- Email: [email protected]
Integrin expression patterns during early limb muscle development in the mouse
Mechanisms of Development 119S (2002) S131–S134 www.elsevier.com/locate/modo
Integrin expression patterns during early limb muscle development in the mouse Fernanda Bajanca a,b, So´lveig Thorsteinsdo´ttir a,b,* a
Department of Zoology, Centre for Environmental Biology, Faculty of Sciences, University of Lisbon, 1749-016 Lisbon, Portugal b Gulbenkian Institute of Science, 2780-156 Oeiras, Portugal Received 7 May 2002; received in revised form 12 August 2002; accepted 14 August 2002
Abstract Cell-extracellular matrix interactions play crucial roles in limb muscle development but practically nothing is known on what integrins are involved before the differentiation of muscle precursor cells (MPCs) in the limb muscle masses. In this study we determine the expression patterns of integrins during early forelimb muscle development in the mouse. a6b1 integrin is downregulated in the lateral dermomyotome when delamination of MPCs occurs. In late E9.5 embryos, a1b1 and a5b1 are expressed in a pattern very similar to pax3, which marks MPCs migrating to the limb bud. After myf5 upregulation in the limb bud, a1b1 and a5b1 expression is maintained and the a4b1 integrin starts being expressed. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Integrins; Mouse; Limb; Muscle; Dermomyotome; Muscle precursor cells; Migration; Early differentiation
1. Results and discussion Limb muscle precursor cells (MPCs) are derived from the ventro-lateral edge of limb-level dermomyotomes. MPCs express pax3, known to induce expression of the c-met receptor which signals in response to scatter factor/hepatocyte growth factor (SF/HGF) secreted by the limb mesenchyme, resulting in MPC scattering at limb level (Bladt et al., 1995; Epstein et al., 1996; Dietrich et al., 1999). In the forelimb, delamination occurs between 24 and 33 somite stage (Houzelstein et al., 1999). The maintenance of MPC motility requires SF/HGF (Scaal et al., 1999) while the orientation into proper migration routes depends partially on Lbx1 (Brohmann et al., 2000; Gross et al., 2000). In the limb bud, MPCs proliferate, form dorsal and ventral muscle masses and initiate differentiation into skeletal muscle (Buckingham et al., 1992). Integrins are heterodimeric transmembrane receptors composed of non-covalently bound a- and b-subunits. They bind extracellular matrix molecules and transmit signals to inside the cell, affecting cell proliferation, differentiation and migration (Hynes, 1992). Several studies have addressed the expression and function of integrins during skeletal muscle development, but information on early * Corresponding author. Tel.: 1351-21-7500-000; fax: 1351-21-7500028. E-mail address: [email protected] (S. Thorsteinsdo´ttir).
stages in vivo is missing (reviewed in Gullberg et al., 1998). Antibodies against the b1 integrin subunit inhibited MPC migration in chick embryos (Jaffredo et al., 1988). However, in chimeras for b1-null and wild-type cells, b1null MPCs migrate to limb buds (Fa¨ssler and Meyer, 1995), but the low percentage of chimerism used in those experiments could have allowed the adhesion (e.g. via N-cadherin; Brand-Saberi et al., 1996) of b1-null cells to wild type cells and their passive transport to the limb (Fa¨ssler et al., 1996). To determine whether b1 integrins are present in the earliest phases of limb muscle development in the mouse, we used in situ hybridisation to detect the expression pattern of a1, a4, a5 and a6 subunits. a1, a4, and a5 pair exclusively with b1 (Hynes, 1992), while a6 also pairs with b4, but only later in development (Thorsteinsdo´ttir et al., 1995). Furthermore, these a subunits are all known to be present in somites and/or in limb muscle masses (Duband et al., 1992; Thorsteinsdo´ttir et al., 1995; Gullberg et al., 1998), making them good candidates to be involved in the early stages of limb muscle development. In contrast, the most prominently expressed a subunit in adult skeletal muscle, the a7 subunit, is only upregulated in limb muscle around E13 (Velling et al., 1996; Brancaccio et al., 1998). Before MPC delamination, the a6 integrin subunit is expressed in epithelial somites and in dermomyotome at all axial levels. By the 26 somite stage, the lateral one third of forelimb dermomyotomes is negative for a6
