1 ALK5-mediated TGF-β signaling in neural crest cells controls craniofacial muscle 2 development via tissue-tissue interactions 3 * 4 Arum Han, Hu Zhao, Jingyuan Li, Richard Pelikan, and Yang Chai 5 6 Center for Craniofacial Molecular Biology, Ostrow School of Dentistry, University of 7 Southern California, Los Angeles, California, 90033, USA 8 9 Competing financial interests 10 The authors declare no competing financial interests 11 12 Running title: TGF-β signaling and tongue development 13 * 14 Author for Correspondence: 15 Dr. Yang Chai 16 George and MaryLou Boone Professor 17 Center for Craniofacial Molecular Biology 18 Ostrow School of Dentistry 19 University of Southern California 20 Tel. (323)442-3480 21 [email protected] 22 23 Key words; myogenesis, TGF-β, BMP, FGF, Alk5, Cranial neural crest cells, Tongue, 24 eye and masticatory muscles 25 Number of characters without space: 31,152 26 27 1 28 Abstract 29 The development of the craniofacial muscles requires reciprocal interactions with 30 surrounding craniofacial tissues that originate from cranial neural crest cells (CNCCs). 31 However, the molecular mechanism involved in the tissue-tissue interactions between 32 CNCCs and muscle progenitors during craniofacial muscle development is largely 33 unknown. In the current study, we address how CNCCs regulate the development of the fl/fl 34 tongue and other craniofacial muscles using Wnt1-Cre;Alk5 mice, in which loss of 35 Alk5 in CNCCs results in severely disrupted muscle formation. We found that Bmp4 is 36 responsible for reduced proliferation of the myogenic progenitor cells in Wnt1- fl/fl 37 Cre;Alk5 mice during early myogenesis. In addition, Fgf4 and Fgf6 ligands were fl/fl 38 reduced in Wnt1-Cre;Alk5 mice and are critical for differentiation of the myogenic 39 cells. Addition of Bmp4 or Fgf ligands rescues the proliferation and differentiation 40 defects in the craniofacial muscles of Alk5 mutant mice in vitro. Taken together, our 41 results indicate that CNCCs play critical roles in controlling craniofacial myogenic 42 proliferation and differentiation through tissue-tissue interactions. 43 44 45 46 2 47 Introduction 48 49 The craniofacial musculature consists of the eye, masticatory, facial, tongue and other 50 head muscles. The development of the craniofacial musculature is distinct from that of 51 the trunk in terms of the origin of the muscles and the genetic programs underlying 52 myogenesis (1). Lineage tracing approaches have indicated that most of the craniofacial 53 muscles originate from cranial paraxial mesoderm, whereas tongue muscles originate 54 from muscle precursors that migrate from the occipital somites and eye muscles originate 55 from mixed populations of paraxial mesoderm and prechordal mesoderm (2,3). 56 Irrespective of this heterogeneity, the migrating myogenic precursors undergo 57 proliferation and differentiation at designated sites where they interact with surrounding 58 tissues during the two-phase process of myogenesis. During primary myogenesis, 59 myogenic precursors proliferate and generate primary myotubes; this occurs in mice 60 during embryonic days E10.5-E12.5. Secondary myofibers are created by the fusion of 61 fetal myoblasts and pre-existing primary myofibers or between primary myofibers at 62 E14.5-E17.5 (4). 63 64 Myogenic cells do not appear to have intrinsic muscle patterning information, but gain 65 this information from interactions with surrounding tissues such as tendons (5). 66 Supporting tissues in the tongue bud primarily originate from cranial neural crest cells 67 (CNCCs), which belong to a migratory multipotent population that gives rise to bones, 68 connective tissues, nerves, glial cells, smooth muscle cells, dentin and tendons in the 69 craniofacial region (6,7). The migration, specification, and survival of CNCCs play a 70 significant role in craniofacial morphogenesis. The role of neural crest cells in 3 71 myogenesis has been investigated in both trunk and craniofacial myogenesis. Neural crest 72 cells induce myogenesis in somite dermomyotomes by secreting Notch ligands that 73 transiently activate Notch signaling in myogenic progenitors (8). Previous studies have 74 demonstrated that tissue-tissue interactions between CNCCs and craniofacial myogenic 75 populations play a role in craniofacial myogenesis using CNC ablation approaches (9- 76 12). Myogenic cells from muscle-less chn mutant zebrafish were able to form normal 77 branchial muscles after being grafted into wild type hosts, suggesting that CNCCs play an 78 instructive role in muscle formation (11). Taken together, these studies indicate that 79 CNCCs control muscle patterning or differentiation; however, the underlying molecular 80 and cellular mechanisms of the CNCC-myogenic interactions remain to be elucidated. 