Review Role of skeletal muscle in mandible development · 2020-03-03 · ossification, but have...

Summary. As a continuation of the previous study onpalate development (Rot and Kablar, 2013), here weexplore the relationship between the secondary cartilagemandibular condyles (parts of the temporomandibularjoint) and the contributions (mechanical and secretory)from the adjacent skeletal musculature. Previousanalysis of Myf5-/-:MyoD-/- mouse fetuses lackingskeletal muscle demonstrated the importance of musclecontraction and static loading in mouse skeletogenesis.Among abnormal skeletal features, micrognathia(mandibular hypoplasia) was detected: small, bent andposteriorly displaced mandible. As an example ofWaddingtonian epigenetics, we suggest that muscle, inaddition to acting via mechanochemical signaltransduction pathways, networks and promoters, alsoexerts secretory stimuli on skeleton. Our goal is toidentify candidate molecules at that muscle-mandibleinterface. By employing Systematic SubtractiveMicroarray Analysis approach, we compared geneexpression between mandibles of amyogenic and wildtype mouse fetuses and we identified up- and down-regulated genes. This step was followed by abioinformatics approach and consultation of web-accessible mouse databases. We searched for individualtissue-specific gene expression and distribution, and forthe functional effects of mutations in a particular gene.The database search tools allowed us to generate a set ofcandidate genes with involvement in mandibular

development: Cacna1s, Ckm, Des, Mir300, Myog andTnnc1. We also performed mouse-to-human translationalexperiments and found analogies. In the light of ourfindings we discuss various players in mandibularmorphogenesis and make an argument for the need toconsider mandibular development as a consequence ofreciprocal epigenetic interactions of both skeletal andnon-skeletal compartments.Key words: Skeletal muscle, Mandible, Mouse,Development, Epigenetics

General introduction

In the past decade and a half our research interestshave been focused on epigenetic interactions at the tissuelevel, and specifically for this review on the interactionsthat occur between the developing muscular and skeletalsystems. This approach was possible because of theexistence of a compound mouse knockout that entirelylacks one basic tissue type, the skeletal muscle.According to the basic (general) histology classification,the mouse (and human) body consists of only four basictissue types: epithelium, connective tissue, nervoustissue and muscle. While there are a number ofconnective tissue types, there are only three types ofmuscle: skeletal (striated), cardiac and smooth. Bymeans of gene targeting, it seems to be very rare toobtain a complete lack of one basic tissue type.Consequently, when employing Myf5:MyoD nulls(Rudnicki et al., 1993), we are in fact employing a“tissue knock-out” (without having to generate a

Review

Role of skeletal muscle in mandible developmentIrena Rot1, Snjezana Mardesic-Brakus2, Willard J. Costain3, Mirna Saraga-Babic2 and Boris Kablar11Department of Medical Neuroscience, Faculty of Medicine, Dallhousie University, Halifax, NS, Canada, 2Department of Anatomy,Histology and Embryology, School of Medicine, University of Split, Split, Croatia and 3Glycosyltransferases and Neuroglycomics,Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada

Histol Histopathol (2014) 29: 1377-1394DOI: 10.14670/HH-29.1377

http://www.hh.um.es

Histology andHistopathologyCellular and Molecular Biology

Offprint requests to: Dr. Boris Kablar, Department of MedicalNeuroscience, Faculty of Medicine, Dalhousie University, 5850 CollegeStreet, PO Box 15000, Halifax, NS, Canada B3H 4R2. e-mail:[email protected]

conditional knockout), as opposed to just a gene (or agroup of genes, i.e., compound) knock-out. There aresome examples of mouse knock-outs that show asignificant (but not necessarily a complete) lack of aparticular cell type (but not of a basic tissue type), or ofa certain connective tissue type; but, again, that tissuetype is not entirely absent. One example is targeteddisruption of Cbfa1 that results in a complete lack ofbone tissue, but osteoblasts are present, while arrested intheir maturation (Komori et al., 1997). Therefore, ourbasic tissue knock-out of skeletal muscle represents aunique and valuable experimental model system, wherecompound mutant embryos and fetuses completely lackone basic tissue type, the skeletal myoblasts andmuscles, from the beginning of their life, and are viableas long as in the womb.

Muscles and bones make one functional system andtheir main interaction is considered to be mechanical(reviewed by Herring, 1994). Mechanical stimuli areproduced by both dynamic and static loading ofmusculature, and the bone’s response to loading isdemonstrated through cell proliferation, differentiationand tissue remodeling. Since bone morphology is aresult of interactions between bone and muscle activity,bone morphogenesis is a typical example of anepigenetic process in the original, broad Waddingtoniansense (Waddington, 1942). Epigenetic interactionsintegrate developmentally heterogeneous tissues, such asbones and skeletal muscle, into a functional systemthrough complex networking of genes and their productsthat will form the phenotype (Waddington, 1942, 1975).

Our model system has been embryonic mouse fetusin which the elimination of two myogenic regulatoryfactors (MRFs), Myf5 and MyoD, resulted in a completeelimination of skeletal muscle (Rudnicki et al., 1993).(N.B., in fact, MRF4 is also eliminated in our fetuses.)(Kassar-Duchossoy et al., 2004.) These amyogeniccompound-mutant mouse embryos consequently developabnormal phenotypes in many organs, such as skeleton(palate, mandible, clavicle, sternum, etc.), lungs, innerear, retina, spinal cord and brain (reviewed in Kablar,2011).

Our earlier anatomical study (Rot-Nikcevic et al.,2006) described development of the embryonic and fetalskeleton in the complete absence of skeletal muscle. Infact, several subsequent studies confirm our findings onmuscle-bone interactions, while expanding the body ofknowledge produced by our systematic analysis ofamyogenic fetuses (Gomez et al., 2007; Nowlan et al,2010a,b; Solem et al., 2011).

We classified our findings into two major categories.One category is about tissue/organ fusion, such as thepalatal fusion and the fusion of the sternum. Anothercategory is about the process of initiation andmaintenance of the secondary cartilage, relevant to thecomplete development of the clavicle and the mandible.We chose two examples, one from each of these majorcategories, to focus on. First, we focused on the role ofmuscle in palate development (Rot and Kablar, 2013). In

the current review, we focus on the interface between themuscle and the secondary cartilage development in thecondyles of the mandible, as this is clinically relevant tothe development of the temporomandibular joint (TMJ).Through this approach it is possible to gain insight intothe molecular players that are responsible for the processof secondary cartilage maintenance that seems to dependheavily on the cues from the skeletal muscle. In fact, ourearlier study (Rot-Nikcevic et al., 2007) revealed that inthe complete absence of skeletal musculature themandible was severely affected, displaying micrognathiaand reduced or absent condyles.

This study summarizes current knowledge ofmandible morphogenesis and its regulation, anddiscusses epigenetic processes that occur duringmandible development, i.e., the developmentalmechanisms that can only be understood in terms ofinteractions that are above the level of the gene.

In the light of our microarray findings of genes thatare up- and down-regulated in mandibles of Myf5:MyoDnull mutant mice fetuses (or double-mutant, compound-mutant, amyogenic, in this review) that completely lackskeletal musculature we discuss the role of muscle as asource of both mechanical and secretory cues inmandible development. Our goal is to identify novelmolecular players with precisely defined functions inmandibular morphogenesis. We hope to elucidatepossible causes of mandibular hypoplasia (micrognathia)characterized by deficiency in mandibular bone growthand determine which features of the phenotype are theresult of the absence of the gene and secreted proteins,and which are due to the absence of mechanical forcesfrom the muscle.Development of the mandible: anatomicalconsiderations

The mammalian mandible is a complexmorphological structure. It develops from neural crestcells that populate the first pharyngeal arch. Neural crestcells differentiate into two cartilaginous rods, theMeckel’s cartilages. The early mandible is formed byintramembranous ossification, and the ossificationproceeds along Meckel’s cartilage. However, secondarycartilages contribute endochondral components at laterstages (Ramesh and Bard, 2003). In other words, thebody of the mandible undergoes intramembranousossification, while secondary cartilages of coronoid,condylar and angular processes appear in the periosteumand are then generated endochondraly (Fang and Hall,1997; Tomo et al., 1997; Ramaesh and Bard, 2003).