0925-4773/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(03)00105-9
S132
F. Bajanca, S. Thorsteinsdo´ttir / Mechanisms of Development 119S (2002) S131–S134
Fig. 1. In situ hybridisation reveals that a6 integrin messenger RNA is downregulated in the ventro-lateral dermomyotome during MPC delamination. (A) E9.5 (26 somite) embryo where a6 is downregulated at the ventro-lateral edge of the dermomyotome in limb level somites, while expression is maintained in all the dermomyotome of younger and older somites. Dotted lines show the level of sections B–E. (B) Section showing a young dermomyotome of a caudal somite, located posterior to the forelimb. All dermomyotomal cells express a6; the arrow points to labelled cells on its ventro-lateral edge. (C) Section showing the dermomyotome of a somite located immediately posterior to the forelimb. All dermomyotomal cells still express a6, the arrow shows labelled cells on its ventro-lateral edge. (D) Section of a somite at limb level, showing that a6 is no longer expressed at the ventro-lateral edge of its dermomyotome (arrows delimit unlabeled area). (E) Section showing a somite at heart level, anterior to the forelimb. The expression of a6 remains in the whole dermomyotome, even though this somite is older than those at forelimb level. a6 is also strongly expressed in the myotome (C–E). Bar ¼ 100 mm.
(compare Fig. 1D with 1B,C,E) while, at the same stage, pax3 expression is still present in the whole epithelial dermomyotome and in MPCs undergoing delamination (Fig. 2D). The a1, a4 and a5 integrin subunits were never detected in the dermomyotome (not shown). After the initiation of delamination, the a1 integrin subunit is detected in the forelimb bud (Fig. 2A) in a pattern very similar to pax3 (Fig. 2B). The expression of a5 (Fig. 2C) is more widespread than pax3 (Fig. 2D), but areas positive for pax3 are also a5-positive. No hybridisation signal for a4 or a6 was detected on these cells (not shown). At E10.5, MPCs in the forelimb are organised in dorsal and ventral muscle masses. Pax3 is still expressed by these cells (Fig. 3B,E,H) and some have already started expressing myf5 (Fig. 3C,F,I). Expression of a4 (Fig. 3A) can now be detected in a pattern very similar to pax3 and myf5 (compare Fig. 3A to B,C). a1 (Fig. 3D) and a5 (Fig. 3G) are also expressed in areas where pax3 (Figs. 3E,H) and myf5 (Figs. 3F,I) are present, although a5 is also expressed in the distal limb (Fig. 3G). Double immunofluorescence localisation of a5 and myf5 proteins (Figs. 3J–L) confirms that the great majority of myf5-positive cells express a5 (Fig. 3L, insert). In summary, we found a previously undescribed dynamic expression pattern for the a6 integrin subunit in limb-level
Fig. 2. a1 and a5 integrins are expressed during MPC migration in E9.5 (26–27 somite) embryos. Pairs of adjacent cryostat sections were exposed to probes against a1 (A) and pax3 (B) or a5 (C) and pax3 (D). (A) a1 is expressed in the forelimb mesenchyme; the arrows point to two areas, one ventral and one dorsal, where expression is strongest. (B) Adjacent section to A, where pax3 positive cells are located in two areas, ventral and dorsal (arrows). (C) a5 is widely expressed in the forelimb mesenchyme; the arrow points to a ventral area, where expression is similar to pax3. (D) Consecutive section to C, where the pax3 positive ventral area is indicated by the arrow. Arrowhead in D indicates ventro-lateral lip of dermomyotome. Bar ¼ 100 mm.
