DEV029264 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Expression of follistatin-like 1 and follistatin-like 2. (A) Expression of these genes was not present. (B) Lateral view, animal pole at the top. (C,D) Lateral views, ventral to the left. (E-H) Dorsal views, with anterior to the top. (I,J) Transverse sections through the trunk of six-somite embryos. (K,L) Lateral views, with anterior towards the left. (M,N) Transverse sections of 24 hpf embryos at the level of somites 10-15.

• Supplemental Figure S2 -

  Fig. S2. Phenotype of translation-blocking fstl1/2 morpholinos. The size of the notochord and tailbud are increased in embryos injected with a single fstl morpholino, and to a greater extent when fstl1/2 morpholinos are injected in combination. Brackets illustrate the domain observed in control embryos.

• Supplemental Figure S3 -

  Fig. S3. BMP attenuation in tBR embryos heat-shocked during late gastrulation. Wild-type and tBR embryos were heat-shocked during late gastrulation and fixed at 30-minute intervals. Antibodies against PSMAD1/5/8 (red) labels cells responding to BMP activity at the times and backgrounds indicated.

• Supplemental Figure S4 -

  Fig. S4. Ventral margin extirpation. (A) Model of the procedure. Approximately 100 cells were removed from the ventral margin (red) of 80% epiboly embryos. All images shown are of embryos with their ventral margins removed. (B-D) flh (B), ntl (C) and admp (D) expression in embryos at 90% epiboly. (E-E′′) Antibodies against PSMAD1/5/8 (red) and BrdU (green) in longitudinal sections of the tail of 14-somite stage embryos. (F) High-magnification view of notochord cells of 24 hpf embryos at the level of the cloaca. (G,H) shha and ptc1 expression in 24 hpf.

• Supplemental Figure S5 -

  Fig. S5. BrdU incorporation of embryos treated with the cell cycle inhibitors hydroxyurea and aphidicolin. Embryos were treated from 80% epiboly to bud stage, then inhibitors were removed by washing. (A,B) BrdU incorporation of DMSO- (A) and hydroxyurea/aphidicolin-treated (B) bud stage embryos. (C-C′′) Antibodies against PSMAD1/5/8 (red) and BrdU (green) in longitudinal sections of the tail of 14-somite hydroxyurea/aphidicolin treated embryos. (D,E) BrdU incorporation of DMSO- (D) and hydroxyurea/aphidicolin-treated (E) 24 hpf embryos.

• Supplemental Figure S6 -

  Fig. S6. Bmp2/7 do not affect notochord morphology. (A,B) gsc and flh expression in 90% epiboly embryos that have had BSA- (A) or Bmp2/7- (B) soaked beads implanted into axial tissue. (C,D) The notochord of 24 hpf embryos implanted with BSA- (C) or Bmp2/7- (D) soaked beads. Beads are located anterior to the region shown in D. (E,F) ntl expression in 27 hpf embryos implanted with BSA- (E) or Bmp2/7- (F) soaked beads. (G,H) High-magnification images of the notochord at the level of the hind yolk extension of 48 hpf embryos that were implanted with BSA- (G) or Bmp2/7- (H) soaked beads. fp, floorplate (A,B) Dorsal views, with anterior towards the top. Bars in A,B indicate the width of chordamesodermal domain at its widest point in the control embryo in A. All other views are lateral views, with anterior towards the left.

• Supplemental Figure S7 -

  Fig. S7. Myotome patterning as a proxy of notochord maturity. (A-C) myod1 expression in 9-somite stage embryos is increased in adaxial and medial fast muscle cells of Fstl1/2 morphants (B), and is reduced in the medial fast muscle domain of tBR embryos heat-shocked at 80% epiboly (C). (D-F) Immunostaining against Myod1 and F-actin reveals an increase in Myod1-positive cells of the medial fast muscle domain of Fstl1/2 morphants (E), and a decrease in Myod1-positive cells in tBR embryos (F). Images are of the anterior-most three somites. The mean number of Myod1-stained cells from five experimental embryos was subjected to one-way ANOVA, followed by a two-tailed, equal variance t-test. Asterisks indicate significance (P<0.001). (G-I) Immunostaining with the F59 antibody, which recognizes mainly slow muscle, reveals that the horizontal myoseptum is increased in 24 hpf Fstl1/2 morphant embryos (H). Muscle fibers are disorganized in tBR embryos (I). (J-L) Immunostaining with antibodies against 4D9, which labels Engrailed-positive cells (red), and Prox1, which labels slow muscle cells (green), reveal an increase in medial fast muscle (red) and slow muscle pioneers (yellow) in 24 hpf Fstl1/2 morphants (K), and a reduction of Engrailed positive cells in tBR embryos (L). (M-O) Sections through the trunk level of 24 hpf embryos confirm the results in J-L. Antibodies against PSMAD1/5/8 (red) and Engrailed (green) reveal an increase in PSMAD1/5/8-positive chondrocytes as well as medial fast muscle fibers and slow muscle pioneers (green) in Fstl1/2 morphants (N). Chondrocytes do not form in tBR embryos, and the domain of medial fast muscle fibers and slow muscle precursors is reduced (O).