Additional file 1 of Transcriptome analyses based on genetic screens for Pax3 myogenic targets in the mouse embryo
Creators: name: Lagha, Mounia
name: Sato, Takahiko
name: Regnault, Béatrice
name: Cumano, Ana
name: Zuniga, Aimée
name: Licht, Jonathan
name: Relaix, Frédéric
name: Buckingham, Margaret
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 1:Supplementary Figures S1-S3. Figure S1: Examples of transcript validation and analysis of the expression of Zic1, negatively regulated by Pax3. (A, B) Examples of the analysis of transcripts in different Pax3-GFP cell populations isolated from Pax3GFP/+ embryos at E10.5. (A) RT-PCR analysis of RNA extracted from FACs sorted GFP positive and GFP negative cells from Pax3GFP/+embryos, as indicated in Figure 1 showing that transcripts for the neural tube markers, Sox2 and Sox10, which are potential Pax3 targets in neural crest, since the transcripts are enriched in whole somite preparations which include neural tissue. (B) RT-PCR analysis of transcripts in GFP positive cells from the hypaxial versus whole somites of Pax3GFP/+embryos. c-Met, Six1, Sim1 and Zic1 transcripts are shown, with HPRT transcripts as a control. Zic1 expression is higher in the whole somite preparation, consistent with a more epaxial location. (C) Immunohistochemistry with Zic1 and Pax3 antibodies on a transverse section of an interlimb somite of an E10.5 embryo, showing Zic1 protein mainly detected in Pax3 positive epithelial cells of the epaxial dermomyotome (Ep), as well as in the dorsal neural tube (NT) and in mesenchymal cells between the neural tube and somites. Zic1 is mainly absent from Pax3 positive migratory neural crest in this region. (D, E) Whole mount in situ hybridization with a Zic1 probe on posterior somites of control Pax3GFP/+ (D) and mutant Pax3GFP/GFP (E) embryos at E11, showing up-regulation in the absence of Pax3. Cell death is extensive in the hypaxial domain of more anterior somites by this stage, but despite some loss of cells in immature posterior somites, Zic1 hybridisation is still higher in the Pax3 mutant. (F, G) Transverse sections of immature posterior somites from Pax3GFP/+ (F) and Pax3GFP/GFP (G) embryos at E11. In this tail region, immature somites have not yet undergone cell death. In the control (F), transcripts are concentrated in the more central and epaxial domain whereas in the Pax3 mutant (G) they are detected throughout the somite. NT, neural tube. Figure S2: The onset of myogenesis in Sprouty1 mutants and reduction of Sprouty2 expression in the absence of Pax3. (A-D) Whole mount in situ hybridisation on Sprouty1lacZ/+ (A, C) and Sprouty1lacZ/- (B, D) embryos at E10 (somite stages (ss) are indicated), using Myf5 (A, B) and MyoD (C, D) probes. FL, forelimb bud. (E, F) In situ hybridisation with a Sprouty2 probe on E9.5 (E), and with a Sprouty4 probe on E10.5 (F), embryos showing interlimb somites and limb buds. There is extensive Sprouty expression in the distal limb buds, but positive Sprouty4 positive myogenic progenitors in the proximal forelimb bud are detectable at E10.5 (arrow). Figure S3: Expression of Gremlin1 and perturbation of MyoD activation in Gremlin1 mutant embryos. (A, B, D-I) Whole mount in situ hybridisation on wild type (A, B), control (PGK-Cre) (D, D'), Pax3PAX3-FKHR/+ (E, E'), Gremlin1+/- (Grem1+/- ) (F, H) and Gremlin1-/- (G, I) embryos at the stages indicated (ss, somite stage), using Gremlin1 (Grem1) (A-E'), Myf5 (F, G) and MyoD (H, I) probes. (C) Transverse section of the embryo shown in (B) at the level indicated by the white bar in (B). Note the expression of Grem1 in the hypaxial domain of the dermomyotome (Hyp). NT, neural tube; DA, dorsal aorta; FL, forelimb bud; HL, hindlimb bud. Arrows and arrowheads point to differences in expression in forelimb buds and more posterior somites, respectively. (PDF 4 MB)