F. Bajanca, S. Thorsteinsdo´ ttir / Mechanisms of Development 119S (2002) S131–S134
dermomyotomes. Furthermore, our results strongly suggest that a1 and a5 subunits are expressed by migrating MPCs and remain present at least until myf5 is upregulated. These data fill an important gap in the knowledge of integrin
S133
expression patterns during MPC delamination and migration (see Gullberg et al., 1998). Finally, a4 was detected as soon as myf5 is upregulated, slightly earlier than previously reported (Rosen et al., 1992; Kil et al., 1998).
Fig. 3. a4, a1 and a5 are expressed after myf5 upregulation in E10.5 forelimbs. In situ hybridisation (A–I) or immunohistochemistry (J–L) on forelimb-level cryostat sections of E10 (C) and E10.5 (A, B, D–L) embryos. Sections D–F and G–I are consecutive. At E10.5, a4 (A) is expressed in a pattern typical for muscle masses, as shown by an identical pattern for pax3 (B). Myf5 expression normally comes up slightly earlier (E10; C). a1 (D) is expressed dorsally and ventrally at E10.5 in a very similar pattern to pax3 (E) and myf5 (F). a5 (G) expression in E10.5 is present in areas positive for pax3 (H) and myf5 (I). Double immunohistochemistry in the same section showing localisation of a5 (J) and myf5 (K) proteins. Merging the two images (L and insert) shows that the great majority of myf5-positive cells express a5. Insert is a high magnification of the ventral muscle mass in L. Bar ¼ 100 mm; bar in insert ¼ 10 mm.
S134
F. Bajanca, S. Thorsteinsdo´ ttir / Mechanisms of Development 119S (2002) S131–S134
Future work should focus on the functional role of these b1 integrins in early limb muscle development and determining whether there is a link between these molecules and the numerous factors known to control MPC delamination, migration and proliferation. 2. Materials and methods In situ hybridisation with digoxigenin-labeled riboprobes was performed in whole mount or over 10 mm cryostat sections essentially as described by Henrique et al. (1995) and revealed with BM Purple (Roche). Following whole mount hybridisation, embryos were embedded in hydroxyethylmethacrylate (Kulzer Histo-Technik 8100) and sectioned. Double immunohistochemistry was performed on 10 mm cryostat sections of embryos fixed in 2% paraformaldehyde. Primary antibodies were rat anti-a5 integrin (clone BIIG-2; Developmental Studies Hybridoma Bank) and rabbit antimyf5 (Santa Cruz), while secondary antibodies were fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rat and anti-rabbit IgG, respectively (Sigma). Acknowledgements This work was supported by PhD grant SFRH/BD/1359/ 2000 to FB and the project PRAXIS XXI/P/PCNA/BIA/ 131/96 (FCT, Portugal). We thank the following researchers for their kind gift of cDNA: Drs J.T. Yang (a4 integrin), R.O. Hynes (a5 integrin), H. Gardner (a1 integrin), M. Buckingham (myf-5) and E. Olson (pax3), and I. Palmeirim for reading the manuscript. The BIIG-2 monoclonal antibody, developed by C. Damsky, was from Developmental Studies Hybridoma Bank developed under the auspices of NICHD and maintained by the University of Iowa. References Bladt, F., Riethmacher, D., Isenmann, S., Aguzzi, A., Birchmeier, C., 1995. Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud. Nature 376, 768–771. Brancaccio, M., Cabodi, S., Belkin, A.M., Collo, G., Koteliansky, V.E., Tomatis, D., Altruda, F., Silengo, L., Tarone, G., 1998. Differential onset of expression of a7 and b1D integrins during mouse heart and skeletal muscle development. Cell Adhes. Commun. 5, 193–205. Brand-Saberi, B., Gamel, A.J., Krenn, V., Muller, T.S., Wilting, J., Christ, B., 1996. N-cadherin is involved in myoblast migration and muscle differentiation in the avian limb bud. Dev. Biol. 178, 160–173.
