Morphogenesis of Adaxial Cells and the Determination of MPs from DV Midline

(A)Time-lapse of the muscle morphogenesis of adaxial cells from segmentation (i) to 140 min after segmentation (viii). Anterior to the left and dorsal to the top in all parasagittal views unless otherwise stated. Timing refers to the relative time after the formation of corresponding somite in all figures unless otherwise stated. White brackets label fully elongated adaxial cells. White dash lines denote somite boundaries unless otherwise stated. Images are taken at somite 18–20.

(B) The timing of elongation of each individual adaxial cell at different positions relative to DV midline (n_Cells = 135, from total of 8 somites taken from 8 embryos, ^∗∗∗p < 0.001, Student’s t test).

(C–Cʹʹ) Mapping of all slow muscles from four contiguous somites back to the PSM with colors denoting distinct cell fates (C), timing of elongation relative to the segmentation of somite S0 (Cʹ) or relative to the segmentation of each somite (Cʹʹ). Black dash lines label the position of notochord. White asterisks in (C) label the adaxial cells lying on the dorsal or ventral margin of the notochord.

(D) Time-lapse of MP differentiation with the live MP cell fate marker eng2a:GFP from PSM (i) to 290 min later (ix). Images are taken at somite 19–20.

(E) 2D representation of the adaxial cell rearrangement during slow muscle myogenesis. The varying colors in each track denote the corresponding time relative to the somite segmentation. Black circles and squares denote to the initial positions of future SSFs and MPs, respectively.

(F) Probability map of MP initial position constructed from adaxial cell positions in S0 (n_Somites = 52, n_Embryos = 16). Left, right, top and bottom refer to anterior, posterior, dorsal and ventral side of adaxial cell population, respectivel.

(G) Distribution of AP-Bias of the quantified 52 somite. (i) The AP-Bias in each somite is defined by log(∑area(A)/∑area(P)). ∑area(A) and ∑area(P) denote the area taken by the future MPs anteriorly and posteriorly in the somite, respectively.

FGF Signaling Participates in the Further Differentiation of Both Slow and Fast Muscle Lineages

(A–C) Fluorescent in situ of FGF downstream activators pea3 (A–Aʹʹ) and erm (B–Bʹʹ) and inhibitor spry4 (C–Cʹʹ). Images are taken at the lateral somitic cells (A–C) and the slow muscle fibers (Aʹ–Cʹ) at 18-somite stage. Constructed transverse images are taken at somite S3 and are displayed in (Aʹʹ–Cʹʹ) with lateral to the top and dorsal to the left. Slow muscles are labeled with Prdm1a:GFP and white short arrows. (Cʹʹʹ) Spry4 expression level per cell along the DV axis. The expression of spry4 is measured cell by cell by calculating the average intensity within the nucleus (n_Cells = 153, from total of 9 somites taken from 5 embryos, ^NSp > 0.05, Student’s t test).

(D–H) Perturbations of FGF signaling change the number of MPs with MPs identified by Eng2a:eGFP expression. Wild-type embryo (D), SU5402 treatment at 60 μM (E), heat shock of hsp70l:dn-fgfr1-eGFP (F), and heat shock of hsp70l:ca-fgfr1 (G) analyzed, all imaged at 28 hpf at muscle segments 16–20, with quantification of MP number shown in (H), where n_Segments = 55, 60, 40, and 40 (from 11, 12, 10, and 10 embryos) for conditions (D–G), respectively.

(I–L) Perturbations of FGF signaling change the number of ECL cells, with ECL cells identified by Pax7 antibody staining. White asterisks label ECL cells with moderate Pax7 intensity. The much brighter cells labeled with white short arrows are neural crest cells. Wild-type embryo (I and Iʹ), SU5402 treatment at 60 μM (J and Jʹ), heat shock of hsp70l:dn-fgfr1 (K and Kʹ) and heat shock of hsp70l:ca-fgfr1 (L and Lʹ) analyzed at 28 hpf at muscle segments 16–20. Transverse views shown in (I–L) with lateral to the top and dorsal to the left.

(M) Quantification of ECL cell number, where n_Segments = 16, 21, 15, and 19 (from 6 embryos in each condition) for conditions (I–L), respectively.

^∗∗∗p < 0.001, Student’s t test.

