type (N,P) embryos at the 20 s stage (M,N, adaxial cell level) and 26 hpf (O,P, anterior tail level). Dorsal is upwards. (M-P) Nuclear pSmad labelling is in green. (M,N) Nuclei are counterstained with DAPI (in blue). (O,P) 4D9 (in red) labels pioneers. The horizontal white lines delimitate the pSmad-negative central domain. n, notochord; nt, neural tube. Scale bar: 50 μm.

Chondroitin sulfate proteoglycans are not involved in the regulation of Engrailed expression. (A-D) Transverse sections of wild-type (A,C) and sly (B,D) embryos at the 20 s stage (A,B, adaxial cell stacking level) and 26 hpf (C,D, anterior tail level). Dorsal is upwards. Anti-CS antibody is in green. In wild type (A,C), CSPGs are found in the basement membrane lining the somite, notochord and ventral neural tube at the 18-20 s stage (n=8/8) and 26 hpf (n=9/9). In sly embryos (B,D), although unaffected at the 18-20 s stage (n=6/6), CSPGs labelling is weak by 26 hpf (n=7/7). (E,I) Lateral views of vehicle solution-injected wild-type (E) and Chondroitinase-injected wild-type (I) embryos at 26 hpf. Anterior is on the left, dorsal is upwards. (F-H,J-L) Magnified views (projection of confocal sections) of the region around somite 10 (F-G,J-K) and somite 18 (H,L) for each type of embryo. (E-L) 4D9 (in green) labels pioneer (arrowheads) and MFF nuclei; Prox1 (in red) labels slow muscle fibre nuclei, including pioneers. Because of the projection, some nuclei artificially overlap. All pioneer nuclei are therefore identified by arrowheads. (E,G,I,K) 4D9 labelling only. After injection at the 10 s stage, Engrailed expression at 26 hpf is normal in the trunk and tail of embryos injected with vehicle solution (n=6/6) (E-H) or Chondroitinase ABC (n=12/12) (I-L). n, notochord; nt, neural tube. Scale bar: 50 μm.

Heparan sulfate and chondroitin sulfate proteoglycans are removed by heparinase III and Chondroitinase ABC treatments, respectively. (A-H) Frontal sections of vehicle solution-injected wild-type (A,B), heparinase III-injected wild-type (C,D), vehicle solution-injected wild-type (E,F) and Chondroitinase ABC-injected wild-type (G,H) embryos at 24 hpf (i.e. 10 hours post-injection) at the levels of anterior trunk (A,C,E,G) and tail (B,D,F,H). Anterior is on the left. (A-D) In addition to its extracellular labelling, anti-HS antibody (in green) labels all nuclei. Compared with injected controls (n=6/6) (A,B), HSPGs are absent from all heparinase III-injected embryos (n=13/13) (C,D), except for a weak labelling (D, red bracket) in presomitic mesoderm cells (n=13/13) and around the notochord and on the somite surface of the last 7-12 formed somites (n=3/13) (D). The weak labelling in the presomitic mesoderm could be due to de novo synthesis of HSPGs if the enzyme activity declines by 10 hours post-injection or to a HSPGs type that is not degraded by heparinase III. (E-H) Anti-CS antibody is in green. Compared with injected controls (n=7/7) (E,F), CSPGs are absent from the majority of Chondroitinase ABC-injected embryos after a 10-hour treatment (n=7/11) (G,H). A minority (n=4/11) shows a weak labelling at the level of the presomitic mesoderm and last formed somites (H). n, notochord. Scale bar: 50 μm.