Brohmann, H., Jagla, K., Birchmeier, C., 2000. The role of Lbx1 in migration of muscle precursor cells. Development 127, 437–445. Buckingham, M., Houzelstein, D., Lyons, G., Ontell, M., Ott, M., Sassoon, D., 1992. Expression of muscle genes in the mouse embryo. Symp. Soc. Exp. Biol. 46, 203–217. Dietrich, S., Abou-Rebyeh, F., Brohmann, H., Bladt, F., Sonnenberg-Riethmacher, E., Yamaai, T., Lumsden, A., Brand-Saberi, B., Birchmeier, C., 1999. The role of SF/HGF and c-met in the development of skeletal muscle. Development 126, 1621–1629. Duband, J., Belkin, A., Syfrig, J., Thiery, J., Koteliansky, V., 1992. Expression of a1 integrin, a laminin-collagen receptor, during myogenesis and neurogenesis in the avian embryo. Development 116, 585–600. Epstein, J.A., Shapiro, D.N., Cheng, J., Lam, P.Y., Maas, R.L., 1996. Pax3 modulates expression of the c-Met receptor during limb muscle development. Proc. Natl. Acad. Sci. USA 939, 4213–4218. Fa¨ ssler, R., Meyer, M., 1995. Consequences of lack of b1 integrin gene expression in mice. Genes Dev. 9, 1896–1908. Fa¨ ssler, R., Georges-Labouesse, E., Hirsch, E., 1996. Genetic analyses of integrin function in mice. Curr. Opin. Cell Biol. 8, 641–646. Gross, M., Rivard, L., Velasquez, T., Nakatsu, M., Jagla, K., Goulding, M., 2000. Lbx1 is required for muscle precursor migration along a lateral pathway into the limb. Development 127, 413–424. Gullberg, D., Velling, T., Lohikangas, L., Tiger, C.F., 1998. Integrins during muscle development and in muscular dystrophies. Front. Biosci. 3, d1039–d1050. Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J., Ish-Horowicz, D., 1995. Expression of a Delta homologue in prospective neurons in the chick. Nature 375, 787–790. Houzelstein, D., Auda-Boucher, G., Che´ raud, Y., Rouaud, T., Blanc, I., Tajbakhsh, S., Buckingham, M., Fontaine-Pe´ rus, J., Robet, B., 1999. The homeobox gene Msx1 is expressed in a subset of somites, and in muscle progenitor cells migrating into the forelimbs. Development 126, 2689–2701. Hynes, R.O., 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 691, 11–25. Jaffredo, T., Horwitz, A.F., Buck, C.A., Rong, P.M., Dieterlen-Lievre, F., 1988. Myoblast migration specifically inhibited in the chick embryo by grafted CSAT hybridoma cells secreting an anti-integrin antibody. Development 103, 431–446. Kil, S.H., Krull, C.E., Cann, G., Clegg, D., Bronner-Fraser, M., 1998. The a4 subunit of integrin is important for neural crest cell migration. Dev. Biol. 202, 29–42. Rosen, G., Sanes, J., LaChance, R., Cunningham, J., Roman, J., Dean, D., 1992. Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis. Cell 69, 1107–1119. Scaal, M., Bonafede, A., Dathe, V., Sachs, M., Cann, G., Christ, B., BrandSaberi, B., 1999. SF/HGF is a mediator between limb patterning and muscle development. Development 126, 4885–4893. Thorsteinsdo´ ttir, S., Roelen, B.A.J., Freund, E., Gaspar, A.C., Sonnenberg, A., Mummery, C.L., 1995. Expression patterns of laminin receptor splice variants a6Ab1 and a6Bb1 suggest different roles in mouse development. Dev. Dyn. 204, 240–258. Velling, T., Collo, G., Sorokin, L., Durbeej, M., Zhang, H.Y., Gullberg, D., 1996. Distinct a7Ab1 and a7Bb1 expression patterns during mouse development: a7A is restricted to skeletal muscle but a7B is expressed in striated muscle, vasculature, and nervous system. Dev. Dyn. 207, 355–371.
