Clu, Clusterin; CM, cutaneous maximus; Etv4, Ets variant gene 4; Gdnf, glial cell line-derived neurotrophic factor; Gfra1, glial cell line-derived neurotrophic factor family receptor alpha 1; MHC, myosin heavy chain; MLC, myosin light chain; MN, motor neuron; Myh1, myosin heavy chain 1; PAX7, Paired box 7; controls CM muscle growth and impacts MN development Ablating in the mesenchyme profoundly disrupts the progression of progenitor migration and subsequently interferes not only with myogenic differentiation but also with axonal growth and complete specification of the cognate MN pool. a side view of entire embryos and indicate the position of higher magnification areas, including that of the top pictures in Fig 1A and 1B as well as the upper forelimb region (with scapulohumeral muscles) and distal forelimb, showing a region where dispersed myocytes accumulate in embryos. The second row images highlight a closer view of the upper thoracic and forelimb region. The dotted lines indicate for each genotype the levels and angle corresponding to the three consecutive sections shown in (C) and (D). (B) Quantification of the number of dispersed myocytes found in orange areas in the forelimb (left plot) and of the area occupied by the ectopic humeral muscle (ectop) appearing between the spinodeltoid (Del) and the triceps brachii (TriBra) muscles (right plot). These data reproduce and Labetalol HCl confirm our own previous results. Underlying data are provided in S1 Data. (C, D) Cross sections of control and mutant E12.5 embryos, featuring three consecutive sections at forelimb levels (Level 1 and Level 2) and upper thoracic level (Level 3), immunostained with Labetalol HCl antibodies against Pax7 (red), Myh1 (green), and neurofilament (white) and with DAPI (blue). Images in (D) represent high-magnification views of the area highlighted with the yellow dotted square in (C). These data confirm (1) the severe reduction in thickness of the CM muscle (Level 3, and higher magnification in [D]), (2) the presence of a robust ectopic muscle next to the triceps brachii (Levels 2 + 3, and higher magnification in [D]), and (3) the presence of dispersed myogenic progenitors and muscle fibers in the ectopic subcutaneous position in the forelimb (the image in [D] shows higher magnification of an area between the digit extensors and the skin). Lack of obvious phenotype in the diaphragm is also shown. CM, Labetalol HCl cutaneous maximus; Del, spinodeltoid; diaph, diaphragm; disp. Myo, dispersed myoblatsts; ectop, ectopic humeral muscle; ext. dig, extensor digitorum; trap, trapezius; TriBra, triceps brachii.(TIF) pbio.2004734.s004.tif (6.6M) GUID:?1FFDB4F8-7A2F-4777-BBD4-A0A48A85EC8B S2 Fig: Analysis of muscle phenotype in embryos. Expression of embryos (right panels). (A) Gdnf expression is usually visualized at three successive anteroposterior levels, showing a hot spot at the brachial plexus (mesenchymal cells around passing nerves), where Gdnf expression is usually drastically reduced by the absence of embryos exhibit a thinner CM with less overall signal. (B) On sections corresponding to the anterior part of the CM muscle, expression of markers of muscle differentiations (embryos exhibit a selective loss of staining in the CM and not other neighboring muscle masses. CM, cutaneous maximus; alters motor innervation of the CM muscle. (A, B) The nerve pattern was analyzed by IHC with antibodies against neurofilament (2H3 antibody) (A) or Labetalol HCl by taking advantage of the Hb9-GFP transgene (S1 Table) (B), which labels motor neurons and their axons. (A) Anti-neurofilament histochemistry on whole-mount wild-type and embryos at E12.0. (B) Hb9-GFP was visualized with antibodies against GFP (top and middle images) or by direct fluorescence imaging in (= 35, same sample set as in controls of Fig 2); red dots: (= 12). Underlying data are provided in S1 Data. BB-BA, benzyl-benzoate/benzyl-alcohol mix; CM, cutaneous maximus; IHC, immunohistochemistry; PFA, paraformaldehyde.(TIF) pbio.2004734.s006.tif (2.1M) GUID:?5CB48043-B96F-4712-85F3-31ED39C504AE S4 Fig: Validation of Fat1 IHC with antibodies against the Fat1-LacZ fusion. (A) Scheme of the protein products of a wild-type allele (full-length Fat1) and of a allele (producing a chimaeric protein with the first 8 cadherin domains of Fat1 extracellular domain name, fused to an exogenous transmembrane domain name in frame with Rabbit Polyclonal to LAMA5 -galactosidase as intracellular domain name). An antibody to Fat1 (Sigma 1869) directed against a portion of the common segment of Fat1 extracellular domain name recognizes both proteins, whereas an antibody to -galactosidase recognizes only the Fat1C-gal fusion protein, most of which is usually sequestered in the Golgi apparatus and not localized at the cell membrane. (B) Comparison of immunohistochemical detection of Fat1 in a embryo using the anti–galactosidase antibody (red), the Fat1-1869 antibody (green), and the pattern of -galactosidase activity revealed by Salmon-Gal staining on cross sections of an E13.5 mouse embryo at lumbar levels where it is possible to detect both the expression in subsets of MNs and in the caudal-most part of the mesenchymal subcutaneous layer, towards which the CM extends. The Fat1C-gal fusion protein is mainly detected by both antibodies as punctae in the Golgi, most likely because the protein is usually misfolded and does not reach the cell membrane. The two stainings perfectly overlap (except for some weak staining of blood vessels in the green channel, mostly due to the secondary antibody) and correspond to the signal detected by Salmon-Gal staining. -gal, -galactosidase; CM, cutaneous maximus; epax, epaxial; MNs, motor.