DEV019331 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Table S1. Selected list of genes expressed in skeletal muscles of wild-type and Myf5-Cre//DTA mice. DNA microarray experiments were performed using RNA isolated from wild-type and Myf5-Cre//DTA embryos at E14.5. Hybridization and analysis of GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix) were carried out using established procedures. Genes were selected based on their expression in skeletal muscle.

• Supplemental Figure S1 -

  Fig. S1. Low-level expression of Myf5 in the unsegemented paraxial mesoderm. Semi-quantitative RT-PCR analysis of Myf5, Myod1, myogenin, Myf6, Pax3 and Pax7 mRNAs at E8.5. RNAs were isolated from the unsegemented caudal part of E8.5 embryos and from the head. GAPDH served as a loading control.

• Supplemental Figure S2 -

  Fig. S2. Formation of skeletal muscles from myogenin-derived cells at E15.5. Expression of lacZ in E15.5 Rosa26lacZ embryos crossed with myogenin-Cre. lacZ staining (blue) reveals myogenin-derived cells in different skeletal muscles but not in sclerotome-derived cells.

• Supplemental Figure S3 -

  Fig. S3. Loss of skeletal muscles in myogenin-Cre//DTA mice at E18.5. Hematoxylin and Eosin staining of sections from Myf5-Cre//DTA (A,D), Myogenin-Cre//DTA (B,E) and wild-type mice (C,F) at E18.5. Skeletal muscle tissue is absent in myogenin-Cre//DTA but not in Myf5-Cre//DTA mice, which show a virtually normal formation of skeletal muscles at E18.5. Sections from wild-type mice are shown for comparison. Scale bars: 200 µm.

• Supplemental Figure S4 -

  Fig. S4. Start of ablation of Myf5-expressing cells in somites of Myf5-Cre//DTA mice at E9.5. Immunofluorescence staining of somites of Myf5-Cre//DTA (A,C) and wild-type embryos (B,D) at E9.5 using a combination of Myf5 and Pax7 antibodies. Somites from the cranial part of embryos are shown. Myf5-expressing cells are reduced in number in Myf5-Cre//DTA, which indicates the initiation of ablation of Myf5-expressing cells. Broken lines indicate the contours of the embryos. Scale bars: 100 µm.

• Supplemental Figure S5 -

  Fig. S5. Induction of apoptosis by Myf5Cre-mediated activation of DTA. TUNEL assay of sections derived from Myf5-Cre//DTA (A-C,G-I) and wild-type (D-F,J-L) embryos at E9.5 and E10.5. Activation of DTA resulted in a significant increase of the number of apoptotic cells in somites of Myf5-Cre//DTA compared with wild-type embryos. Inlets depict enlarged areas of somites. Arrowheads indicate apoptotic cells. Scale bars: 200 µm.

• Supplemental Figure S6 -

  Fig. S6. Efficient DTA-mediated ablation of Myf5-derived cells at E10.5. In situ hybridization of Myf5-Cre//DTA (B,D,H,J,F,L) and wild-type (A,C,G,I,E,K) embryos at E10.5 with probes against Myf5 (A,B,G,H), Myod1 (C,D,I,J) and myogenin (E,F,K,L). Sections were derived from more mature cranial (A-F) and more immature caudal (G-L) somites of embryos. All cells that expressed Myf5 and Myod1 were successfully ablated in the cranial parts of Myf5-Cre//DTA embryos, while this process was still under way in the caudal parts at this stage. Residual amounts of myogenin mRNA were detected in the cranial and caudal parts of Myf5-Cre//DTA embryos. Scale bars: 100 µm.