The Direct and Indirect Roles of FGF Signaling in the Further Differentiation of Fast and Slow Lineages, Respectively

(A–D) Mosaic FGF perturbations in slow muscle cells driven by smyhc1:gal4;UAS:dn-fgfr1-p2a-mCherry (A) or smyhc1:gal4;UAS:ca-fgfr1-p2a-mCherry (Aʹ), respectively. (B and C) Mosaic FGF inhibition (B) or over-activation (C) in the slow muscle fibers is identifiable through mCherry expression. Images are taken at 28 hpf at muscle segments 13–16 and projected along ML axis. (Bʹ–Cʹ) Transverse views of (B and C) made at the sites of dash lines with lateral to the left and dorsal to the top. (D) Fraction of mCherry positive MPs among mCherry positive slow muscles in control group (60/296), FGF inhibited group (52/262), and FGF over-activated group (45/214).

(E–G) Mosaic FGF perturbations regardless of cell types with the expression of dn-fgfr1-mCherry (E) or ca-fgfr1-p2a-mCherrry (F) driven by heat shockpromoter hsp70l. The perturbed cells are identifiable through membrane localized dn-fgfr1-mCherry (E) or uniformly distributed mCherry, respectively (F), and labeled with white short arrows. (Eʹ–Fʹ) Transverse views of (E and F) made at the sites of dash lines with lateral to the left and dorsal to the top. (G) Fraction of mCherry positiveECL cells among the total population of mCherry positive cells in fast muscle lineage (both fast muscle fibers and ECL cells) in control group (104/495), FGF inhibited group (373/777), and FGF over-activated group (41/696). Heat shock was performed at 16-somite stage. The quantification of cell fates was performed at muscle segments 16–22 at 30 hpf.

AP Polarity of Fast Muscle Myogenesis and the Roles of FGF Signaling in the AP Patterning

(A) Time-lapse of the earliest fast muscle elongation and the displacement of slow muscles. Yellow, white, blue, and magenta labels the contours of elongating fast muscle progenitors. Slow muscles are labeled with Prdm1a:GFP.

(B–E) Time-lapse of the cell rearrangement and differentiation of lateral somitic cells throughout the primary myogenesis at coronal view (B) with lateral to the top and anterior to the left and parasagittal optical views (C–E). Yellow, white, and blue circles label cells from posterior (C), central (D) and anterior (E) part of somite S2. In (C)–(E), the z-plane selected corresponds to the position of the center of each cell being tracked; corresponding z-position is clear in (B). White arrows label the elongating fast muscle progenitors.

(F) Maps of the timing of fast muscle elongation constructed at somite stage S2 (i) at parasagittal planes 20 μm (ii) or 36 μm (iii) away from the notochord. Cells labeled with gray color did not elongate throughout the primary myogenesis.

(G) The timing of elongation of fast muscle progenitors quantified from the rectangle region of (Fii) along the AP axis in wild-type embryo (blue, n_Cells = 57 from total of 7 somites taken from 6 embryos), embryos under heat shock of hsp70l:dn-fgfr1-eGFP (green, n_Cells = 35 from total of 6 somites taken from 5 embryos), or heat shock of hsp70l:ca-fgfr1 (red, n_Cells = 45 from total of 6 somites taken from 5 embryos). The ellipse represents the minimum-volume covering of data points in each group.

(H and I) Maps of timing of fast muscle elongation (H–Hʹʹ) and cell fates (I–Iʹʹ) at somite stage S2 in in wild-type embryo (H and I), embryos under heat shock of hsp70l:dn-fgfr1-eGFP (Hʹ and Iʹ), or heat shock of hsp70l:ca-fgfr1 (Hʹʹ and Iʹʹ). Images above are taken at somite 16–18.

FGF Signaling Determines MP Cell Fate by Regulating Slow Muscle Migration

(A–C) Time lapses of Eng2a:eGFP expression during MP differentiation in wild-type embryos (A), embryos under treatment of dorsomorphin (DM) at 50 μM (B), or SU5402 at 60 μM (C). White short arrows denote the onset of Eng2a:eGFP expression that can be identified. (Aʹ–Cʹ) The corresponding temporal expression profile of Eng2a:eGFP for individual MP under same conditions of (A–C). All the MPs in each group are sorted along the y axis according to the timing of initial Eng2a:eGFP expression. The color map represents differential intensity of Eng2a:eGFP for each cell. White and red lines label the start and end of the time window for the onset of Eng2a:eGFP expression. (Aʹ) (n_Cells = 22 from total of 6 somites taken from 5 embryos); (Bʹ) (n_Cells = 28 from total of 6 somites taken from 4 embryos); (Cʹ) (n_Cells = 34 from total of 7 somites taken from 5 embryos).

(D–F) Time lapses of Eng2a:eGFP expression in MPs and Eng2a:eGFP+ SSFs in wild-type embryos (D), embryos under treatment of SU5402 at 60 μM (E), and heat shock of hsp70l:ca-fgfr1 (F). White short arrows label Eng2a:eGFP+ SSFs.