Integrin expression patterns during early limb muscle development in the mouse Fernanda Bajanca a,b, So´lveig Thorsteinsdo´ttir a,b,* a
Department of Zoology, Centre for Environmental Biology, Faculty of Sciences, University of Lisbon, 1749-016 Lisbon, Portugal b Gulbenkian Institute of Science, 2780-156 Oeiras, Portugal Received 7 May 2002; received in revised form 12 August 2002; accepted 14 August 2002
Abstract Cell-extracellular matrix interactions play crucial roles in limb muscle development but practically nothing is known on what integrins are involved before the differentiation of muscle precursor cells (MPCs) in the limb muscle masses. In this study we determine the expression patterns of integrins during early forelimb muscle development in the mouse. a6b1 integrin is downregulated in the lateral dermomyotome when delamination of MPCs occurs. In late E9.5 embryos, a1b1 and a5b1 are expressed in a pattern very similar to pax3, which marks MPCs migrating to the limb bud. After myf5 upregulation in the limb bud, a1b1 and a5b1 expression is maintained and the a4b1 integrin starts being expressed. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Integrins; Mouse; Limb; Muscle; Dermomyotome; Muscle precursor cells; Migration; Early differentiation
1. Results and discussion Limb muscle precursor cells (MPCs) are derived from the ventro-lateral edge of limb-level dermomyotomes. MPCs express pax3, known to induce expression of the c-met receptor which signals in response to scatter factor/hepatocyte growth factor (SF/HGF) secreted by the limb mesenchyme, resulting in MPC scattering at limb level (Bladt et al., 1995; Epstein et al., 1996; Dietrich et al., 1999). In the forelimb, delamination occurs between 24 and 33 somite stage (Houzelstein et al., 1999). The maintenance of MPC motility requires SF/HGF (Scaal et al., 1999) while the orientation into proper migration routes depends partially on Lbx1 (Brohmann et al., 2000; Gross et al., 2000). In the limb bud, MPCs proliferate, form dorsal and ventral muscle masses and initiate differentiation into skeletal muscle (Buckingham et al., 1992). Integrins are heterodimeric transmembrane receptors composed of non-covalently bound a- and b-subunits. They bind extracellular matrix molecules and transmit signals to inside the cell, affecting cell proliferation, differentiation and migration (Hynes, 1992). Several studies have addressed the expression and function of integrins during skeletal muscle development, but information on early * Corresponding author. Tel.: 1351-21-7500-000; fax: 1351-21-7500028. E-mail address: [email protected] (S. Thorsteinsdo´ttir).
stages in vivo is missing (reviewed in Gullberg et al., 1998). Antibodies against the b1 integrin subunit inhibited MPC migration in chick embryos (Jaffredo et al., 1988). However, in chimeras for b1-null and wild-type cells, b1null MPCs migrate to limb buds (Fa¨ssler and Meyer, 1995), but the low percentage of chimerism used in those experiments could have allowed the adhesion (e.g. via N-cadherin; Brand-Saberi et al., 1996) of b1-null cells to wild type cells and their passive transport to the limb (Fa¨ssler et al., 1996). To determine whether b1 integrins are present in the earliest phases of limb muscle development in the mouse, we used in situ hybridisation to detect the expression pattern of a1, a4, a5 and a6 subunits. a1, a4, and a5 pair exclusively with b1 (Hynes, 1992), while a6 also pairs with b4, but only later in development (Thorsteinsdo´ttir et al., 1995). Furthermore, these a subunits are all known to be present in somites and/or in limb muscle masses (Duband et al., 1992; Thorsteinsdo´ttir et al., 1995; Gullberg et al., 1998), making them good candidates to be involved in the early stages of limb muscle development. In contrast, the most prominently expressed a subunit in adult skeletal muscle, the a7 subunit, is only upregulated in limb muscle around E13 (Velling et al., 1996; Brancaccio et al., 1998). Before MPC delamination, the a6 integrin subunit is expressed in epithelial somites and in dermomyotome at all axial levels. By the 26 somite stage, the lateral one third of forelimb dermomyotomes is negative for a6