In embryonic mice Meckel’s cartilages form at aboutembryonic day (E) 12.5 and the two rods join at E13.5.At this stage the developing mandibles are thin plates ofcondensed mesenchymal cells, lateral to Meckel’scartilage. At E14.5 mandibles elongate alongsideMeckel’s cartilages and future secondary condylar andangular cartilages are seen at the end of the mandible.By E15.5 Meckel’s cartilage is fully differentiated and is

1378Skeletal muscle and the mandible

beginning to be surrounded by the mandible. Thecondylar and angular cartilages begin to chondrify.Meckel’s cartilage starts to degenerate at E16, and itdisintegrates as the mandible develops (see Ramaesh andBard, 2003 and references therein).

Secondary cartilage is the tissue that forms afterossification begins and it participates in the processes ofgrowth. It occurs at articulations, sutures and muscleattachments and provides growth sites during bone andjoint morphogenesis. In mice it develops in mandibles(Fang and Hall, 1997 and references therein) andclavicles (Hall, 2001 and references therein) and requiresmechanical stimulation for its maintenance (Tran andHall, 1989; Rot-Nikcevic et al., 2007). In mice andhumans secondary cartilage is seen in all threemandibular processes, condylar, coronoid and angular(Fang and Hall, 1997). The condylar cartilage especiallyplays an important role as condyle contributes to theformation of the TMJ. Genetic analyses of thedeveloping mouse TMJ confirmed the expression ofmany genes known to be involved in endochondralossification, but have additionally revealed that TMJforms using a distinct molecular program from othersynovial joints (Hall, 2005; Purcell et al., 2009).Regulation of mandibular development: moleculesand networks

Mandibular growth takes place under strict geneticcontrol. Different genes are involved in regulation ofdevelopmental processes, such as migration ofmesenchymal cells derived from the neural crest,chondrocyte and osteoblast differentiation, andorganogenesis of cartilage and bones. Some of theessential genes in mandible development include genesencoding homeobox-containing transcription factorssuch as goosecoid (gsc), Dlx, Lhx, Msx1, Wnt (Rivera-Perez et al., 1999; Tucker et al., 1999; Chai et al., 2000;Depew et al, 2002; Sharpe and Cobourne, 2003; Chaiand Maxson, 2006) as well as cytokines and growthfactors such as BMP4 (St Amand et al., 2000; Zhang etal., 2002; Liu et al., 2005), fgf8 (Trumpp et al., 1999)and Tgfα (Miettinen et al., 1999). CHD and NOG,proteins that bind BMPs and act as their extracellularantagonists, are, for example, individually necessary fornormal development of the first branchial archderivatives, and their double-mutants displaymicrognathia (Bachilller et al., 2000; Stottman et al.,2001). The complexity of molecular regulation ofmandible development could be illustrated through theexample of the Dlx code (reviewed in Depew et al.,2005). In mice, six Dlx genes have been detected, andthey are organized in three linked pairs in the genome(Dxl1/2, Dxl3/4 and Dxl5/6). They are differentiallyexpressed in a regional, nested pattern in theectomesenchyme along the proximodistal axis of thebranchial arch (Qiu et al., 1995, 1997). Inactivation ofDlx1 and/or Dlx2 causes abnormalities in the upper jaw(Qiu et al., 1995, 1997; Depew et al., 2005), while

simultaneous inactivation of Dlx5 and Dlx6 results intransformation of the lower jaw into the upper jaw(Beverdam et al., 2002; Depew et al., 2002). Dlx3/4expression is restricted to a small domain within themandibular arch and its expression is dependent onDlx5/6 (Depew et al., 2002). Therefore, it has beenhypothesized that a combinatorial code of these genescontrols the proximodistal growth and patterning of thebranchial arch units and that the combinations, i.e., thenumber and type of Dlx alleles (“code”), define themorphology (Depew et al., 2005 and references therein).Moreover, recent evidence suggests that jaw, hyoid andgill arch cartilages are in fact serially homologous andpatterned by a common Dlx blueprint (Gillis et al.,2013). Mechanical role of skeletal muscle in mandibulardevelopment

Apart from the molecular regulation, it has beendocumented that individual components of themammalian dentary, which constitutes the bony skeletonof the lower jaw, respond to epigenetic interactions fromdifferent tissues. For example, areas of the dentaryassociated with the teeth respond to movementassociated with developing teeth. (N.B., in this article weuse the term “mandible” when referring to the bonealone, i.e., the dentary. This is more consistent with ourprevious work, Rot-Nikcevic et al., 2007, but also withthe anatomical terminology to be employed for mouse-to-human translation. Therefore, we are not referring tothe entire lower jaw that would include the Meckel’scartilage, dentary and muscles.) More importantly, theposterior mandibular processes (coronoid, condylar, andangular), onto which muscles insert (attach), requiremechanical action from muscles that attach to them fortheir proper development. The muscle contractionproduces the strongest loading at muscle attachmentsites and these sites are usually characterized byformation of bony processes (e.g., coronoid process ofmandible). These processes respond to muscle action inways that are specific to sets of muscles and theprocesses with which they interact (Atchley and Hall,1991; Atchley, 1993). The most powerful of jaw musclesis the masseter that attaches to the large area on thelateral aspect of the angle of the mandible, extending tothe angular process, whereas temporalis muscle,attaching to the coronoid process, is weaker. Medial andlateral pterygoid muscles attach to the angular processand the condyle, respectively. Our previous studies haveshown that mandibles in Myf5:MyoD null fetuses weresignificantly altered in shape when compared to wildtype; they were smaller, the condylar process wasslender, and angular and coronoid processes weresignificantly reduced or absent (Rot-Nikcevic et al.,2007). As mentioned, all three processes of the mandiblerequire secondary cartilage for their formation.Formation of secondary cartilage itself is dependent onmechanical stimulation from the skeletal muscle


(Herring, 1994). Our previous findings conclude thatsecondary cartilage formation in mouse mandibles canbe initiated in the absence of striated muscle, but theamount of tissue is reduced (with the exception of theangular process of the mandible that cannot be initiated).On the other hand maintenance of secondary cartilage isimpossible in the absence of musculature, since all theprocesses were either reduced or completely absent(Rot-Nikcevic et al., 2007). Secretory role of skeletal muscle in mandibulardevelopment

Of special interest to us is that, in addition toproducing a mechanical load, muscle can also be asource of secretory proteins. Secretory, but especially theparacrine, role of the muscle is achieved throughsecretory cues (proteins) produced by the muscle, whichinduce a response in adjacent tissues, such as cartilageand bone, by affecting levels of cell proliferation, death,differentiation and tissue fusion. Muscle is shown to becapable of expressing and secreting cytokines that act assignaling molecules in intercellular communication(Pedersen and Febbraio, 2008). Muscle is viewed as asecretory organ, and its products, the cytokines (or“myokines”), can have autocrine, paracrine andendocrine effects (Pedersen and Febbraio, 2008;Henningsen et al., 2010). Here we propose that anotherlikely reason for micrognathia is a lack of secretory cuesfrom the adjacent muscle. In fact, recent work identifiedmuscle as a source of paracrine secretion of growthfactors essential for bone tissue formation (Hamrick etal., 2010). Unfortunately, in our study, none of therelevant up- or down-regulated genes encodes forgrowth factors. Molecular networking that connects genotype andphenotype

Our approach to analyzing mandible development isone of systems biology that focuses on processes, i.e.,functional outputs of protein networks, as they are the

level at which the genotype affects the phenotype (Bard,2013). The tissues do not form in isolation but are eachother’s environment and part of a larger unit. A mutationin a gene results in an altered protein (or proteinregulation) that, in turn, alters the network that drivesmorphogenetic process; altered process alters the trait,i.e., the phenotype. In most cases the trait is formedthrough the output properties of more than one network,and mutations in any of these proteins can affect thenetwork outputs (Bard, 2010). Molecular networks areoften referred to as pathways, but, as Bard (2013)argued, network is a more appropriate term asassemblages of proteins often include alternate routes.

