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Molecular Mechanisms Governing Early Myogenesis of Mouse Tongue
J. Oral Biosci. 49 （3）：211−215, 2007
Recent Studies of Craniofacial Muscles MINI−REVIEW 2
Molecular Mechanisms Governing Early Myogenesis of Mouse Tongue Yuji Taya and Takaaki Aoba§ Department of Pathology, School of Life Dentistry at Tokyo, Nippon Dental University 1−9−20 Fujimi, Chiyoda−ku, Tokyo 102−8159, Japan 〔Received on March 20, 2007；Accepted on April 27, 2007〕 Key words：mouse tongue／developmental morphogenesis／cell migration Abstract：The tongue muscle develops from myogenic precursor cells that undergo long−range migration from the occipital somites to tongue primordium. In this article, we aimed to overview cellular and molecular events taking place during the migration of tongue myogenic precursor cells in embryonic mice. Immunohistochemical techniques were used to determine the chronology and positions of desmin expression during early tongue development. Based on the three−dimensional（3D）reconstruction of serial desmin−immunostained images, we defined the discrete migration pathway of myogenic precursor cells as follows：occipital somites→mesenchyme of trunk→ventral areas of the fourth→third and second branchial arches before arriving at the median mandibular arches around E10.3. The migrating myogenic precursor cells were characterized as desmin−positive／MyoD−negative cells with the development of lamellipodia and expression of Rho family genes. Once within the tongue primordium, myogenic precursor cells were ready to switch off their migratory phenotypes and express myogenic regulatory factors（MRFs）. Although multiple signaling cues have been supposed to exert effects on the migration and terminal differentiation of tongue myogenic precursor cells, the exact molecular mechanisms and cellular interactions remain to be elucidated.
Introduction Tongue development involves cells from multiple origins, i. e., the first, second, third, and fourth branchial arches, and the occipital somites1）. The tongue musculature is uniquely derived from the occipital somites. Tongue myogenic precursor cells undergo long−range migration from the occipital somites to the putative area of the tongue primordium, where they follow the morphogenic program of differentiation into myoblasts and myotubes under the guidance of the MyoD family of basic helix−loop−helix factors, §
Corresponding author E−mail：patho−[email protected]
termed myogenic regulatory factors（MRFs）1,2）. Our long−term goal is to gain a comprehensive understanding of the cellular and molecular mechanisms regulating tongue morphogenesis and muscle development. In this article, we provide a brief overview regarding the migration pathway of myogenic precursor cells from the occipital somites to tongue primordium, and the phenotypic features of migrating precursor cells in embryonic mice. Spatiotemporal Histomorphologic Events Linked to Early Tongue Myogenesis In order to formulate the spatiotemporal events of early tongue development, ICR mouse embryos at 9.5 through 12.5 days postcoitus （called herein E9.5
212
Y. Taya & T. Aoba：Embryonic Tongue Myogenesis in Mice
through E12.5, respectively） were subjected to analysis. Studies included histomorphologic including three−dimensional（3D）reconstruction of serial histologic views, and immunohistochemistry and real−time PCR for the localization and expression analysis of muscle−specific markers and putative regulators in the tongue primordium and circumferential regions. Immunohistochemistry proved that desmin expression, i. e., a phenotypic marker for myogenic commitment and differentiation, occurs in very early developmental stages, followed by the departure and migration of desmin−positive／MyoD−negative cells from the occipital somites into the branchial arch around E9.8. The migrating myogenic precursor cells were characterized as desmin−positive／MyoD−negative cells with the formation of cellular protrusions（e. g., 3） lamellipodia）at their leading edge . Particularly, we aimed at monitoring the spatiotemporal localization of migrating myogenic precursor cells with the aid of 3D reconstruction of anti−desmin immunostained views. As illustrated in Fig. 1, the results validated that the tongue myogenic cells take a long−range migration route as follows：occipital somites→mesenchyme of trunk→ventral areas of the fourth→third and second branchial arches3）. A leading group of the desmin− positive／MyoD−negative cells first reached the medial portion of mandibular arches around E10.3. Within the mandibular arches between E10.3 and E11.0, the progenitor cells remained MyoD−negative, but their migratory phenotypes altered markedly into those of non−migratory cells, as evidenced by a loss of lamellipodia and their polygonal morphology with elongated cellular processes. The lateral lingual swellings, i. e., the earliest morphogenic feature of the tongue primordium, became discernible in histologic views around E11.0. At that developmental stage, the migrated myogenic cells contacted with the epithelium underneath the median sulcus of lateral lingual swellings and created a massive cellular community at the central circumferential region of the primordium3）. Notably, MyoD−positive myoblasts started to differentiate within the population in contact with their progenitor cells, supporting the concept of“community effects” , that is, myoblast differentiation requires interactions between a certain
Fig. 1 Schematic transverse view showing the migration route of myogenic precursor cells from occipital somites to the tongue primordium at E10.7. MA：mandibular arches, BA2−4： branchial arches 2−4, MT：mesenchyme of trunk.