0925-4773/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(03)00105-9
S132
F. Bajanca, S. Thorsteinsdo´ttir / Mechanisms of Development 119S (2002) S131–S134
Fig. 1. In situ hybridisation reveals that a6 integrin messenger RNA is downregulated in the ventro-lateral dermomyotome during MPC delamination. (A) E9.5 (26 somite) embryo where a6 is downregulated at the ventro-lateral edge of the dermomyotome in limb level somites, while expression is maintained in all the dermomyotome of younger and older somites. Dotted lines show the level of sections B–E. (B) Section showing a young dermomyotome of a caudal somite, located posterior to the forelimb. All dermomyotomal cells express a6; the arrow points to labelled cells on its ventro-lateral edge. (C) Section showing the dermomyotome of a somite located immediately posterior to the forelimb. All dermomyotomal cells still express a6, the arrow shows labelled cells on its ventro-lateral edge. (D) Section of a somite at limb level, showing that a6 is no longer expressed at the ventro-lateral edge of its dermomyotome (arrows delimit unlabeled area). (E) Section showing a somite at heart level, anterior to the forelimb. The expression of a6 remains in the whole dermomyotome, even though this somite is older than those at forelimb level. a6 is also strongly expressed in the myotome (C–E). Bar ¼ 100 mm.
(compare Fig. 1D with 1B,C,E) while, at the same stage, pax3 expression is still present in the whole epithelial dermomyotome and in MPCs undergoing delamination (Fig. 2D). The a1, a4 and a5 integrin subunits were never detected in the dermomyotome (not shown). After the initiation of delamination, the a1 integrin subunit is detected in the forelimb bud (Fig. 2A) in a pattern very similar to pax3 (Fig. 2B). The expression of a5 (Fig. 2C) is more widespread than pax3 (Fig. 2D), but areas positive for pax3 are also a5-positive. No hybridisation signal for a4 or a6 was detected on these cells (not shown). At E10.5, MPCs in the forelimb are organised in dorsal and ventral muscle masses. Pax3 is still expressed by these cells (Fig. 3B,E,H) and some have already started expressing myf5 (Fig. 3C,F,I). Expression of a4 (Fig. 3A) can now be detected in a pattern very similar to pax3 and myf5 (compare Fig. 3A to B,C). a1 (Fig. 3D) and a5 (Fig. 3G) are also expressed in areas where pax3 (Figs. 3E,H) and myf5 (Figs. 3F,I) are present, although a5 is also expressed in the distal limb (Fig. 3G). Double immunofluorescence localisation of a5 and myf5 proteins (Figs. 3J–L) confirms that the great majority of myf5-positive cells express a5 (Fig. 3L, insert). In summary, we found a previously undescribed dynamic expression pattern for the a6 integrin subunit in limb-level
Fig. 2. a1 and a5 integrins are expressed during MPC migration in E9.5 (26–27 somite) embryos. Pairs of adjacent cryostat sections were exposed to probes against a1 (A) and pax3 (B) or a5 (C) and pax3 (D). (A) a1 is expressed in the forelimb mesenchyme; the arrows point to two areas, one ventral and one dorsal, where expression is strongest. (B) Adjacent section to A, where pax3 positive cells are located in two areas, ventral and dorsal (arrows). (C) a5 is widely expressed in the forelimb mesenchyme; the arrow points to a ventral area, where expression is similar to pax3. (D) Consecutive section to C, where the pax3 positive ventral area is indicated by the arrow. Arrowhead in D indicates ventro-lateral lip of dermomyotome. Bar ¼ 100 mm.