The mammalian mandible is a classic model systemfor studying integration, as well as complexmorphologies (Atchley, 1983; Atchley and Hall, 1991;Klingenberg et al., 2001; Zelditch and Swiderski, 2011and references therein), and as such it can provide aninsight into the complex gene networking that connectsgenotype and phenotype. Systematic subtractive microarray analysisapproach (SSMAA)

As previously described in the publications from ourlaboratory (Rot and Kablar, 2013, and referencestherein), using a stereomicroscope the mandibular bone(the dentary) was dissected out, the total RNA wasisolated, and the Affymetrix GeneChip cDNAmicroarray analysis was performed (Ottawa GenomeCentre) to obtain the expression ratios and fold changesbetween the wild type and double-mutant fetalmandibles at E18.5. An arbitrary cut off value for log2(ratio) of 3.0 (i.e. -3.0≤log2 (ratio)≥3.0) was chosen as amean of determining the up- and down-regulatedprobesets. Here, our analysis starts by gettinginformation from microarrays to reveal differentiallyexpressed genes in the mutant tissues. Amyogenic Myf5-/-:MyoD-/- fetuses develop various skeletalabnormalities in the absence of musculature, one of thembeing mandibular hypoplasia (Rot-Nikcevic et al., 2006;2007). Following these findings we employed SSMAA


Table 1. Genes up-regulated ≥3-fold in Myf5-/-:MyoD-/- mouse mandible, sorted by fold change.

Gene Fold change Gene title Molecular function

Spt1 5.56 salivary protein 1 / mucin-like 1 Not specifiedProl1 5.38 proline rich, lacrimal 1 Peptidase inhibitor activityPip 4.23 prolactin induced protein Protein bindingEif2s3y 3.80 eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked GTP bindingUty 3.53 ubiquitously transcribed tetratricopeptide repeat gene, Y chromosome Metal ion bindingCar6 3.42 carbonic anhydrase 6 Carbonate dehydratase activitySmgc 3.36 submandibular gland protein C Not specifiedDdx3y 3.35 DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked ATP bindingLpo 3.29 lactoperoxidase Peroxidase activityPrpmp5 3.26 proline-rich protein MP5 Not specifiedPsp 3.24 parotid secretory protein Not specifiedSrsy 3.17 serine-rich, secreted, Y-linked Not specified

to reveal a profile of genes involved in mandibledevelopment and to explore muscle’s contribution interms of molecular cues. The SSMAA is used to revealthe difference in gene expression patterns between thewild type and the compound-mutant mandible. Wehypothesized that the difference in gene expressionpatterns between the control (fully developed mandibleand its secondary cartilage condyles) and the compound-mutant mandible (as described in Rot-Nikcevic et al.,2007) would be related to the described mandibularphenotype and its causes. Our Affymetrix Gene ChipcDNA microarray analysis revealed a profile of geneswith a potential role in mandible development inrelationship to the skeletal muscle. (N.B., the microarrayapproach was preferred to the proteomics for theconsistency reasons described in Kablar, 2011, inaddition to other well discussed/known advantages anddisadvantages). With a cut-off value of 3-fold, weidentified 13 up-regulated and 132 down-regulatedprobesets in mutant mandibles, of which 12 and 107,respectively, are identified named genes. The lists of up-and down-regulated genes are given in Tables 1 and 2,respectively.

In addition, the analysis of functional interactions(FI) for the entire dataset was performed by employingthe same double-mutant Myf5-/-:MyoD-/- mandibleprobesets, but with a cut-off value of 2-fold, and with theinclusion of linking genes. The purpose of the FIanalysis is to cluster genes of related function within a

larger dataset. We employed Reactome FI plugin inCytoscape 2.7.0., which collects and aggregates pathwayinformation from a variety of public sources. Wegenerated the entire network of interactions with FIsclustered into correlated nodes distinguished by nodecolor. There were more than 10 different, and somewhatoverlapping, node clusters. However, because of thedifficulties interpreting such a large body of information,we could not show the entire network. Instead, toillustrate our analysis, we show the top node (pathway)that we identified, and that was concerned with themuscle contraction pathway (Fig. 1). Bioinformatics approach

The microarray findings were followed by abioinformatics approach (Bard, 1999, 2002a,b), and adetailed consultation of the web-accessible mousedatabases was performed. Mouse Genome Informatics(MGI) (http://www.informatics.jax.org) holds, amongvarious other features, information on spatial andtemporal expression of genes, on distribution patterns ofproteins, and on genetically modified mice phenotypes.Databases were searched for individual gene expressionand distribution in the tissues of interest and for afunction in mandibular development. In particular, foreach gene of interest, we searched for the effects ofmutations that a genetically modified mouse would havein relationship to the mandibular development, such as


Fig. 1. Analysis of FunctionalInteractions (FIs). Analysis ofFIs was performed employingthe Reactome FI plugin inCytoscape 2.7.0. The entirenetwork of interactions withFIs clustered into correlatednodes distinguished by nodecolor is not shown, because ofthe diff iculties interpretingsuch a large body of infor-mation. However, the topnode/pathway (in blue)identified and shown here wasconcerned with the musclecontraction pathway. (N.B.,Note two overlapping nodes,one in green, with five genesidentified, and one in yellow,with one gene identified.) Themuscle contraction pathway(in blue) identif ied by FIanalysis consisted of 18

genes, which were largely structural components of muscle tissue (DES, desmin; DMD, dystrophin muscular dystrophy; MYBPC1, myosin bindingprotein C, slow type; MYBPC2, myosin binding protein C, fast type; MYH3, myosin heavy chain 3; MYH8, myosin heavy chain 8; MYL1, myosin lightchain 1; MYL2, myosin light chain 2; MYL3, myosin light chain 3; MYL4, myosin light chain 4; NEB, nebulin; TCAP, titin-cap; TMOD1, tropomodulin 1;TNNC1, troponin C type 1; TNNI1, troponin I type 1; TNNI2, troponin I type 2; TNNT1, troponin T type 1; TNNT2, troponin T type 2; TNNT3, troponin Ttype 3; TPM2, tropomyosin 2 beta; TPM3, tropomyosin 3 gamma; TTN, titin). Note that several molecules identified in this pathway, such as: TNNC1,ACTN2, TNNT3, are also visible in Table 5, indicating a potential role for these molecules in skeletal muscle-mandible secretory relationship. Note:color code denotes node/pathway (e.g., in blue, green and yellow); lines and arrows represent the direction of interactions between the molecules;dotted line indicates a less documented possibility of an interaction within a pathway (node).

micrognathia or mandibular hypoplasia. Our goal was todetermine whether any of the listed genes (in Tables 1and 2) was expressed/distributed in mandible (or theadjacent tissues, potentially signaling/relating to it), andwhether any of the differentially expressed genes in thedouble-mutant mandible has already been shown tocause micrognathia through the study of their genetically

modified mice. The database search tools allowed us toidentify a set of candidate genes that are likely involvedin the mandible morphogenesis, as described below.

Only one of the down-regulated genes in double-mutant mandible, Cacna1s, was previously associatedwith the micrognathia (Pai, 1965). Originally, the mousemutant was named muscular dysgenesis (mdg). It is a


Table 2. Genes down-regulated ≥3-fold in Myf5-/-:MyoD-/- mouse mandible, sorted by fold change (FC). (N.B., certain probesets lack completespecificity for their gene and this is reflected in the Gene title, where some genes have more than one name separated by ///.)

