Table 1.
Sequence of oligomorpholinos.
Figure 1.
Myod is required for lateral myogenesis.
(A) Western blot with anti-flag and anti-tubulin antibodies of gastrula embryos injected bilaterally at the two-cell stage with 300 pg of 5utrMyf5F, 5utrMyodF or 70 pg of 5utrMrf4F synthetic mRNAs alone or with oligomorpholinos. Arrows point out the specific signal. Lane 1: synthetic mRNA alone, 2: synthetic mRNA+moControl, 3: synthetic mRNA+specific mo (moMyf5, moMyod1 or moMrf4-1 with 5utrMyf5F, 5utrMyodF or 5utrMrf4F respectively). (B) Whole-mount in situ hybridization of embryos unilaterally injected with 20 ng of moMyf5, moMyod1 or moMrf4-1 and fixed at stages 14 or 18/19. β-galactosidase mRNA (blue) was co-injected to identify the injected side, indicated by an asterisk (*). Dorsal views. The anterior side of the embryos is on the left; st., stage. (C) Transverse sections of the morphants at stage 26. (D) Transverse section of the Myod morphant submitted to whole-mount immunohistochemistry with the 12/101 antibody. (E) Rescue experiments: Embryos were injected unilaterally with 20 ng of moMyod1 alone or co-injected with synthetic mRNA coding for MyodF (150 pg) and probed with MyhE3. Nc, notochord. Probes are in a framed box and indicated for each panel. For complete statistical data, see supporting information, figure S2.
Figure 2.
Mef2d drives lateral Myod expression.
(A) In situ hybridization of Myod and Mef2d at gastrula and neurula stages realized from time-course experiment with embryos of the same fecondation pool. Brackets show initial posterior Myod expression. (B) Single and double in situ hybridization with Mef2d (blue) and Myod (purple) show that Mef2d precedes Myod expression in the posterolateral domain of presomitic mesoderm (arrows) at stage 12.5/13. (C) Embryos injected unilaterally with 400 pg of Mef2dF mRNA. (D) Western blot with anti-flag and anti-tubulin antibodies of late gastrula embryos injected bilaterally either with 300 pg of 5utrMef2dF synthetic mRNA alone (lane1) or with oligomorpholinos: moControl (lane 2), moMef2d1 (lane3), or moMef2d2 (lane 4). (E) Embryos injected unilaterally with 20 ng of moMef2d1 and submitted to in situ hybridization to detect either Myod or Desmin mRNA. (F) Co-injection of Mef2dF mRNA (200 pg) with moMef2d1 was able to rescue the phenotype of moMef2d1 embryos. (G) Unilateral injection of MyodF mRNA (300 pg) or moMyod1 and detection of Actc and Mef2d expression at stage 16. β-galactosidase mRNA (blue) was co-injected to identify the injected side, indicated by an asterisk (*). Vertical lines define the limit between anterior and trunk regions. Probes are in a framed box and indicated for each panel. Dorsal views. the anterior side of the embryos is on the left; st., stage. For complete statistical data, see supporting information, figure S2.
Figure 3.
Mef2d is necessary for the activating effect of Fgf on Myod expression at the neurula stage.
(A) Embryos were injected unilaterally either with Fgf8b synthetic mRNA alone (5 pg), or with moMyod1 or with moMef2d1, fixed at the gastrula stage and submitted to in situ hybridization for Xbra, Myod or Mef2d. Co-injection of Mef2dF mRNA with moMef2d1 was able to rescue the phenotype of moMef2d1 embryos (+Mef2dF). (B) Unilateral injection of Fgf8b mRNA induced the lateral expansion of Mef2d expression domain at stage 16. (C) Embryos were injected unilaterally with 20 ng of XIMOF8 and fixed at stage 16. Co-injection with Mef2dF mRNA was able to rescue the phenotype of XIMOF8 embryos (+Mef2dF). (D) Embryos were injected at stage 11.5/12 with DMSO or 5 mM SU5402, an inhibitor of Fgf signaling, and fixed at stage 16. A first unilateral injection of Mef2dF mRNA at the two-cell stage was able to rescue the phenotype of treated embryos (+Mef2dF). Injected side (*) at the bottom except in (A), on the left. Bracket indicates the position of lateral myogenic cells. Probes are in a framed box and indicated for each panel. For complete statistical data, see supporting information, figure S2.