F. Bajanca, S. Thorsteinsdo´ ttir / Mechanisms of Development 119S (2002) S131–S134
dermomyotomes. Furthermore, our results strongly suggest that a1 and a5 subunits are expressed by migrating MPCs and remain present at least until myf5 is upregulated. These data fill an important gap in the knowledge of integrin
S133
expression patterns during MPC delamination and migration (see Gullberg et al., 1998). Finally, a4 was detected as soon as myf5 is upregulated, slightly earlier than previously reported (Rosen et al., 1992; Kil et al., 1998).
Fig. 3. a4, a1 and a5 are expressed after myf5 upregulation in E10.5 forelimbs. In situ hybridisation (A–I) or immunohistochemistry (J–L) on forelimb-level cryostat sections of E10 (C) and E10.5 (A, B, D–L) embryos. Sections D–F and G–I are consecutive. At E10.5, a4 (A) is expressed in a pattern typical for muscle masses, as shown by an identical pattern for pax3 (B). Myf5 expression normally comes up slightly earlier (E10; C). a1 (D) is expressed dorsally and ventrally at E10.5 in a very similar pattern to pax3 (E) and myf5 (F). a5 (G) expression in E10.5 is present in areas positive for pax3 (H) and myf5 (I). Double immunohistochemistry in the same section showing localisation of a5 (J) and myf5 (K) proteins. Merging the two images (L and insert) shows that the great majority of myf5-positive cells express a5. Insert is a high magnification of the ventral muscle mass in L. Bar ¼ 100 mm; bar in insert ¼ 10 mm.
S134
F. Bajanca, S. Thorsteinsdo´ ttir / Mechanisms of Development 119S (2002) S131–S134
Future work should focus on the functional role of these b1 integrins in early limb muscle development and determining whether there is a link between these molecules and the numerous factors known to control MPC delamination, migration and proliferation. 2. Materials and methods In situ hybridisation with digoxigenin-labeled riboprobes was performed in whole mount or over 10 mm cryostat sections essentially as described by Henrique et al. (1995) and revealed with BM Purple (Roche). Following whole mount hybridisation, embryos were embedded in hydroxyethylmethacrylate (Kulzer Histo-Technik 8100) and sectioned. Double immunohistochemistry was performed on 10 mm cryostat sections of embryos fixed in 2% paraformaldehyde. Primary antibodies were rat anti-a5 integrin (clone BIIG-2; Developmental Studies Hybridoma Bank) and rabbit antimyf5 (Santa Cruz), while secondary antibodies were fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rat and anti-rabbit IgG, respectively (Sigma). Acknowledgements This work was supported by PhD grant SFRH/BD/1359/ 2000 to FB and the project PRAXIS XXI/P/PCNA/BIA/ 131/96 (FCT, Portugal). We thank the following researchers for their kind gift of cDNA: Drs J.T. Yang (a4 integrin), R.O. Hynes (a5 integrin), H. Gardner (a1 integrin), M. Buckingham (myf-5) and E. Olson (pax3), and I. Palmeirim for reading the manuscript. The BIIG-2 monoclonal antibody, developed by C. Damsky, was from Developmental Studies Hybridoma Bank developed under the auspices of NICHD and maintained by the University of Iowa. References Bladt, F., Riethmacher, D., Isenmann, S., Aguzzi, A., Birchmeier, C., 1995. Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud. Nature 376, 768–771. Brancaccio, M., Cabodi, S., Belkin, A.M., Collo, G., Koteliansky, V.E., Tomatis, D., Altruda, F., Silengo, L., Tarone, G., 1998. Differential onset of expression of a7 and b1D integrins during mouse heart and skeletal muscle development. Cell Adhes. Commun. 5, 193–205. Brand-Saberi, B., Gamel, A.J., Krenn, V., Muller, T.S., Wilting, J., Christ, B., 1996. N-cadherin is involved in myoblast migration and muscle differentiation in the avian limb bud. Dev. Biol. 178, 160–173.