81 82 TGF-β signaling in both myogenic precursors and CNCCs is important for tongue 83 myogenesis. Specifically, our previous study has shown that loss of Smad4 in Myf5- 84 positive muscle precursors caused defective muscle differentiation and fusion during 85 tongue development without affecting cell migration or survival (13). We have also 86 shown that loss of Tgfbr2 in CNCCs results in microglossia due to defects in myogenic 87 cell proliferation and differentiation via tissue-tissue interactions (12). However, these 88 muscle defects were not detectable at early myogenic stages, during which CNCCs guide 89 migrating myogenic precursors for muscle growth and patterning. The signaling cascade 90 downstream of TGF-β that controls the early primary myogenesis of tongue muscles is 91 still poorly understood. 92 4 93 In this study, we investigated three different groups of craniofacial muscles, namely the 94 tongue, eye and masticatory muscles, to study the molecular mechanism of tissue-tissue 95 interactions between CNCCs and myogenic precursors. We show that the early formation fl/fl 96 of craniofacial muscles is severely affected in Wnt1-Cre;Alk5 mice. We found that the 97 Alk5-mediated TGF-β signaling pathway in CNCCs regulates the gene expression of 98 Bmps and Fgfs during craniofacial primary myogenesis. Exogenous Bmp4 and Fgfs can 99 rescue proliferation and differentiation defects in primary cell culture in vitro. Loss of 100 Alk5 receptors in CNCCs also results in impaired tendon development and decreased 101 Scleraxis expression, suggesting the Scleraxis expression in CNCCs is downstream of 102 Alk5-mediated TGF-β signaling. 103 104 Materials and methods 105 fl/fl 106 Generation of Wnt1-Cre;Alk5 mice 107 108 Wnt1-Cre transgenic mice have been described previously (7). We crossed Wnt1- fl/+ fl/fl fl/fl 109 Cre;Alk5 with Alk5 mice to generate Wnt1-Cre;Alk5 mice. Genotyping was carried 110 out using PCR primers as previously described (14). Mice expressing ZsGreen Cre 111 reporter were obtained from Jackson Laboratory. 112 113 Histological analysis and immunostaining 114 5 115 Hematoxylin and eosin (H&E) and immunofluorescence staining were performed 116 following standard procedures. The following antibodies were used for immunostaining: 117 mouse anti-myosin heavy chain (DSHB); mouse anti-MyoD1 (Abcam); rabbit anti- 118 phospho-histone-H3 (Santa Cruz Biotechnology); rabbit anti-active Caspase-3 (Abcam); 119 rabbit anti-phospho-Smad1/5/8 (Cell Signaling); mouse anti-Pax7 (DSHB); rabbit anti- 120 Desmin (Abcam); mouse anti-Myogenin (Abcam). After MHC immunostaining, 121 immunofluorescent images were acquired after analyzing ten fields from each condition. 122 Results were assessed for statistical significance using Student’s t-test. 123 124 In situ hybridization 125 126 In situ hybridization was performed following standard procedures. Digoxigenin-labeled 127 antisense probes were generated from mouse cDNA clones that were kindly provided by 128 several laboratories: Bmp4 (Malcolm Snead, University of Southern California, USA); 129 Pitx2 (Marina Campione, Albert Einstein College of Medicine, USA); Fgf4 and Fgf6 130 (Pascal Maire, Institute Cochin, France); Scleraxis (Eric N. Olson, University of Texas 131 Southwestern Medical Center, USA). 132 133 Quantitative RT-PCR 134 135 The mRNA levels of Bmp4, Fgf4, Fgf6 and Scleraxis were analyzed by quantitative real- 136 time RT-PCR (Bio-Rad iCycler system). Tongue primordium was dissected at E11.5, 137 E12.5 and E13.5 and total RNA was subsequently extracted. The mRNAs were reverse 6 138 transcribed into cDNAs using RNeasy Mini and QuantiTect Reverse Transcription kits 139 (Qiagen), followed by real-time PCR with specific primers. Gene-specific primer 140 sequences were obtained from the Primer Bank (15). Real-time PCR was performed 141 using SYBR Super Mix kits (Bio-Rad). Values were normalized against Gapdh using the ∆∆Ct 142 2 method (16). Global gene expression analysis was performed as previously 143 described (17). Data are shown as mean ± standard deviation (SD). 144 145 Cell culture 146 147 Tongue primordium, eye, and masticatory muscle tissues were dissected from E12.5 or 148 E13.5 embryos and cut into small pieces. Tissue blocks were cultured in Dulbecco's 149 modified Eagle's medium containing 10% fetal bovine serum at 37°C as previously 150 described (12). Cultures were treated with recombinant mouse Bmp4 (15 ng/ml; R&D 151 systems) for two days for the proliferation assays. Cultures were switched to 152 differentiation medium supplemented with 2% horse serum for one week for the 153 differentiation assays. Where indicated, recombinant mouse Fgf4 or Fgf6 (10 ng/ml; 154 R&D systems) or recombinant mouse Bmp4 (15 ng/ml; R&D systems)