Gene FC Gene title Molecular function

Trdn -7.85 triadin Not specifiedMyl1 -7.79 myosin, light polypeptide 1 Calcium ion bindingAtp1b4 -7.35 ATPase, (Na+)/K+ transporting, beta 4 polypeptide Protein bindingAtp2a1 -7.13 ATPase, Ca++ transporting, cardiac muscle, fast twitch 1 Calcium ion bindingMyh8 -7.12 myosin, heavy polypeptide 8, skeletal muscle, perinatal Actin binding, ATP bindingNeb -6.97 nebulin Not specifiedMyh3 -6.91 myosin, heavy polypeptide 3, skeletal muscle, embryonic Actin binding, ATP bindingTtn -6.79 titin Actin filament bindingCasq1 -6.70 calsequestrin 1 Calcium ion bindingTnnt3 -6.65 troponin T3, skeletal, fast Actin bindingActa1 -6.65 actin, alpha 1, skeletal muscle ATP binding, nucleotide bindingMybpc1 -6.58 myosin binding protein C, slow-type Not specifiedMyot -6.51 myotilin Actin bindingArhgap36 -6.48 Rho GTPase activating protein 36 GTPase activator activityUbe2c -6.44 ubiquitin-conjugating enzyme E2C /// troponin C2, fast Acid-amino acid ligase activityActn2 -6.41 actinin alpha 2 Actin bindingMyoz2 -6.34 myozenin 2 Actin bindingMyoz1 -6.32 myozenin 1 FATZ binding, protein bindingKbtbd10 -6.27 kelch repeat and BTB (POZ) domain containing 10 Not specifiedCkm -6.23 creatine kinase, muscle ATP bindingCasq2 -5.96 calsequestrin 2 Calcium ion bindingApobec2 -5.95 apolipoprotein B mRNA editing enzyme, catalytic polypeptide 2 Catalytic activityTnni2 -5.84 troponin I, skeletal, fast 2 Actin bindingMylk4 -5.57 myosin light chain kinase family, member 4 ATP binding, kinase activitySmpx -5.56 small muscle protein, X-linked Not specifiedMyl4 -5.50 myosin, light polypeptide 4 Actin filament bindingMyom2 -5.47 myomesin 2 Not specifiedKlhl31 -5.44 kelch-like 31 (Drosophila) Not specifiedAsb12 -5.41 ankyrin repeat and SOCS box-containing 12 Not specifiedPgam2 -5.20 phosphoglycerate mutase 2 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase activityRps13 -5.09 ribosomal protein S13 mRNA bindingHfe2 -5.06 hemochromatosis type 2 (juvenile) (human homolog) Coreceptor activityCox8b -5.02 cytochrome c oxidase, subunit VIIIb Cytochrome-c oxidase activityMylpf -5.01 myosin light chain, phosphorylatable, fast skeletal muscle Calcium ion bindingRyr1 -5.00 ryanodine receptor 1, skeletal muscle Calcium channel activityPvalb -4.98 parvalbumin Calcium ion bindingTmem182 -4.96 transmembrane protein 182 Not specifiedLdb3 -4.95 LIM domain binding 3 Metal ion bindingDes -4.95 desmin Cytoskeletal protein bindingPygm -4.95 muscle glycogen phosphorylase AMP bindingTmod4 -4.93 tropomodulin 4 Actin bindingSacs -4.90 sacsin /// sarcoglycan, gamma (dystrophin-associated glycoprotein) ATP bindingFitm1 -4.86 fat storage-inducing transmembrane protein 1 Not specifiedPpp1r3a -4.70 protein phosphatase 1, regulatory (inhibitor) subunit 3A Protein serine/threonine phosphatase activityMybpc2 -4.67 myosin binding protein C, fast-type Actin bindingItgb1bp2 -4.65 integrin beta 1 binding protein 2 Calcium ion bindingMyf6 -4.62 myogenic factor 6 DNA bindingAmpd1 -4.57 adenosine monophosphate deaminase 1 AMP deaminase activityCkmt2 -4.56 creatine kinase, mitochondrial 2 ATP bindingSmyd1 -4.55 SET and MYND domain containing 1 DNA bindingMyh1 -4.37 myosin, heavy polypeptide 1, skeletal muscle, adult Actin binding

To be continued.

spontaneous mutation, and all homozygous mutantsshow a short mandible with an abnormally pronouncedcurvature, as well as smaller condylar and coronoidprocesses. Mutants show consistent bone abnormalities,but the most affected bone appears to be mandible. Thestriking characteristic of all mutants is general andsevere deficiency of skeletal musculature. The mutation

is lethal probably due to degeneration of the muscles ofrespiration (Pai, 1965). Importantly, several subsequentstudies employing mdg (and Cacna1s) have established amechanistic basis for tendon-skeleton regulatoryinteractions during the development of musculoskeletalsystem and the process of secondary patterning of bones(Brent et al., 2003; Blitz et al., 2009; Sharir et al., 2013).


Continuation.

Tmod1 -4.33 tropomodulin 1/ thiosulfate sulfurtransferase-like domain containing 2 Actin bindingTmem8c -4.32 transmembrane protein 8C Not specifiedSrl -4.28 sarcalumenin GTPase activityFsd2 -4.24 fibronectin type III and SPRY domain containing 2 Not specifiedHabp2 -4.23 hyaluronic acid binding protein 2 /// nebulin-related anchoring protein Catalytic activityCsrp3 -4.19 cysteine and glycine-rich protein 3 Actinin bindingXirp2 -4.15 xin actin-binding repeat containing 2 Actin bindingMypn -4.12 myopalladin Actin bindingPdlim3 -4.07 PDZ and LIM domain 3 Cytoskeletal protein bindingSypl2 -4.04 synaptophysin-like 2 Transporter activityGm5105 -4.03 predicted gene 5105 Not specifiedTecrl -4.01 trans-2,3-enoyl-CoA reductase-like Oxidoreductase activityMurc -3.95 muscle-related coiled-coil protein Rho GTPase activator activityMstn -3.92 myostatin Cytokine activityCpt1b -3.91 carnitine palmitoyltransferase 1b muscle/ Chkb-Cpt1b read through transcript Carnitine O-palmitoyl transferase activityLmod3 -3.91 leiomodin 3 (fetal) Not specifiedTnnt2 -3.90 troponin T2, cardiac Actin binding, ATPase activityTnnc1 -3.84 troponin C, cardiac/slow skeletal Actin filament bindingMyog -3.79 myogenin DNA bindingCacna1s -3.79 calcium channel, voltage-dependent, L type, alpha 1S subunit Calcium channel activityChrna1 -3.78 cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle) Acetylcholine bindingEno3 -3.77 enolase 3, beta muscle Lyase activityTnni1 -3.77 troponin I, skeletal, slow 1 Actin bindingCap2 -3.77 CAP, adenylate cyclase-associated protein, 2 (yeast) Actin bindingAlpk3 -3.71 alpha-kinase 3 ATP bindingMylk2 -3.70 myosin, light polypeptide kinase 2, skeletal muscle ATP bindingXirp1 -3.69 xin actin-binding repeat containing 1 Actin bindingCacng1 -3.65 calcium channel, voltage-dependent, gamma subunit 1 Calcium channel activitySynpo2l -3.65 synaptopodin 2-like Not specifiedSync -3.59 syncoilin / retinoblastoma binding protein 4 Protein bindingHspb7 -3.58 heat shock protein family, member 7 (cardiovascular) Filamin bindingTrim72 -3.53 tripartite motif-containing 72 Metal ion bindingMyo18b -3.51 myosin XVIIIb ATP bindingLmod2 -3.47 leiomodin 2 (cardiac) Actin bindingBves -3.44 blood vessel epicardial substance Protein bindingCox6a2 -3.40 cytochrome c oxidase, subunit VI a, polypeptide 2 Cytochrome-c oxidase activityMyom3 -3.36 myomesin family, member 3 Protein homodimerization activityTxlnb -3.34 taxilin beta Syntaxin bindingLrrc39 -3.32 leucine rich repeat containing 39 /// coiled-coil domain containing 76 Not specifiedMustn1 -3.31 musculoskeletal, embryonic nuclear protein 1 Not specifiedCmya5 -3.29 cardiomyopathy associated 5 Protein bindingHhatl -3.28 hedgehog acyltransferase-like Not specifiedGatm -3.27 glycine amidinotransferase (L-arginine:glycine amidinotransferase) Amidinotransferase activityMyom1 -3.26 myomesin 1 Not specifiedMlf1 -3.25 myeloid leukemia factor 1 DNA bindingNexn -3.24 nexilin Actin bindingMir300 -3.18 microRNA 300 Not specifiedTrim55 -3.17 tripartite motif-containing 55 Metal ion bindingCox17 -3.17 cytochrome c oxidase, subunit XVII assembly protein homolog (yeast) Copper ion bindingTnnt1 -3.06 troponin T1, skeletal, slow Tropomyosin bindingTpm2 -3.04 tropomyosin 2, beta Actin bindingJph1 -3.03 junctophilin 1 Not specifiedMir377 -3.02 microRNA 377 Not specifiedGjd4 -3.01 gap junction protein, delta 4 Not specifiedChrnd -3.01 cholinergic receptor, nicotinic, delta polypeptide Acetylcholine bindingMir369 -3.00 microRNA 369 Not specified

1384

Fig. 2. Mouse-to-Human Translation Experiments. Distribution of: TGF1 (A-C), CTGF (D-F), VEGF (G-I) and FN (J-L). Developing human mandible at5th (A, D, G, J), 7th (B, E, H, K) and 10th (C, F, I, L) developmental week. Week 5 (A, D, G, J): human mandibular arch consists of surface epithelium(e) and underlying ectomesenchyme (em), at places containing blood vessels (bv). Cells showing positive reaction to the specific antibody (arrows andinsets in A, D, G, J): in the epithelium (A, D), ectomesenchyme (G, J). Week 7 (B, E, H, K): human mandible containing epithelium (e),ectomesenchyme (em), Meckel’s cartilage (Mc) and ossification zone (oz). Cells showing moderate to strong reaction to specific antibodies within theossification zone, primarily osteoblasts (arrows, insets in B, E, H, K). Week 10: (C, F, I, L): human mandible containing surface skin epithelium (e),ectomesenchyme (em), Meckel’s cartilage (Mc) and ossification zone (oz). Cells showing positive reaction (arrows) in the ectomesenchyme andosteoblasts of the ossification zone (arrows and insets in C, F, I, L). Scale bar: 50 µm; insets x100.