Brohmann, H., Jagla, K., Birchmeier, C., 2000. The role of Lbx1 in migration of muscle precursor cells. Development 127, 437–445. Buckingham, M., Houzelstein, D., Lyons, G., Ontell, M., Ott, M., Sassoon, D., 1992. Expression of muscle genes in the mouse embryo. Symp. Soc. Exp. Biol. 46, 203–217. Dietrich, S., Abou-Rebyeh, F., Brohmann, H., Bladt, F., Sonnenberg-Riethmacher, E., Yamaai, T., Lumsden, A., Brand-Saberi, B., Birchmeier, C., 1999. The role of SF/HGF and c-met in the development of skeletal muscle. Development 126, 1621–1629. Duband, J., Belkin, A., Syfrig, J., Thiery, J., Koteliansky, V., 1992. Expression of a1 integrin, a laminin-collagen receptor, during myogenesis and neurogenesis in the avian embryo. Development 116, 585–600. Epstein, J.A., Shapiro, D.N., Cheng, J., Lam, P.Y., Maas, R.L., 1996. Pax3 modulates expression of the c-Met receptor during limb muscle development. Proc. Natl. Acad. Sci. USA 939, 4213–4218. Fa¨ ssler, R., Meyer, M., 1995. Consequences of lack of b1 integrin gene expression in mice. Genes Dev. 9, 1896–1908. Fa¨ ssler, R., Georges-Labouesse, E., Hirsch, E., 1996. Genetic analyses of integrin function in mice. Curr. Opin. Cell Biol. 8, 641–646. Gross, M., Rivard, L., Velasquez, T., Nakatsu, M., Jagla, K., Goulding, M., 2000. Lbx1 is required for muscle precursor migration along a lateral pathway into the limb. Development 127, 413–424. Gullberg, D., Velling, T., Lohikangas, L., Tiger, C.F., 1998. Integrins during muscle development and in muscular dystrophies. Front. Biosci. 3, d1039–d1050. Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J., Ish-Horowicz, D., 1995. Expression of a Delta homologue in prospective neurons in the chick. Nature 375, 787–790. Houzelstein, D., Auda-Boucher, G., Che´ raud, Y., Rouaud, T., Blanc, I., Tajbakhsh, S., Buckingham, M., Fontaine-Pe´ rus, J., Robet, B., 1999. The homeobox gene Msx1 is expressed in a subset of somites, and in muscle progenitor cells migrating into the forelimbs. Development 126, 2689–2701. Hynes, R.O., 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 691, 11–25. Jaffredo, T., Horwitz, A.F., Buck, C.A., Rong, P.M., Dieterlen-Lievre, F., 1988. Myoblast migration specifically inhibited in the chick embryo by grafted CSAT hybridoma cells secreting an anti-integrin antibody. Development 103, 431–446. Kil, S.H., Krull, C.E., Cann, G., Clegg, D., Bronner-Fraser, M., 1998. The a4 subunit of integrin is important for neural crest cell migration. Dev. Biol. 202, 29–42. Rosen, G., Sanes, J., LaChance, R., Cunningham, J., Roman, J., Dean, D., 1992. Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis. Cell 69, 1107–1119. Scaal, M., Bonafede, A., Dathe, V., Sachs, M., Cann, G., Christ, B., BrandSaberi, B., 1999. SF/HGF is a mediator between limb patterning and muscle development. Development 126, 4885–4893. Thorsteinsdo´ ttir, S., Roelen, B.A.J., Freund, E., Gaspar, A.C., Sonnenberg, A., Mummery, C.L., 1995. Expression patterns of laminin receptor splice variants a6Ab1 and a6Bb1 suggest different roles in mouse development. Dev. Dyn. 204, 240–258. Velling, T., Collo, G., Sorokin, L., Durbeej, M., Zhang, H.Y., Gullberg, D., 1996. Distinct a7Ab1 and a7Bb1 expression patterns during mouse development: a7A is restricted to skeletal muscle but a7B is expressed in striated muscle, vasculature, and nervous system. Dev. Dyn. 207, 355–371.