mass of cells or a variety of environmental cues through multiple cell interactions. During the period of E11.4 through E12.5, the tissue volume of the tongue primordium increased rapidly by accompanying the locational shift of the myogenic cell population comprising multistep−differentiated cells of precursors and myoblasts with and without proliferation activities. Phenotypic Features of Migrating Myogenic Lineage As shown in the schematic diagram of migrating cells（Fig. 2）, a migrating cell is, in general, characterized by its intrinsic morphologic polarity with the development of protrusions such as lamellipodia and filopodia at the leading edge4）. Cell migration requires the sequentially coordinated modulation of cellular morphology and functions：creation of membrane extensions at the leading edge, establishment of new focal adhesions, contraction of the cytoplasm, breaking of adhesions at the trailing edge, and translocation of the cell to allow cell movement. Rho family GTPases, i. e., Rac1, Cdc42, and RhoA, are known to play central roles in the cellular directed migration through the regulation of the organization of actin filaments：specifically, Rac1 stimulates lamellipodia for-
213
Fig. 2 Schematic diagram of migrating cell
mation by activating WAVE and Filamin via PAK and is essential for forward movement；Cdc42 binds to N−WASP, mediating filopodia formation via Filamin； and RhoA regulates the formation of actin stress fibers and focal adhesions4）. Immunofluorescence analysis with anti−Rac1 and anti−desmin antibodies showed that some of the desmin−positive cells localized in the migration pathway display a polarized cell morphology with the localization of Rac1 at the leading edge（Fig. 3）. It is pertinent that in some of the desmin−positive cells, the Rac1−positive leading edge pointed toward their destination, i. e., mandibular arch5）. In parallel experiments using real−time RT−PCR in combination with laser capture microdissection, we also verified expressions of Rac1, Cdc42, and RhoA, as well as Lbx1, Pax3, and cMet as migratory cell markers, in the cell population of mouse secondary branchial arches at E10.55）. Restriction of Cell Differentiation for Migrating Myogenic Precursors In addition to the determination of the migration pathway of myogenic precursor cells, we focused on whether the migrating myogenic lineage is under tight control for proliferation and differentiation during their long−range migration period, because premature myogenic differentiation would interfere with the migration of tongue myogenic precursor cells. Immunohistochemical analysis showed that a significant fraction of desmin−positive cells localized in the migration route were Ki67−positive and／or MyoD−
Fig. 3 Immunofluorescence localization of Rac1 （green） in a desmin−positive （red）muscle precursor cell（second branchial arch, E10.5）. The nucleus is stained with DAPI. The merged image shows the localization of Rac1 at the leading edge of the desmin− positive cell toward the mandibular arch. Arrow indicates the direction of the mandibular arch. Bar：20 μm.
positive3）. These observations indicated that these myogenic cells possess the potential for mitosis and differentiation into myoblasts in the migration route.