In addition, we identified a set of 5 genes, down-regulated in the double-mutant mandible, that have beenshown to be expressed in the normal mouse mandible.Their differential expression in wild type versus double-mutant mandibles, as well as the identified expression inthe normal mandible, establishes them as themicrognathia candidate genes. In other words, according

to the current database content, these 5 genes arepotentially the most responsible for the mandibularphenotype previously described (Rot-Nikcevic et al.,2007) and employed in this study. The results are shownin Table 3. For three of these candidate genes (Ckm, Desand Myog) knockouts have been reported, but there wereno reports regarding mandibular phenotype. For the


Table 3. Genes down-regulated ≥3 fold in double-mutant mouse mandible with a potential role in mandibular development, sorted by fold change (FC).

Gene Gene title FC Molecular function Gene Expression Knock-out phenotypes

Ckm creatine kinase, muscle -6.23 ATP binding Lower jaw Abnormalities in function and energyutilization of skeletal and cardiac muscle1

Des desmin -4.95 Cytoskeletal proteinbinding Lower jaw Defects of cardiac, skeletal and smooth

muscle; necrosis of myocardium2

Tnnc1 troponin C, cardiac/slow skeletal -3.84 Actin filament binding Lower jaw Not reported

Myog myogenin -3.79 DNA binding Lower jaw, Mandibleprimordium

Severe reduction in muscle mass, defectsof thoracic skeleton, perinatal death3

Mir300 microRNA 300 -3.18 Not specified Lower jaw, Meckel’s cartilage Not reported1van Deursen et al., 1993; 2Milner et al., 1996; 3Hasty et al., 1993.

Table 4. Genes reported to date (May, 2013) with knockouts displaying micrognathia or mandibular hypoplasia phenotype.

Gene Gene title Gene Gene title

1 Acvr1 activin A receptor, type 1 35 Lamp1 lysosomal-associated membrane protein 12 Acvr2a activin receptor IIA 36 Lmna lamin A3 Atr ataxia telangiectasia and Rad3 related 37 M3sapc mutation 3, Sabine Cordes4 Baz1b bromodomain adjacent to zinc finger domain, 1B 38 Mks1 homeobox, msh-like 15 Cacna1s calcium channel, voltage-dependent, L type, alpha 1S subunit 39 Nabp2 nucleic acid binding protein 26 Cep290 centrosomal protein 290 40 Ndst1 N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 17 Cnbp cellular nucleic acid binding protein 41 Otx2 orthodenticle homolog 2 (Drosophila)8 Cyp26b1 cytochrome P450, family 26, subfamily b, polypeptide 1 42 Pbx1 pre B cell leukemia homeobox 19 Cyp51 cytochrome P450, family 51 43 Pdk1 pyruvate dehydrogenase kinase, isoenzyme 1

10 Dlg1 discs, large homolog 1 (Drosophila) 44 Pdss2 prenyl (solanesyl) diphosphate synthase, subunit 211 Dlx5 distal-less homeobox 5 45 Pgap1 post-GPI attachment to proteins 112 Dync2h1 dynein cytoplasmic 2 heavy chain 1 46 Pitx1 paired-like homeodomain transcription factor 113 Ece1 endothelin converting enzyme 1 47 Prdm16 PR domain containing 1614 Edn1 endothelin 1 48 Prrx1 paired related homeobox 115 Ednra endothelin receptor type A 49 Pta persistent truncus arteriosus16 Eya1 eyes absent 1 homolog (Drosophila) 50 Ptpn11 protein tyrosine phosphatase17 Fbln1 fibulin 1 51 Ptprf protein tyrosine phosphatase, receptor type, F18 Fgfrl1 fibroblast growth factor receptor-like 1 52 Rpgrip1 Rpgrip1-like; targeted mutation 119 Fuz fuzzy homolog (Drosophila) 53 Satb2 special AT-rich sequence binding protein 220 Gli2 GLI-Kruppel family member GLI2 54 Sc5d sterol-C5-desaturase (fungal ERG3, delta-5-desaturase) homolog21 Gpc3 glypican 3 55 Sh3pxd2b SH3 and PX domains 2B22 Gpg1 gasping 1 56 Six1 sine oculis-related homeobox 123 Gpg2 gasping 2 57 Slc27a4 solute carrier family 2724 Gpg3 gasping 3 58 Smad2 SMAD family member 225 Gpg4 gasping 4 59 Sox11 SRY-box containing gene 1126 Gpg5 gasping 5 60 Sox9 SRY-box containing gene 927 Gpg6 gasping 6 61 Srn siren28 Gsc goosecoid homeobox 62 Sufu suppressor of fused homolog (Drosophila)29 Hand2 heart and neural crest derivatives expressed transcript 2 63 Tbx1 T-box 130 Hpmd hypoplastic mandible 64 Tcof1 Treacher Collins Franceschetti syndrome 1, homolog31 Hspb11 heat shock protein family B (small), member 11 65 Trps1 transgene insertion 1, Andrew K Groves32 Itgb1 integrin beta 1 (fibronectin receptor beta) 66 Zeb1 zinc finger E-box binding homeobox 133 Kat6a K(lysine) acetyltransferase 6A 67 Zic5 zinc finger protein of the cerebellum 534 Kif15 kinesin family member 15 68 Zmpste24 zinc metallopeptidase, STE24


Table 5. Genes down-regulated ≥3 fold in double-mutant mouse mandible with a potential role in muscle-mandible secretory relationship, sorted by foldchange (FC).

Gene Gene title FC Molecular function Gene Expression/Distribution

Myl1 Myosin, light polypeptide -7.85 Calcium ion binding Skeletal muscle, tongueAtp2a1 ATPase, Ca++ transporting, cardiac muscle, fast twitch 1 -7.13 Calcium ion binding Skeletal muscle, tongueMyh8 myosin, heavy polypeptide 8, skeletal muscle, perinatal -7.12 Actin binding, ATP binding Skeletal muscleActc1 Actin, alpha, cardiac muscle 1 -6.92 ATPase activity TongueMyh3 myosin, heavy polypeptide 3, skeletal muscle, embryonic -6.91 Actin binding, ATP binding Skeletal muscle, tongueTtn titin -6.79 Actin filament binding Skeletal muscle, tongueActa1 actin, alpha 1, skeletal muscle -6.65 ATP binding, nucleotide binding Skeletal muscleTnnt3 Troponin T3, skeletal, fast -6.65 Actin binding Skeletal muscle, tongueMybpc1 myosin binding protein C, slow-type -6.58 Not specified Skeletal muscleMyot myotilin -6.51 Actin binding Skeletal muscleArhgap36 Rho GTPase activating protein 36 -6.48 GTPase activator activity Skeletal muscle, tongueActn2 actinin alpha 2 -6.41 Actin binding Skeletal muscleMyoz2 myozenin 2 -6.34 Actin binding Skeletal muscleMyoz1 myozenin 1 -6.32 FATZ binding, protein binding Skeletal muscleCkm Creatine kinase, muscle -6.23 ATP binding Masseter muscle, skeletal muscle, tongueApobec2 apolipoprotein B mRNA editing enzyme, catalytic polypeptide 2 -5.95 Catalytic activity TongueTnni2 Troponin I, skeletal, fast 2 -5.84 Actin binding Masseter muscle, skeletal muscle, tongueSmpx small muscle protein, X-linked -5.56 Not specified Skeletal muscleMyl4 myosin, light polypeptide 4 -5.50 Actin filament binding Skeletal muscleMyom2 myomesin 2 -5.47 Not specified Skeletal muscleKlhl31 kelch-like 31 (Drosophila) -5.44 Not specified Skeletal musclePgam2 Phosphoglycerate mutase 2 -5.20 2,3-bisphospho-glycerate-dependent Skeletal muscle, tongue, tooth