Figure 4.
Mef2d is required for dermomyotome formation.
(A) Embryos injected unilaterally with 400 pg of Mef2dF mRNA or 20 ng of moMef2d1 were fixed at stage 26 and submitted to staining with the specific muscle 12/101 antibody (red). (B) Expression of Pax7 and Pax3 mRNA at stage 17/18 (dorsal view or transverse sections at the trunk level). Co-staining of Pax3 mRNA (purple) and differentiated muscle cells detected by 12/101 antibody (blue). Dotted lines indicate the position of the medial and lateral population of myogenic cells [5]. (C) Expression of Pax3 mRNA on a transverse section at stage 22. (D) Pax3 mRNA expression on lateral view (left) or on transverse section (right) at the tailbud stage after unilateral injection of Mef2dF or moMef2d1. Rescue experiments restored the phenotype of moMef2d1. Arrows indicate the sites of lateral ectopic expression of Pax3. (*) Injected side. Probes are in a framed box and indicated for each panel. Nc, notochord. For complete statistical data, see supporting information, figure S2.
Figure 5.
Paraxis and Mef2d have a cooperative effect on dermomyotome formation.
(A) Expression of Paraxis mRNA during neurulation. Costaining of Mef2d (blue) and Paraxis (purple) mRNA at stage 13 in comparison with Mef2d expression alone. Rounded brackets indicate the region of colocalization of Paraxis and Mef2d. Dorsal views. The anterior side of the embryos is on the left; st., stage. Vertical lines define the limit between anterior and trunk region. (B) Transverse sections of the costained embryos compared to Mef2d staining at stage 13 (upper panels). Expression of Paraxis and Mef2d at stage 17/18 (lower panels). Dotted lines indicate the position of the medial and lateral population of myogenic cells. (C) Expression of Paraxis mRNA on a transverse section at stage 23. (D) Western blot with anti-flag and anti-tubulin antibodies of late gastrula embryos injected bilaterally either with 300 pg of 5utrParaxis synthetic mRNA alone (lane1) or with oligomorpholinos: moControl (lane 2) or moParaxis1 (lane3). (E) Embryos injected unilaterally with 20 ng of moParaxis1 were submitted to in situ hybridization with Pax3 antisense probe at the tailbud stage (lateral view or transverse section). A co-injection of ParaxisF’ mRNA (150 pg) with moParaxis1 was able to rescue the phenotype of moParaxis1 injected embryos (lateral view). (F) Unilateral injection of ParaxisGRF (150 pg) induced an increase of Pax3 mRNA expression at the tailbud stage. Pax3 expression after co-injection of ParaxisF+Mef2dF or ParaxisGRF+Mef2dF. ParaxisGRF injection with moMef2d1 had no effect on Pax3 expression. (*) Injected side. Probes are in a framed box and indicated for each panel. Nc, notochord. For complete statistical data, see supporting information, figure S2.
Figure 6.
Lateral presomitic cells expressing Meox2 are the dermomytome progenitors.
(A) Expression of Meox2 mRNA in the most lateral region (rounded brackets) of presomitic mesoderm at stage 14 (dorsal view and transverse section), 16 (dorsal view), 17/18 (dorsal view and transverse section) and 23 (transverse section). Dotted lines indicate the position of the medial and lateral population of myogenic cells. Vertical lines define the limit between anterior and trunk region. Nc, notochord. (B) Lineage tracing experiments: embryos were injected with WGA-rhodamine (red) in the most lateral presomitic mesoderm (a) or co-injected with WGA-fluorescein (green) in the lateral presomitic mesoderm (c) at stage 13. Transverse sections of the embryo at the trunk level and at stages 18 or 23; embryo injected with WGA-rhodamine and submitted to indirect immunofluorescence with 12/101 antibody followed by secondary Alexa fluor 488 anti-mouse antibody (green) (b) or injected with the both tracers (d, e and f). WGA-rhodamine fluorescence (d), WGA-fluorescein fluorescence (e), merge (f). Dotted lines indicate the bilateral symmetry plan. In (a) and (c): Vertical lines define the limit between anterior and trunk region. (a) and (c) dorsal views, anterior side on the left. (C, D and E). Ablation experiments of presomitic mesoderm at stage 14. After microdissection experiments, embryos were fixed at stage 19 or at the tailbud stage to analyze meox2 and pax3 expression respectively. (C) sham-operated embryos, ectoderm was incised at the lateral level (line), ectoderm and mesoderm were separated from each other on the lateral side, but mesoderm was not removed. (D) Same operation on the mediolateral level but superficial mesoderm was removed (hatched zone) (E) Same operation on the lateral level but superficial mesoderm was removed (hatched zone). Brackets show the axis level corresponding to incision. For complete statistical data, see supporting information, figure S2.