214
Y. Taya & T. Aoba：Embryonic Tongue Myogenesis in Mice
In spite of their potential capability to readily express MyoD, it is of importance that tongue myogenic cells first reaching the median sides of the mandibular arches remained MyoD−negative. Collectively, we consider that all desmin−positive cells in the migration route comprise discrete subgroups, and that some of the migrated cells at any position may undergo myogenic commitment in response to patterning signals that may arise from adjacent tissues. Transcriptional regulation of MRF genes is likely regulated by a variety of environmental cues including cell interactions. Multiple signals emanating from adjacent tissues, such as sonic hedgehog（Shh）, may converge to dictate the timing and positional specification of phenotypes and differentiation of the myogenic lineage, giving rise to the formation of the appropriate muscles at the distinctly coded branchial arch regions6）. On the other hand, the precursor state, marked as desmin−positive but MyoD−negative, can be maintained during the migration process. This segregation of certain precursors from the inducible environmental cues may be counteracted by functions of certain transcriptional repressors. Bmp4 and Msx1 are candidates for key inhibitory molecules of skeletal myogenesis7）. Our preliminary studies using real− time RT−PCR supported the involvement of myogenic regulatory genes, e. g., BMP4, Follistatin, Shh, Notch1, and TGFβ in the guidance mechanism and myoblast differentiation in the branchial arches. At present, much still remains to be elucidated about the molecular network governing muscle development, but it is a hypothesis to be tested that the cohort of inductory or inhibitory signals regulates myogenic differentiation of the migrating myogenic lineage with only a subpopulation of premyogenic cells becoming committed at any position and timing. Conclusive Remarks The tongue muscle develops from myogenic precursor cells that have originated from the occipital somites and have undergone long−range migration toward the tongue primordium. We defined a discrete cell migration pathway initiating in somites and terminating in the lateral lingual swellings of the tongue
primordium based upon desmin expression patterns in E9−11 embryos. Their migrating cells reached the second branchial arches at E9.8, mandibular arches at E10.3, and, terminally, the lateral lingual swellings at E11.4. During their migration through the branchial mesenchyme, the phenotype and differentiation potential of myogenic precursor cells is restricted under the guidance of various molecular cues within a mesenchymal population including branchial mesodermal and neural crest cells. After arriving at the mandibular arches, tongue muscle precursor cells began to aggregate in contact with the epithelium and, within the formed cell community, underwent marked changes in their morphology and phenotype before their terminal differentiation. The timing and positional specification of precursors to myoblast differentiation via multicellular community interactions need to be further elucidated. Acknowledgement This study was supported in part by Grants−in−Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan （16591842 and 17390489）. References
1）Noden, D. M. and Francis−West, P.：The differentiation and morphogenesis of craniofacial muscles. Dev. Dyn. 235：1194―1218, 2006. 2）Yamane, A., Mayo, M., Shuler, C., Crowe, D., Ohnuki, Y., Dalrymple, K. and Saeki, Y.：Expression of myogenic regulatory factors during the development of mouse tongue striated muscle. Arch. Oral Biol. 45： 71―78, 2000. 3）Fujitake, T., Taya, Y., Uchikawa, Y. and Aoba, T.： Migration and phenotype−transition of myogenic lineage in the development of the mouse tongue. Jpn. J. Ped. Dent. 43：403―416, 2005. 4）Burridge, K. and Wennerberg, K.：Rho and Rac take center stage. Cell 116：167―179, 2004. 5）Taya, Y. and Aoba, T.：Molecular networks governing mouse tongue morphogenesis and myogenesis. J. Oral Biosci. 48（Suppl）：94, 2006. 6）Dale, K., Sattar, N., Heemskerk, J., Clarke, J. D.,
215 Placzek, M. and Dodd, J.：Differential patterning of ventral midline cells by axial mesoderm is regulated by BMP7 and chordin. Development 126：397―408, 1999.
7）Parker, M. H., Seale, P. and Rudnicki, M. A.：Looking back to the embryo：defining transcriptional networks in adult myogenesis. Nat. Rev. Genet. 4：497 ―507, 2003.