phosphoglycerate mutase activityCox8b cytochrome c oxidase, subunit VIIIb -5.02 Cytochrome-c oxidase activity TongueMylpf myosin light chain, phosphorylatable, fast skeletal muscle -5.01 Calcium ion binding Skeletal muscleRyr1 Ryanodine receptor 1, skeletal muscle -5.00 Calcium channel activity TonguePvalb parvalbumin -4.98 Calcium ion binding Skeletal muscle, facial bone primordiumPygm muscle glycogen phosphorylase -4.95 AMP binding Skeletal muscle, temporalis, tongueDes Desmin -4.95 Cytoskeletal protein binding Lower jaw, masseter muscle, tongueLdb3 LIM domain binding 3 -4.95 Metal ion binding Skeletal muscleMyh1 Myosin, heavy polypeptide 1, skeletal muscle, adult -4.37 Actin binding Skeletal muscle, tongueMybpc2 myosin binding protein C, fast-type -4.67 Actin binding Skeletal muscleItgb1bp2 integrin beta 1 binding protein 2 -4.65 Calcium ion binding Skeletal muscleAmpd1 adenosine monophosphate deaminase 1 -4.57 AMP deaminase activity TongueCkmt2 creatine kinase, mitochondrial 2 -4.56 ATP binding TongueKlhl31 kelch-like 31 (Drosophila) -4.43 Not specified Skeletal muscleTmod1 tropomodulin 1/ thiosulfate sulfurtransferase-like domain containing 2 -4.33 Actin binding Skeletal muscleSrl sarcalumenin -4.28 GTPase activity Skeletal muscle, tongueMypn myopalladin -4.12 Actin binding Skeletal muscle, tongueLmod3 leiomodin 3 (fetal) -3.91 Not specified Skeletal muscleTnnt2 troponin T2, cardiac -3.90 Actin binding, ATPase activity Skeletal muscleTnnc1 Troponin C, cardiac/slow skeletal -3.84 Actin filament binding Lower jaw, masseter, tongueCacna1s Calcium channel, voltage-dependent, L type, alpha 1S subunit -3.79 Calcium channel activity Skeletal muscleMyog Myogenin -3.79 DNA binding Lower jaw, mandible primordium, masseter

muscle, skeletal muscle, tongueChrna1 cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle) -3.78 Acetylcholine binding Skeletal muscleTnni1 Troponin I, skeletal, slow I -3.77 Actin binding Tongue, lower jaw molarEno3 enolase 3, beta muscle -3.77 Lyase activity Skeletal muscle, facial bones primordiaAlpk3 alpha-kinase 3 -3.71 ATP binding Skeletal muscleXirp1 xin actin-binding repeat containing 1 -3.69 Actin binding Skeletal muscleSynpo21 synaptopodin 2-like -3.65 Not specified Skeletal muscleMyo18b myosin XVIIIb -3.51 ATP binding Skeletal muscleLmod2 leiomodin 2 (cardiac) -3.47 Actin binding Skeletal muscleCox6a2 cytochrome c oxidase, subunit VI a, polypeptide 2 -3.40 Cytochrome-c oxidase activity Skeletal muscleMyom3 myomesin family, member 3 -3.36 Protein homodimerization activity Skeletal muscleMustn1 musculoskeletal, embryonic nuclear protein 1 -3.31 Not specified Skeletal muscleCmya5 cardiomyopathy associated 5 -3.29 Protein binding Skeletal muscleGatm glycine amidinotransferase (L-arginine:glycine amidinotransferase) -3.27 Amidinotransferase activity Skeletal muscle, tongueMyom1 myomesin 1 -3.26 Not specified Skeletal muscleTtn titin -3.25 Actin filament binding Skeletal muscle, tongueNexn nexilin -3.24 Actin binding TongueMir300 microRNA 300 -3.18 Not specified Meckel’s cartilage, mandibleCox17 cytochrome c oxidase, subunit XVII assembly -3.17 Copper ion binding Skeletal muscle

protein homolog /popeye domain containing 2Tnnt1 troponin T1, skeletal, slow -3.06 Tropomyosin binding Skeletal muscleTpm2 tropomyosin 2, beta -3.04 Actin binding Skeletal muscleJph1 junctophilin 1 -3.03 Not specified Skeletal muscleChrnd cholinergic receptor, nicotinic, delta polypeptide -3.01 Acetylcholine binding Skeletal muscle, tongueGjd4 gap junction protein, delta 4 -3.01 Not specified Skeletal muscle, tongue

other two genes, Tnnc1 and Mir300, knockouts have notbeen generated. The generation of knockout mice forthese two candidate genes, and subsequent analysis ofmandibular development in search for mandibularhypoplasia in all five knockouts, would elucidate theprecise role in mandible development of these fivecandidates.

We also used mouse databases to search for genesinvolved in a specific phenotype, such as micrognathiaor mandibular hypoplasia. The search returned 68 genes(listed in Table 4). Interestingly, only one gene,Cacna1s, appeared in both the micrognathia genes listobtained through web-database and our down-regulatedor up-regulated genes list.

Finally, we have identified a large set of 66 down-regulated genes (Table 5) in double-mutant mandiblewhose expression/distribution is normally restricted toskeletal muscle (including, in some cases, specificreports on expression in the masseter muscle, temporalismuscle or tongue). This is a very interesting finding andit contributes to our hypothesis of muscle’s secretoryrole. Since skeletal muscle is completely eliminated indouble-mutants, these genes (and, in turn, their products,potentially secreted by the muscle during mandibulardevelopment) could represent a subset of secretory,likely paracrine, cues from muscle that regulatesmandible development.

In Table 6 we listed all the genes (50) that are down-regulated in double-mutant mandible for whichknockouts were generated, but for which an abnormalmandible phenotype has not been reported. These genesare potential redundancy candidates, in case themandibles were analyzed, but the phenotype was absent.Indeed, many gene knockouts in mice result in normalphenotype (Davies, 1999), because the gene is probablypart of the secondary pathway and will be redundantunder normal circumstances (Bard, 2010). Alternatively,however, for many of these gene knockouts, themandibular development was not analyzed, andtherefore could be the next step in this endeavor.

In conclusion, so far, many of the follow-up stepswill depend on the new data availability via theInternational Mouse Phenotyping Consortium (IMPC). Itwould be impractical and unrealistic to generate such alarge number of knockouts in an individual laboratory,just to search for the mandibular phenotype. However,since the genes are redeployed, and numerous scientistsare generating knockouts in their laboratories fordifferent reasons, and with the additional help from theunifying IMPC pipeline, it is very likely that theinformation on the functional involvement in mandibulardevelopment for a variety of genes of interest identifiedin this study will become available in the near future. Skeletal muscle and the mandible: model forsecretory and mechanical integration

Anomalies of the mutant mandibles involve areasthat are sites of muscle attachment and are probably

secondary effects of the absence of the respectivemuscles. We suggest that not only mechanical, but alsoparacrine cues from adjacent musculature play a role inregulating development of neighboring tissues andorgans, and in this case the mandible. The load due tomuscle contraction is the most concentrated at themuscle attachment site, and usually results in formationof processes, such as the coronoid process of themandible, to which the temporalis muscle attaches. If thetemporalis muscle were removed the muscle load wouldbe removed, as well as possible paracrine cues, i.e.,signaling proteins from the muscle. In our study, genesexpressed in muscles attaching to the mandible wereaffected, and therefore it is possible that paracrinecontributions from muscle were affected too. This couldhave an influence on mandibular development, inaddition to static and dynamic loading stimuli from themusculature. Our discovery of a large set of down-regulated genes in the double-mutant mandible, whoseexpression and/or distribution is normally restricted tothe muscle, supports this hypothesis. Further researchshould attempt to determine if a knockout for each of ourproposed candidate genes would result in abnormalmandible phenotype. This would prove that the productof the gene in question, and not the lack of themusculature (or, at least, not only the lack of themusculature), produced the phenotype. Alternatively, itis possible that the compound mutant possessesmyogenic progenitor cells that erroneously migrated tothe mandible (Kablar et al., 1999; Sambasivan et al.,2009).

The paracrine cues could originate from variousneighboring tissues. Knockout mice for endothelin-converting-enzyme-1 lack Meckel’s cartilage and have aseverely hypoplastic mandible (Yanagisawa et al., 1998).Ramaesh and Bard (2003) suggested that mandibulargrowth and morphogenesis could be directed andconstrained by paracrine signaling from Meckel’scartilage and tooth buds. However, there is no evidencethat Meckel’s cartilage secretes growth factors.Interestingly, our results revealed one down-regulatedgene in the double-mutant mandible, Mir300, which is,indeed, expressed in Meckel’s cartilage. Mir300 is atranscription factor, and therefore it could be involved inregulating expression of other genes. It is also possiblethat both muscle and Meckel’s cartilage secrete cues thatregulate mandibular morphogenesis and growth.