Figure 7.
Paraxis and Mef2d targets Meox2 gene in the most lateral part of the presomitic mesoderm.
(A) Embryos injected unilaterally with 20 ng of moParaxis1 or moMef2d1 were submitted to in situ hybridization with Meox2 antisense probe at the neurula stage. Injection of either ParaxisF’ mRNA with moParaxis1 or Mef2dF with moMef2d1 was able to rescue the phenotype. (B) Unilateral injection of either ParaxisGRF or Mef2dGRF induced an increase of Meox2 mRNA expression at the neurula stage after induction by dexamethasone (DXM) at stage 12.5. A cooperative effect was observed after co-injection of ParaxisGRF and Mef2dF. A treatment by cycloheximid (CHX) followed by induction by dexamethasone (DXM) at stage 12.5 indicated that Meox2 is a direct target gene of Paraxis and Mef2d. (*) Injected side. Probes are in a framed box and indicated for each panel. (C) COS7 cells were transfected with pmeox2-luc alone, or co-transfected with either Paraxis, Mef2d-V5/His or both and luciferase activity was determined 48 h after transfection. * P<0.01 (D) Protein extracts from COS7 cells transfected with Mef2d-V5/His alone or with either empty vector (Gal4) or Gal4-Paraxis construct were immunoprecipitated (IP) with Ni-NTA beads and subjected to Western blot (WB) using an anti-Gal4 antibody (upper panel). Input control experiments with anti-V5 (lower panel) or anti-Gal4 (mid panel) antibodies. For complete statistical data, see supporting information, figure S2.
Figure 8.
Meox2 acts downstream of Paraxis and Mef2d on dermomyotome formation.
(A). Western blot with anti-flag and anti-tubulin antibodies of late gastrula embryos injected bilaterally either with 300 pg of 5utrMeox2F synthetic mRNA alone (lane1) or with oligomorpholinos: moControl (lane 2) or moMeox2-1 (lane3). (B) Embryos injected unilaterally with 20 ng of moMeox2-1 were submitted to in situ hybridization with Pax3 antisense probe at the tailbud stage (lateral view or transverse section). A co-injection of Meox2F mRNA with moMeox2-1 was able to rescue the phenotype of moMeox2-1 (lateral view). (C) Pax3 mRNA expression on lateral views and section of embryos at the tailbud stage after unilateral injection of Meox2F (200 pg). (D, E) Pax3 mRNA expression on lateral view of embryos at the tailbud stage after unilateral injection of moMef2d1(D) or moparaxis1 (E). Rescue experiments by Meox2F (100 pg) restored the phenotype of moMef2d1 (D) and moparaxis1 (E) morphants. Arrows indicate hypaxial expression of Pax3. (*) Injected side. Nc, notochord. For complete statistical data, see supporting information, figure S2.
Figure 9.
Schematic representation of myotome and dermomyotome formation in Xenopus.
(A) The most lateral Meox2 expressing cells of the paraxial mesoderm give rise to the dermomyotome (De) whereas the lateral myogenic cells give rise to a dorsomedial (DoMe) and a ventrolateral (VeLa) cell population. The medial myogenic cells differentiate first and remain associated with the notochord [5]. Arrows design the movement of lateral paraxial mesoderm. Dotted arrows design the movement of invagination of neurectodermal cells. NT, neural tube. Nc, notochord. (B) Mef2d couples lateral myogenesis to dermomyotome formation: In lateral presomitic cells, Mef2d transactivates the Myod gene and in the most lateral presomitic cells Mef2d and Paraxis transactivates the Meox2 gene.