Recent Studies of Craniofacial Muscles MINI−REVIEW 2
Molecular Mechanisms Governing Early Myogenesis of Mouse Tongue Yuji Taya and Takaaki Aoba§ Department of Pathology, School of Life Dentistry at Tokyo, Nippon Dental University 1−9−20 Fujimi, Chiyoda−ku, Tokyo 102−8159, Japan 〔Received on March 20, 2007；Accepted on April 27, 2007〕 Key words：mouse tongue／developmental morphogenesis／cell migration Abstract：The tongue muscle develops from myogenic precursor cells that undergo long−range migration from the occipital somites to tongue primordium. In this article, we aimed to overview cellular and molecular events taking place during the migration of tongue myogenic precursor cells in embryonic mice. Immunohistochemical techniques were used to determine the chronology and positions of desmin expression during early tongue development. Based on the three−dimensional（3D）reconstruction of serial desmin−immunostained images, we defined the discrete migration pathway of myogenic precursor cells as follows：occipital somites→mesenchyme of trunk→ventral areas of the fourth→third and second branchial arches before arriving at the median mandibular arches around E10.3. The migrating myogenic precursor cells were characterized as desmin−positive／MyoD−negative cells with the development of lamellipodia and expression of Rho family genes. Once within the tongue primordium, myogenic precursor cells were ready to switch off their migratory phenotypes and express myogenic regulatory factors（MRFs）. Although multiple signaling cues have been supposed to exert effects on the migration and terminal differentiation of tongue myogenic precursor cells, the exact molecular mechanisms and cellular interactions remain to be elucidated.
Introduction Tongue development involves cells from multiple origins, i. e., the first, second, third, and fourth branchial arches, and the occipital somites1）. The tongue musculature is uniquely derived from the occipital somites. Tongue myogenic precursor cells undergo long−range migration from the occipital somites to the putative area of the tongue primordium, where they follow the morphogenic program of differentiation into myoblasts and myotubes under the guidance of the MyoD family of basic helix−loop−helix factors, §
Corresponding author E−mail：patho−[email protected]
termed myogenic regulatory factors（MRFs）1,2）. Our long−term goal is to gain a comprehensive understanding of the cellular and molecular mechanisms regulating tongue morphogenesis and muscle development. In this article, we provide a brief overview regarding the migration pathway of myogenic precursor cells from the occipital somites to tongue primordium, and the phenotypic features of migrating precursor cells in embryonic mice. Spatiotemporal Histomorphologic Events Linked to Early Tongue Myogenesis In order to formulate the spatiotemporal events of early tongue development, ICR mouse embryos at 9.5 through 12.5 days postcoitus （called herein E9.5
212
Y. Taya & T. Aoba：Embryonic Tongue Myogenesis in Mice
through E12.5, respectively） were subjected to analysis. Studies included histomorphologic including three−dimensional（3D）reconstruction of serial histologic views, and immunohistochemistry and real−time PCR for the localization and expression analysis of muscle−specific markers and putative regulators in the tongue primordium and circumferential regions. Immunohistochemistry proved that desmin expression, i. e., a phenotypic marker for myogenic commitment and differentiation, occurs in very early developmental stages, followed by the departure and migration of desmin−positive／MyoD−negative cells from the occipital somites into the branchial arch around E9.8. The migrating myogenic precursor cells were characterized as desmin−positive／MyoD−negative cells with the formation of cellular protrusions（e. g., 3） lamellipodia）at their leading edge . Particularly, we aimed at monitoring the spatiotemporal localization of migrating myogenic precursor cells with the aid of 3D reconstruction of anti−desmin immunostained views. As illustrated in Fig. 1, the results validated that the tongue myogenic cells take a long−range migration route as follows：occipital somites→mesenchyme of trunk→ventral areas of the fourth→third and second branchial arches3）. A leading group of the desmin− positive／MyoD−negative cells first reached the medial portion of mandibular arches around E10.3. Within the mandibular arches between E10.3 and E11.0, the progenitor cells remained MyoD−negative, but their migratory phenotypes altered markedly into those of non−migratory cells, as evidenced by a loss of lamellipodia and their polygonal morphology with elongated cellular processes. The lateral lingual swellings, i. e., the earliest morphogenic feature of the tongue primordium, became discernible in histologic views around E11.0. At that developmental stage, the migrated myogenic cells contacted with the epithelium underneath the median sulcus of lateral lingual swellings and created a massive cellular community at the central circumferential region of the primordium3）. Notably, MyoD−positive myoblasts started to differentiate within the population in contact with their progenitor cells, supporting the concept of“community effects” , that is, myoblast differentiation requires interactions between a certain
Fig. 1 Schematic transverse view showing the migration route of myogenic precursor cells from occipital somites to the tongue primordium at E10.7. MA：mandibular arches, BA2−4： branchial arches 2−4, MT：mesenchyme of trunk.