As previously discussed, secondary cartilageformation can be initiated in the absence of muscle butthe amount of tissue is reduced. Secondary cartilage is,however, poorly maintained in the absence of muscleand all the processes are affected: the condylar process isseverely affected, but the coronoid and angular processesare absent (Rot-Nikcevic et al., 2007). Therefore, anadequate development of TMJ is not possible if thesecondary cartilage cannot be maintained. The condylarprocess is a muscle attachment site, but, in addition, itforms an articulation with the temporal bone to form aTMJ. This provides the condylar process with additional



Table 6. Genes down-regulated ≥3 fold in double-mutant mouse mandible for which knockouts were generated but abnormal mandible phenotype wasnot reported. Genes are sorted by fold change (FC).

Gene FC Gene title Deletion mutants exhibit:

Trdn -7.85 triadin loss of transverse orientation of triads within skeletal muscle cells (Shen et al., 2007)Atp2a1 -7.13 ATPase, Ca++ transporting,

cardiac muscle, fast twitch 1respiratory distress, progressive cyanosis; lung tissues and the diaphragm muscle show aberrantmorphology (Pan et al., 2003)

Neb -6.97 nebulin severe skeletal muscle weakness, depressed contractility (Witt et al., 2006)Ttn -6.79 titin vascular, cardiac and skeletal muscle defects causing growth retardation, muscle weakness,

abnormal posture, and premature death (Gotthardt et al., 2003)Acta1 -6.65 actin, alpha 1, skeletal muscle reduced body weight/size, atrophy of brown adipose tissue, depleted glycogen stores of the liver

and skeletal muscles, muscle weakness, and scoliosis (Crawford et al., 2002)Myot -6.51 myotilin normal skeletal and cardiac muscle morphology and function, growth rate, survival, and internal

organ morphology (Moza et al., 2007)Myoz2 -6.34 myozenin 2 an excess of skeletal muscle fibers and chronically activated hypertrophic gene program despite

the absence of hypertrophy (Frey et al., 2004)Myoz1 -6.32 myozenin 1 reduced body weight and fast-twitch muscle mass, increased exercise capacity (Frey et al., 2008)Ckm -6.23 creatine kinase, muscle abnormalities in function and energy utilization of both skeletal and cardiac muscle (van Deursen et al., 1993)Casq2 -5.96 calsequestrin 2 impaired intracellular calcium regulation in cardiac myocytes (Knollmann et al., 2006)

Apobec2 -5.95 apolipoprotein B mRNA editingenzyme, catalytic polypeptide 2 growth retardation and decreased bone mineralization and density (Mikl et al., 2005)

Smpx -5.56 small muscle protein, X-linked defects in heart, skeletal muscle morphology (Palmer et al., 2001)Hfe2 -5.06 hemochromatosis type 2 (juvenile)

(human homolog) lack of hepcidin expression, severe iron overload and male sterility (Huang et al., 2005)

Mylpf -5.01 myosin light chain, phospho-rylatable, fast skeletal muscle complete lack of skeletal muscle; mutants die immediately after birth (Wang et al., 2007)

Ryr1 -5.00 ryanodine receptor 1, skeletalmuscle

rounded body shape, edema, thin and misshapen ribs, abnormal muscle fibers; mutants dieperinatally (Takeshima et al., 1994)

Pvalb -4.98 parvalbumin abnormal muscle contractility and Purkinje cell mitochondrial morphology (Schwaller et al., 1999)Ldb3 -4.95 LIM domain binding 3 myopathy, dysphagia, heart vascular congestion, dilated heart ventricles, cyanosis, and respiratory

distress (Huang et al, 2003)Des -4.95 desmin histologically detectable defects of cardiac, skeletal, and smooth muscle (Millner et al., 1996)Ppp1r3a -4.70 protein phosphatase 1, regulatory

(inhibitor) subunit 3A reduced levels of skeletal muscle glycogen (Suzuki et al., 2001)

Itgb1bp2 -4.65 integrin beta 1 binding protein 2 contractile dysfunction of the heart and dilated cardiomyopathy when subjected to pressureoverload (Brancaccio et al., 2003)

Myf6 -4.62 myogenic factor 6 variable rib abnormalities, abnormal intercostal muscle morphology, reduced expression of Myf5,postnatal mortality proportional to the severity of the rib defect (Keller et al., 2004)

Ckmt2 -4.56 creatine kinase, mitochondrial 2 hypertrophic and dilated left ventricles and functional abnormalities (Steeghs et al., 1997)Smyd1 -4.55 SET and MYND domain containing 1 enlarged heart and developmental abnormalities of the right ventricle (Gottlieb et al., 2002)Myh1 -4.37 myosin, heavy polypeptide 1,

skeletal muscle, adultreduced growth, muscular weakness, kyphosis, and abnormal kinetics of muscle contraction andrelaxation (Acakpo-Satchivi et al., 1997)

Tmod1 -4.33 tropomodulin 1/ thiosulfate sulfurtrans-ferase-like domain containing 2 aborted heart development and consequent embryonic lethality (Chu et al., 2003)

Srl -4.28 sarcalumenin impaired calcium store functions in skeletal and cardiac muscle cells resulting in slow contractionand relaxation phases (Yoshida et al., 2005)

Csrp3 -4.19 cysteine and glycine-rich protein 3 disrupted cardiomyocyte organization that results in premature death, left ventricle dilation,hypertrophy, decreased contractility, and fibrosis (Arber et al., 1997)

Xirp2 -4.15 xin actin-binding repeat containing 2 abnormal heart shape, ventricular septal defects, a failure of mature intercalated disc formation,severe growth retardation, and postnatal lethality (McCalmon et al., 2010)

Pdlim3 -4.07 PDZ and LIM domain 3 no major defects in skeletal muscle (Jo et al., 2001)Sypl2 -4.04 synaptophysin-like 2 reduced body weight, abnormal skeletal muscle membranes and irregular skeletal muscle

contractility (Nishi et al., 1999)Mstn -3.92 myostatin increased size of striated muscle due to both hyperplasia and hypertrophy, reduced adiposity, and

increased bone mineral density (Welle et al., 2006)

Cpt1b -3.91carnitine palmitoyltransferase 1bmuscle/ Chkb-Cpt1b readthroughtranscript

in utero death prior to E9.5 (Chick et al., 2005)

Tnnt2 -3.90 troponin T2, cardiac embryonic lethality during and prior to organogenesis and abnormal heart development (Ahmad et al., 2008)Myog -3.79 myogenin severe reduction in muscle mass associated with delayed primary myogenesis and very little secondary

myofiber formation, defects of the thoracic skeleton, and perinatal death (Hasty et al., 1993)Cacna1s -3.79 calcium channel, voltage-dependent, L

type, alpha 1S subunitedema and failure of myoblast differentiation by E13; perinatal death; muscle degeneration,secondary anomalies of the skeleton, short jaw, cleft palate (Skarnes et al., 2011)

Chrna1 -3.78 cholinergic receptor, nicotinic,alpha polypeptide 1 (muscle)

neonatal lethality, kyphosis, carpotosis, increased motor neuron number, abnormal neuromuscularsynapse (An et al., 2010)

To be continued.

mechanical stimulation from the bone, which couldpossibly explain why it is less affected by the absence ofmusculature than the coronoid or angular processes. It isalso worth mentioning that the muscle that attaches tothe condyle, the lateral pterygoid muscle, belongs to thegroups of muscles of mastication, but its role issomewhat different. Investigation of myofiber propertiesof the adult mouse masticatory muscles suggests thatmasseter and temporalis muscles are involved with rapidmasticatory movement, characteristic for mice, whilelateral pterygoid muscle is involved in functions notprimarily related to movement, but functions that requireforce, such as retention of jaw position (Abe et al.,2008). These differences in muscle dynamics and thepull they exert on bone could result in the differentlevels to which the three processes are affected in theabsence of the muscle.

A combination of muscle loading, type of itsattachment and muscle activity, and a set of its signalingproteins present a complex environment in which otherstructures form. Change in one of the components of thisenvironment affects the final result, i.e., the phenotype.