(G and Gʹ) The number of MPs and Eng2a:eGFP+ SSFs (Gʹ) per muscle segments in conditions of (D)–(F) (n_Segments = 31, 22, 28, n_Embryos = 7, 7, 5, ^∗∗∗p < 0.001, Student’s t test).

(H–J) Time lapses of slow muscle migration in wild-type embryos (H), embryos under treatment of SU5402 at 60 μM (I), or heat shock of hsp70l:ca-fgfr1 (J). Slow muscles are identified by the expression of Prdm1a:GFP. (Hʹ–Jʹ) The reconstructed transverse view of (H)–(J) with medial to the left and dorsal to the top. White arrows denote the breaking of the slow muscle monolayer by fast muscle elongation. Dash lines label the area at the lateral side of slow muscles. White asterisk in (Iʹ iii) denotes the ectopic ECL cells under SU5402 treatment.

(K) Timing of the initiation of lateral migration in wild-type embryos (n_Somites = 15 from 5 embryos), embryos under dorsomorphin (DM) treatment at 50 μM (n_Somites = 12 from 5 embryos), heat shock of hsp70l:ca-fgfr1 (n_Somites = 12 from 5 embryos), SU5402 treatment at 60 μM (n_Somites = 15 from 5 embryos), and heat shock of hsp70l:dn-fgfr1-eGFP (n_Somites = 15 from 5 embryos) (^∗∗∗p < 0.001, ^NSp > 0.05, Student’s t test).

(L) The relative size of lateral area (labeled with dash lines in Figures 6H–6J) throughout the lateral migration of SSFs. Blue, green, and red denote the profiles under conditions of (H)–(J) (total of 5 somites from 5 separate embryos in each condition). ^∗∗∗p < 0.001, Student’s t test. Images taken at muscle segments 16–18.

Reciprocal Interactions of Fast and Slow Muscle Fibers Guides Their Differentiation

(A–C) The lateral migration of SSFs (A–C) and MPs specification (Aʹ–Cʹ) in wild-type embryos (A), Ripply1 morphants (B), and tbx6^−/− mutants (C). Slow muscles are either labeled with Prdm1a:GFP (A and B) or Smyhc1:lyn_eGFP (C). MPs are either labeled with Eng2a:eGFP (Aʹ and Bʹ) or 4D9 antibody (Cʹ). Images are taken at muscle segments 12–16 at 30 hpf.

(D) The timing of elongation of individual fast muscle progenitors along the DV midline from a region corresponding to four adjacent somites. Shaded regions represent individual somites in wild-type embryos. Blue, orange, and violet dots denote cells from wild-type embryos (n_Embryos = 5), Ripply1 morphants (n_Embryos = 5), and tbx6^−/−mutants (n_Embryos=5), respectively. Cells with ECL cell fate are placed at the top of the panel.

(E) AP dependence of the timing of elongation relative to somite segmentation of individual fast muscle progenitors along the DV midline in wild-type embryos (n_Cells = 57 from total of 7 somites taken from 6 embryos), prdm1a^−/− (n_Cells = 70 from total of 6 somites taken from 6 different embryos), and smo^−/− mutants (n_Cells = 42, from total of 4 somites taken from 4 different embryos). The ellipse represents the minimum-volume covering of data points in each group.

(F) Time-lapse of the patterning of fast muscle fibers in wild-type embryos at muscle segments 16–18. The fusing fast muscle progenitors are labeled with blue and yellow asterisks. White short arrows label the dissolving membrane between the fusing cells. The intercalated slow and fast muscles are labeled with long white arrows.

(G–H) Parasagittal optical view of wild-type embryos and prdm1a^−/− mutant labeled with membrane marker lyn-td tomato at muscle segments 12–15. Transverse views of (G) and (H) are displayed in (Gʹ) and (Hʹ) with dorsal to the left and lateral to the top and manually segmented in (Gʹʹ) and (Hʹʹ).

(I) Distribution of the transverse size of fast muscle fibers in wild-type embryo (n_Cells = 1246 from total of 15 muscle segments taken from 8 embryos) and prdm1a^−/− mutant (n_Cells = 1126 from total of 12 muscle segments taken from 6 embryos) (^∗∗∗p < 0.001, Student’s t test). (Iʹ) Probability density of (I).

(J) Schematic presentation of how FGF signaling regulates MPs differentiation in wild-type embryos by precisely controlling the timing of SSFs migration and hence their exposure to Shh and BMP signaling. (Jʹ) In the absence of FGF signaling, ectopic MPs are induced not only at the expense of eng+ SSFs but also in an extended time window due to the delay of lateral migration.