mass of cells or a variety of environmental cues through multiple cell interactions. During the period of E11.4 through E12.5, the tissue volume of the tongue primordium increased rapidly by accompanying the locational shift of the myogenic cell population comprising multistep−differentiated cells of precursors and myoblasts with and without proliferation activities. Phenotypic Features of Migrating Myogenic Lineage As shown in the schematic diagram of migrating cells（Fig. 2）, a migrating cell is, in general, characterized by its intrinsic morphologic polarity with the development of protrusions such as lamellipodia and filopodia at the leading edge4）. Cell migration requires the sequentially coordinated modulation of cellular morphology and functions：creation of membrane extensions at the leading edge, establishment of new focal adhesions, contraction of the cytoplasm, breaking of adhesions at the trailing edge, and translocation of the cell to allow cell movement. Rho family GTPases, i. e., Rac1, Cdc42, and RhoA, are known to play central roles in the cellular directed migration through the regulation of the organization of actin filaments：specifically, Rac1 stimulates lamellipodia for-
213
Fig. 2 Schematic diagram of migrating cell
mation by activating WAVE and Filamin via PAK and is essential for forward movement；Cdc42 binds to N−WASP, mediating filopodia formation via Filamin； and RhoA regulates the formation of actin stress fibers and focal adhesions4）. Immunofluorescence analysis with anti−Rac1 and anti−desmin antibodies showed that some of the desmin−positive cells localized in the migration pathway display a polarized cell morphology with the localization of Rac1 at the leading edge（Fig. 3）. It is pertinent that in some of the desmin−positive cells, the Rac1−positive leading edge pointed toward their destination, i. e., mandibular arch5）. In parallel experiments using real−time RT−PCR in combination with laser capture microdissection, we also verified expressions of Rac1, Cdc42, and RhoA, as well as Lbx1, Pax3, and cMet as migratory cell markers, in the cell population of mouse secondary branchial arches at E10.55）. Restriction of Cell Differentiation for Migrating Myogenic Precursors In addition to the determination of the migration pathway of myogenic precursor cells, we focused on whether the migrating myogenic lineage is under tight control for proliferation and differentiation during their long−range migration period, because premature myogenic differentiation would interfere with the migration of tongue myogenic precursor cells. Immunohistochemical analysis showed that a significant fraction of desmin−positive cells localized in the migration route were Ki67−positive and／or MyoD−
Fig. 3 Immunofluorescence localization of Rac1 （green） in a desmin−positive （red）muscle precursor cell（second branchial arch, E10.5）. The nucleus is stained with DAPI. The merged image shows the localization of Rac1 at the leading edge of the desmin− positive cell toward the mandibular arch. Arrow indicates the direction of the mandibular arch. Bar：20 μm.
positive3）. These observations indicated that these myogenic cells possess the potential for mitosis and differentiation into myoblasts in the migration route.