The head is viewed as a complex region integratedduring development through numerous epigeneticinteractions, both between and within its units(Lieberman, 2011). The concept of functional matrixhypothesis (FMH) is not a new one (Van der Klaauw,1948-1952; Moss and Young, 1960; Moss, 1968) and itproposes that the head comprises of a series offunctional matrices, i.e., genetically determined andfunctionally maintained soft tissues and the spaces theyoccupy (Moss, 1968; reviewed in Lieberman, 2010).Muscle is one example of a functional matrix that isenclosed by skeletal tissue, or it encloses the skeletaltissue (depending on its anatomical location), whereskeletal tissue is another functional cranial component.Moss (1968) hypothesized that each functionalcomponent derives its shape from the shape and/or

functions of the soft tissue and spaces it encloses. Inaddition, as Lieberman (2010) argues, a more integratedhypothesis should be considered, the one that includesreciprocal epigenetic interactions between skeletal andnon-skeletal (e.g., muscle) components. We suggest thatthis functional matrix model should be considered whenanalyzing and describing complex mandibulardevelopment and phenotype, as it is a result of reciprocalepigenetic interactions among skeletal and non-skeletaltissues. Future plans: mouse-to-human translation

Our use of the web-based mouse databases was alsoaimed at identifying whether Cacna1s or any of our fivecandidate genes from Table 3 have homologues inhumans. Human homologues were identified for five ofthem (Cacna1s, Ckm, Des, Tnnc1 and Myog), and three(Cacna1s, Des and Tnnc1) have human diseasesassociated with them (MGI). However, none of thediseases involves mandibular hypoplasia or affectscraniofacial skeleton (MGI). Meanwhile, recent studiesin humans point out developmental interactions betweenthe mandible, the teeth and the muscle insertion sites(Coquerelle et al., 2013).

In fact, recently, we (and our collaborators) havebeen studying expression and distribution patterns of theregulators of early human mandible (Brakus et al., 2010)and palate (Vukojevic et al., 2012; Hall and Precious,2013) development. Indeed, as a collaborative effortbetween our laboratories, we employed human mandibletissues and examined whether any of the moleculesdetected using mouse tissues apply to human tissues, asa part of the mouse-to-human translation effort. Ourimmediate goal is to improve our understanding ofmolecules’ distribution patterns, and ultimately identifymarkers for predicting micrognathia, and TMJ disordersassociated with the poor maintenance of the secondary


Continuation.

Mylk2 -3.70 myosin, light polypeptide kinase 2,skeletal muscle impaired skeletal muscle twitch tension response to tetanic stimulation (Zhi et al., 2005)

Xirp1 -3.69 xin actin-binding repeat containing 1 cardiac hypertrophy and a disruption of cardiac intercalated disc structure and myofilamentabnormalities (Otten et al., 2010)

Cacng1 -3.65 calcium channel, voltage-dependent, gamma subunit 1 abnormal muscle calcium currents (Freise et al., 2000)

Sync -3.59 syncoilin /retinoblastoma bindingprotein 4

reduced generation of isometric stress in skeletal muscle; impaired contractility and increasedskeletal muscle damage under a forced exercise regime (McCullagh et al., 2008)

Trim72 -3.53 tripartite motif-containing 72 muscle pathologies that develop with age (Cai et al., 2009)Bves -3.44 blood vessel epicardial substance delayed muscle regeneration following induced injury (Andree et al., 2002)Cox6a2 -3.4 cytochrome c oxidase, subunit VI

a, polypeptide 2cardiac dysfunction as a result of abnormal ventricular filling or diastolic dysfunction under maximalcardiac load (Radford et al., 2002)

Trim55 -3.17 tripartite motif-containing 55 increased heart and muscle to body weight ratios and cardiac hypertrophy (Witt et al., 2008)Cox17 -3.17 cytochrome c oxidase, subunit XVII

assembly protein homolog (yeast)retarded growth, die between E8.5 and E10, severe reductions in cytochrome c oxidase activity atE6.5 (Takahashi et al., 2002)

Jph1 -3.03 junctophilin 1 failure to suckle; die shortly after birth; deficiencies of triad junctions and contraction in skeletalmuscle (Takeshima et al., 2000)

Gjd4 -3.01 gap junction protein, delta 4 accelerated muscle regeneration following BaCl2 injection (von Maltzahn et al., 2011)

cartilage tissue. Please note that at this time we providethe human translation of previously discovered mousedata. It is our future plan to translate the mouse datarevealed by our current analysis to the human data.

During embryonic development, TGFβ1(transforming growth factor beta 1) participates in cellmigration to the place of future skeletogenesis, and inthe epithelial-mesenchymal interactions and cellularcondensations (Kanaan and Kanaan, 2006). Expressionand function analysis of one of the TGFβ isoforms,TGFβ2, during mouse mandible development, identifiesthe role of TGFβ2 in proliferation of osteoprogenitor andchondroprogenitor cells. TGFβ2 knockout mice haveanomalies of the lower jaw (Oka et al., 2007b, 2008).TGFβ1 strongly stimulates the expression of CTGF(connective tissue growth factor) and FN (fibronectin),especially at the sites of mesenchymal condensations inthe future mouse Meckel’s cartilage (Ito et al., 2002;Shimo et al., 2004; Oka et al., 2007b). There are noprevious data describing its distribution in early humanmandible development (Fig. 2A-C).

CTGF, on the other hand, mediates cell-cellinteractions and aggregations in the first branchial archand stimulates mesenchymal cell differentiation intochondrocytes (Shimo et al., 2004). Studies with mutantmice showed that this factor has impact not just onchondrocyte differentiation, but also on theirproliferation in Meckel’s cartilage (Oka et al., 2007a).CTGF regulates osteoblast cell morphology, viability,migration, and proliferation (Kanaan et al., 2006). Infact, CTGF is present in periosteal cells andhypertrophic chondrocytes during bone healing inexperimental animals. The functions of this protein onangiogenesis, mesenchymal cells, chondrocytes andosteoblasts are mutually overlapping and as such have animportant role in the development and healing of bones.CTGF knockout mice show bone dimorphism, i.e.,bending of long bones, ribs and shortening of themandible (Ivkovic et al., 2003). There are no previousdata describing its distribution in early human mandibledevelopment (Fig. 2D-F).

Analysis of VEGF (vascular endothelial growthfactor) knockout mice pointed at its role in the initiationof ingrowing blood vessels into hypertrophic cartilage,and therefore in the endochondral ossification (Zelzer etal., 2004). This factor is important for the survival ofchondrocytes during bone formation, for normaldifferentiation of immature progenitor cells intochondrocytes, osteoblasts, osteoclasts and endothelialcells (Maes et al., 2002; Zelzer et al., 2004). VEGF wasfound to stimulate the activity of osteoclasts, increasebone resorption and positively influence the activity ofosteoblasts (Dai and Rabie, 2007). There are no previousdata describing its distribution in early human mandibledevelopment (Fig. 2G-I).

Finally, FN is a multifunctional glycoprotein of theextracellular matrix that binds to membrane receptors,integrins, and to collagen, fibrin, and proteoglycans. Itmediates cell adhesion, growth, migration and

differentiation. In addition to its role in wound healing, itis also important in embryogenesis, angiogenesis,inflammation and tumorigenesis. Connection of FN tointegrins is essential for the survival of osteoblasts, theirproliferation, bone matrix mineralization and ossification(Wierzbicka-Patynowski and Schwarzbauer, 2003 andreferences therein; Garcia and Reyes, 2005). Theinteraction of FN with specific integrins, α5β1, wasimportant for early bone formation in mice (Moursi etal., 1997). There are no previous data describing itsdistribution in early human mandible development (Fig.2J-L).

In conclusion, together with the animal studies data,the described distribution patterns of TGF1, CTGF,VEGF and FN, visible in Fig. 2, support the role of thestudied molecules in mandibular development. Acknowledgements. We are grateful to Heather Angka and Asja Mileticfor their expert technical assistance. This work was funded by anoperating grant from the National Science and Engineering ResearchCouncil of Canada (NSERC), and the infrastructure grants from theCanada Foundation for Innovation (CFI) and the Dalhousie MedicalResearch Foundation (DMRF) to BK. This work has also beensupported by the Ministry of Science, Education and Sports of theRepublic of Croatia to MSB.

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Accepted May 27, 2014


Review Role of skeletal muscle in mandible development · 2020-03-03 · ossification, but have...

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