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First published online 14 January 2004
doi: 10.1242/dev.00967

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Ectopic Myf5 or MyoD prevents the neuronal differentiation program in addition to inducing skeletal muscle differentiation, in the chick neural tube

Marie-Claire Delfini^* and Delphine Duprez

Biologie du Développement, UMR 7622, Université P. et M. Curie, 9 Quai Saint-Bernard, Bât. C, 6^eE, Case 24, 75252 Paris Cedex 05, France

View larger version (106K): [in a new window]   Fig. 1. MyoD and Myogenin but not Myf5 are upregulated in neural tissues under the influence of ectopic mouse Myf5 and MyoD, 1 day after electroporation. Adjacent transverse sections, at the forelimb level of embryos electroporated with either mouse Myf5 (A-D) or MyoD (E-H), 1 day after electroporation were hybridised with the DIG-labelled antisense probes for mouse Myf5 (A), MyoD (E), (chick) Myf5 (B,F), (chick) MyoD (C,G) and (chick) Myogenin (D,H). Myf5 is never induced (B,F), although ectopic expression of MyoD (C,G) and Myogenin (D,H) is observed in the neural tube and in neural crest cells, where ectopic mouse Myf5 (A) or MyoD (E) are detected. Ectopic (chick) MyoD is not observed in neural crest cells in G because of the level of the section. Endogenous expression of chick Myf5 (B,F), MyoD (C,G) and Myogenin (D,H) is normally detected in myotomes.

View larger version (128K): [in a new window]   Fig. 2. Ectopic mouse Myf5 or MyoD expression in neural tube leads to skeletal muscle differentiation. Transverse sections of HH28 embryos transfected with either mouse Myf5 (A-C) or MyoD (D-H) were hybridised 4 days after electroporation with the DIG-antisense probes (blue) for mouse Myf5 (A-C) and MyoD (D-F) and then incubated with the MF20 antibody (brown) recognising all isoforms of myosin heavy chains or only incubated with the MF20 antibody (G,H). (G,H) Pictures of a different embryo from that of D-F. (B,C) Successive higher magnifications of the region below the DRG, framed in A. (E,F) Successive higher magnifications of the ventral part of the DRG framed in D. H is a higher magnification of the ventral part of the neural tube framed in G.

View larger version (139K): [in a new window]   Fig. 3. Ectopic muscle differentiation perturbs the axonal projections. Four days after electroporation of embryos with mouse MyoD, transverse sections with mouse MyoD, were successively incubated with the monoclonal antibodies, MF20 (green, A) and 3A10 (red, B), in order to recognise differentiated muscle cells and axonal projections, respectively. (C,D) Merged pictures from A and B showing the absence of overlap between the two antibodies.

View larger version (101K): [in a new window]   Fig. 4. Ectopic mouse MyoD expression in the neural tube switches off the endogenous neuronal differentiation program, 4 days after electroporation. Transverse sections from embryos electroporated with mouse MyoD, 4 days after electroporation were hybridised with the DIG-labelled antisense probe for mouse MyoD (A,B) to show the location of ectopic mouse MyoD. Adjacent sections hybridised with the neural markers, SCG10 (C,D) and BEN (E,F) and then incubated with the MF20 antibody (D-F) show that neuronal markers are downregulated in the regions where are visualised muscle differentiation, assayed by MF20 labelling. (B,D,F) Higher magnifications of the ventral regions of the neural tube, framed in A,C,E, respectively. One day after electroporation, transverse adjacent sections (G,H), hybridised with the DIG-labelled antisense probe for mouse MyoD (G) and SCG10 (H) show a downregulation of SCG10 expression on the electroporated (right) side compared with the control (left) side (H).

View larger version (140K): [in a new window]   Fig. 5. Consequences of forced expression of mouse Myf5 or MyoD in the neural tube for negative regulators of myogenesis. Adjacent transverse sections of embryos electroporated with mouse MyoD 1 day after electroporation were hybridised with the DIG-labelled antisense probes for mouse MyoD (A,E). The corresponding adjacent sections were hybridised with the ligands Delta1 (B), Serrate2 (C), the receptor Notch1 (D) and the HLH transcription factor Id2 (F). The framed regions in C,D show the right sides of the electroporated-neural tubes, where the expression of Serrate2 (C) and Notch1 (D) is upregulated compared with the control left sides of the neural tubes. The arrows in E,F show the areas where Id2 expression is upregulated (F) corresponding to the regions of the ectopic mouse MyoD (E).

View larger version (156K): [in a new window]   Fig. 6. Location of ectopic Notch receptor and ligand, 4 days after electroporation. Transverse sections from embryos electroporated with mouse MyoD (A-E) were hybridised 4 days after electroporation with the DIG-labelled antisense probe for mouse MyoD (C), Notch1 (A,B,D) and Serrate2 (E) followed by an immunohistochemistry using MF20 (A-D). The arrowhead in B shows the ectopic expression of Notch1 outside the MF20-positive cells. Arrows indicate the ectopic MF20-positive cells (B,D) and the muscle cells expressing the ligand Serrate2 (E).

View larger version (115K): [in a new window]   Fig. 7. Consequences for different muscle markers of forced expression of mouse Myf5 or MyoD in the neural tube. Transverse sections from different embryos electroporated with either mouse MyoD (A,B,E-H) or Myf5 (C,D) were hybridised 1 day after electroporation with the DIG-labelled antisense probe for mouse MyoD (A,E) and Myf5 (C). The corresponding adjacent transverse sections were hybridised with different muscle markers: Mef2c (B), Pax3 (D), Six1 (F), Mox1 (G), Mox2 (H). Ectopic MRF upregulates the expression of Mef2c (A,B) but not that of the other genes. The sections corresponding to three distinct embryos are grouped according to embryo.

View larger version (116K): [in a new window]   Fig. 8. Mouse MyoD activates the expression of FgfR4 in the chick neural tube. One day after electroporation with mouse MyoD, transverse sections were hybridised with the DIG-labelled antisense probe for mouse MyoD (A) and FgfR4 (B). FgfR4 expression is clearly upregulated in the electroporated (right) side of the neural tube. Six hours after electroporation of mouse MyoD, FgfR4 expression is ectopically detected in a region similar to that of GFP location (C,D), whereas electroporation of GFP alone does not induce any FgfR4 expression in the neural tube (E,F).