214
Y. Taya & T. Aoba：Embryonic Tongue Myogenesis in Mice
In spite of their potential capability to readily express MyoD, it is of importance that tongue myogenic cells first reaching the median sides of the mandibular arches remained MyoD−negative. Collectively, we consider that all desmin−positive cells in the migration route comprise discrete subgroups, and that some of the migrated cells at any position may undergo myogenic commitment in response to patterning signals that may arise from adjacent tissues. Transcriptional regulation of MRF genes is likely regulated by a variety of environmental cues including cell interactions. Multiple signals emanating from adjacent tissues, such as sonic hedgehog（Shh）, may converge to dictate the timing and positional specification of phenotypes and differentiation of the myogenic lineage, giving rise to the formation of the appropriate muscles at the distinctly coded branchial arch regions6）. On the other hand, the precursor state, marked as desmin−positive but MyoD−negative, can be maintained during the migration process. This segregation of certain precursors from the inducible environmental cues may be counteracted by functions of certain transcriptional repressors. Bmp4 and Msx1 are candidates for key inhibitory molecules of skeletal myogenesis7）. Our preliminary studies using real− time RT−PCR supported the involvement of myogenic regulatory genes, e. g., BMP4, Follistatin, Shh, Notch1, and TGFβ in the guidance mechanism and myoblast differentiation in the branchial arches. At present, much still remains to be elucidated about the molecular network governing muscle development, but it is a hypothesis to be tested that the cohort of inductory or inhibitory signals regulates myogenic differentiation of the migrating myogenic lineage with only a subpopulation of premyogenic cells becoming committed at any position and timing. Conclusive Remarks The tongue muscle develops from myogenic precursor cells that have originated from the occipital somites and have undergone long−range migration toward the tongue primordium. We defined a discrete cell migration pathway initiating in somites and terminating in the lateral lingual swellings of the tongue
primordium based upon desmin expression patterns in E9−11 embryos. Their migrating cells reached the second branchial arches at E9.8, mandibular arches at E10.3, and, terminally, the lateral lingual swellings at E11.4. During their migration through the branchial mesenchyme, the phenotype and differentiation potential of myogenic precursor cells is restricted under the guidance of various molecular cues within a mesenchymal population including branchial mesodermal and neural crest cells. After arriving at the mandibular arches, tongue muscle precursor cells began to aggregate in contact with the epithelium and, within the formed cell community, underwent marked changes in their morphology and phenotype before their terminal differentiation. The timing and positional specification of precursors to myoblast differentiation via multicellular community interactions need to be further elucidated. Acknowledgement This study was supported in part by Grants−in−Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan （16591842 and 17390489）. References
1）Noden, D. M. and Francis−West, P.：The differentiation and morphogenesis of craniofacial muscles. Dev. Dyn. 235：1194―1218, 2006. 2）Yamane, A., Mayo, M., Shuler, C., Crowe, D., Ohnuki, Y., Dalrymple, K. and Saeki, Y.：Expression of myogenic regulatory factors during the development of mouse tongue striated muscle. Arch. Oral Biol. 45： 71―78, 2000. 3）Fujitake, T., Taya, Y., Uchikawa, Y. and Aoba, T.： Migration and phenotype−transition of myogenic lineage in the development of the mouse tongue. Jpn. J. Ped. Dent. 43：403―416, 2005. 4）Burridge, K. and Wennerberg, K.：Rho and Rac take center stage. Cell 116：167―179, 2004. 5）Taya, Y. and Aoba, T.：Molecular networks governing mouse tongue morphogenesis and myogenesis. J. Oral Biosci. 48（Suppl）：94, 2006. 6）Dale, K., Sattar, N., Heemskerk, J., Clarke, J. D.,
215 Placzek, M. and Dodd, J.：Differential patterning of ventral midline cells by axial mesoderm is regulated by BMP7 and chordin. Development 126：397―408, 1999.
7）Parker, M. H., Seale, P. and Rudnicki, M. A.：Looking back to the embryo：defining transcriptional networks in adult myogenesis. Nat. Rev. Genet. 4：497 ―507, 2003.