Volume 36, Issue 5, Pages 550-561 (March 2016) Localized LoxL3-Dependent Fibronectin Oxidation Regulates Myofiber Stretch and Integrin-Mediated Adhesion  Ortal Kraft-Sheleg, Shelly Zaffryar-Eilot, Olga Genin, Wesal Yaseen, Sharon Soueid-Baumgarten, Ofra Kessler, Tatyana Smolkin, Gal Akiri, Gera Neufeld, Yuval Cinnamon, Peleg Hasson  Developmental Cell  Volume 36, Issue 5, Pages 550-561 (March 2016) DOI: 10.1016/j.devcel.2016.02.009 Copyright © 2016 Elsevier Inc. Terms and Conditions

Developmental Cell 2016 36, 550-561DOI: (10.1016/j.devcel.2016.02.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 LoxL3 Is Expressed in the Extremities of Differentiated Myofibers (A–A″) Whole-mount staining of LoxL3 (A, red in merge) and MHC (A′, green in merge) in an E10.0 embryo. Arrows highlight myofibers that have not reached the somitic boundaries yet express LoxL3 at their tips (A″). (B–C″) Transverse sections through an E10.5 mature somite at the somitic boundary (B) or at the somite's central domain (C) as shown in insets. Somites were immunostained for LoxL3 (B, C, and red in merge) and myosin heavy chain (B′, C′; green, MHC). Whereas LoxL3 is co-expressed with MHC at the ends of the somite (B, white arrows), it is not present in the center of the somite (C, empty arrows). (D–D″) Lateral section through an E11.5 embryo stained for LoxL3 (D) and Pax3 (D′) demonstrating that LoxL3 is not expressed in Pax3-expressing dermomyotomal cells. (E–E″) LoxL3 RNA in situ hybridization at E10.5 demonstrates that LoxL3 mRNA is also localized to somitic boundaries (E, black arrows). Somite myofibers are marked by MHC (E′, green). Developmental Cell 2016 36, 550-561DOI: (10.1016/j.devcel.2016.02.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 LoxL3 Mutant Mice (A) Cassette used to generate the LoxL3 mutant allele (adapted from https://www.mousephenotype.org/data/alleles/MGI:1337004/tm1a(EUCOMM)Wtsi/). (B) qRT-PCR demonstrates that no transcript is present in mutant embryos. RQ, relative quantity of LoxL3 to Hprt. (C) Western blot analysis for LoxL3 protein from wt, heterozygous, and homozygous mutant embryos demonstrates reduced amounts of LoxL3 protein in heterozygous embryos and no protein in LoxL3−/− embryos. (D) Table summarizing genotyping results from LoxL3 heterozygous intercrosses. Developmental Cell 2016 36, 550-561DOI: (10.1016/j.devcel.2016.02.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Myogenesis Is Affected in LoxL3 Mutants (A) qRT-PCR analysis of MyoD demonstrates no significant changes between wt and LoxL3 mutant E10.5 embryos. Data represent mean ± SD. (B and C) RNA in situ hybridization for MyoD demonstrates defective somitogenesis in LoxL3 mutant embryos; somites are smaller and have a triangular rather than rectangular shape in mutants. (D–G) Whole-mount MHC immunostaining in a wild-type (D) or Scx-GFP background (F) reveals the two main observed phenotypes displayed in LoxL3 mutant embryos: wavy and unstretched myofibers that anchor at an adjacent somite (E) or at the center of the somite (G, arrow). Tendon progenitors are also disorganized in mutants (F″ and G″). (D′–G′) are higher magnifications of the boxed areas in (D–G), respectively. (H and I) Immunostaining for CD31/PECAM reveals that intersomitic vessels are not affected by the loss of LoxL3. Developmental Cell 2016 36, 550-561DOI: (10.1016/j.devcel.2016.02.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Lox Enzymatic Activity Regulates Myofiber Anchorage at the Somitic Boundary Inhibition of chick Lox family members' enzymatic activity following the administration of BAPN results in wavy and unstretched (arrowhead) myofibers that disobey the somitic boundaries (arrows) as determined by MHC immunostaining at HH 18. (A′) and (B′) show higher magnifications of boxed areas in (A) and (B). Developmental Cell 2016 36, 550-561DOI: (10.1016/j.devcel.2016.02.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Integrin Signaling Is Reduced in LoxL3 Mutant Myofibers In vitro differentiated myofibers derived from wt (A, B, D, F, and F′) or LoxL3−/− (C, E, G, and G′) SCs. LoxL3 is expressed at myofiber termini (red; arrowheads; composite image from two stitched images; white line) (A). Straight and tense fibers are derived from wt SCs (B) but not from mutant SCs (C). F-actin staining reveals that the cytoskeletal arrangement is abnormal in LoxL3 mutant myofibers (D and E). Phosphorylated Paxillin (p-Pax) marking integrin-dependent focal adhesions is strongly reduced in LoxL3−/− myofibers; two examples of each genotype are shown (F and G). Arrows in (F-G′) mark myofiber termini. Note the reduced size and number of p-Pax foci in mutant myofibers. Arrowheads in (G) and (G′) mark p-Pax staining in nearby fibroblasts demonstrating the validity of the immunostaining procedure. Developmental Cell 2016 36, 550-561DOI: (10.1016/j.devcel.2016.02.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 LoxL3 Directly Oxidizes Fibronectin to Activate Integrin Signaling (A and B) Immunoprecipitation of LoxL3 reveals that Laminin (A) and FN (B) are co-precipitated. (C) In vitro oxidation assay demonstrates that FN, but not Laminin, is oxidized by LoxL3, an oxidation which is inhibited by BAPN. (D) Western blot analysis shows that MCF7LOXL3 cells have augmented levels of p-Pax when compared with the control cells (MCF7Vector; n = 3). (E and F) Significant enhancement of p-Pax, relative to total Pax, in MCF7 cells is induced following the pretreatment of FN matrix with CMLOXL3 (E, n = 3) or with purified recombinant LOXL3 (F, n = 6). (G–I′) Representative images demonstrating a higher number of p-Pax foci observed in myofibers (wt and LoxL3−/−) when differentiated on FN matrix that was pretreated with CM containing LOXL3. WT SCs plated on FN matrix pretreated with CMVector (G and G′) or CMLOXL3 (H and H′). LoxL3−/− SCs plated on FN matrix pretreated with CMLOXL3 (I and I′). White arrowheads in (G), (H), and (I) mark prominent intrafiber p-Pax foci, whereas empty arrows mark p-Pax foci at myofiber tips. In (G′), (H′), and (I′) p-Pax alone is shown. (J) Quantification of the number of intrafiber p-Pax following the distinct treatments. Data in (E), (F), and (J) represent mean ± SD. ∗p < 0.05, ∗∗∗p < 0.001. N.S., not significant. Developmental Cell 2016 36, 550-561DOI: (10.1016/j.devcel.2016.02.009) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Assembly of Fibronectin Matrix Depends on LoxL3 Fibronectin matrix (red) forms along the somitic boundaries in wild-type E10.5 embryos (A) but is abnormal in LoxL3 mutants (B). This effect is most pronouncedly observed in locations where myofibers (green) cross the somitic borders (arrows in B). Developmental Cell 2016 36, 550-561DOI: (10.1016/j.devcel.2016.02.009) Copyright © 2016 Elsevier Inc. Terms and Conditions