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Connexin43 expression during Xenopus development
Mechanisms of Development 108 (2001) 217–220 www.elsevier.com/locate/modo
Gene expression pattern
Connexin43 expression during Xenopus development Marcel A.G. van der Heyden a,b, Liesbeth Roeleveld b, Josi Peterson b, Olivier H.J. Destre´e b,* a
Department of Medical Physiology, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands b Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands Received 6 June 2001; received in revised form 11 July 2001; accepted 18 July 2001
Abstract The spatio-temporal expression of connexin43 in Xenopus laevis embryos was studied by in situ hybridization. Cx43 expression is first detected at stage 25 in the developing eye. In stage 32, expression was found in the margin of the lens placode, the cement gland, notochord, and in stage 37 in the branchial arches. Early limb buds show strong expression of Cx43 distally while later on expression is confined to sites of precartilage condensation. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Connexin; Xenopus laevis; Xenopus tropicalis; Embryo; Lens; Limb; Notochord; Neural crest; Branchial arch; Cartilage
1. Results In mammals, Cx43 is one of more than 15 cloned connexin genes (Bruzzone et al., 1996; Goodenough et al., 1996). Cx43 transcription is responsive to developmentally important signals like Wnt, PTH and FGF (van der Heyden et al., 1998; Nadarajah et al., 1998; Schiller et al., 1997). Only four connexins were described so far in amphibians, i.e. Cx30, Cx38, Cx41 and Cx43 for Xenopus laevis (Gimlich et al., 1988, 1990; Yoshizaki and Patin˜o, 1995). In contrast to mammals, Cx43 becomes expressed relatively late during development in X. laevis (Gimlich et al., 1990) and is expressed in many adult tissues (Gimlich et al., 1990; Yoshizaki and Patin˜o, 1995). Detailed expression patterns of Cx43 during Xenopus development have not been described. Recently, Xenopus tropicalis was introduced as a system for amphibian genetics (Amaya et al., 1998). We isolated the Cx43 gene from X. tropicalis; the coding region shows 95% identity with that of X. laevis Cx43 at the nucleotide level and 99% at the amino acid level (Fig. 1A). Protein similarity is high with human, mouse, rat, bovine, chicken and fish (Table 1). Southern blot hybridizations demonstrate that in X. tropicalis Cx43 is a single copy gene (Fig. 1B). 1.1. Cx43 is expressed in the developing lens, notochord, cement gland and limbs Cx43 expression was first detected by in situ hybridiza* Corresponding author. Tel.: 131-30-2121975; fax: 131-30-2516464. E-mail address: [email protected] (O.H.J. Destre´e).
tion in the developing eye of stage 25 embryos (Fig. 2A,B). Expression levels in the eye increase, showing a double ring pattern at stage 32 (Fig. 2C,D). Sectioning reveals that Cx43 expression is confined to the anterior and lateral lens epithelium (Fig. 3A–C), and at low levels to the ciliary margin (Fig. 3A, arrowheads). The peripheral ring of expression in the eye (Fig. 2D, arrowheads) is hardly visible in sections, but is located at the border between head mesenchyme and retina (Fig. 3A). Expression is also found in the notochord (Fig. 2C,F), posterior part of the cement gland (Fig. 2D,E) and branchial arches (Fig. 2F,G). Expression in the notochord posteriorizes (compare Fig. 2C,F). Abundant expression of Cx43 in the notochord is also found in chick (Dealy et al., 1994) but not in the mouse. Staining in the branchial arches seen in whole mounts (Fig. 2G) is hardly visible in sections and presumably represents expression in derivatives of the neural crest as found in mammals (Ruangvoravat and Lo, 1992). Staining in the brain and otic vesicle is very weak and could not be ascertained in sections when compared to negative sense controls. In lenses isolated from stage 52/53 embryos, Cx43 is expressed in anterior and stronger in lateral epithelial cells (Fig. 3D,E). In the mouse, Cx43 is expressed in the lens placode (Yancey et al., 1992) and during fetal stages in the anterior and lateral lens epithelium (Beyer et al., 1989; Yancey et al., 1992; Gao and Spray, 1998). Similar expression patterns in the lens were found in chick (Musil et al., 1990) and sheep (Yang and Louis, 2000). Strong expression of Cx43 is found in the distal part of early limb buds (Fig. 4A,B). Expression becomes restricted to the developing digits and to regions of precartilage
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00490-7
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M.A.G. van der Heyden, L. Roeleveld / Mechanisms of Development 108 (2001) 217–220
Fig. 2. Cx43 is expressed in the eye, cement gland, notochord and branchial arches. Cx43 is expressed as a ring in the eye of stage 25 embryos (A,B arrow). Cx43 expression in the eye (C,D, arrow indicate inner ring, arrowhead indicate outer ring), part of the notochord (nt) (C) and in the posterior part of the cement gland (cg) (C,E) in stage 32 embryos. Cx43 expression in the lens region of the eye (F,G), in the posterior notochord (F) and in the branchial arches (arrowheads) (G) of stage 37 embryos.
Fig. 1. (A) Comparison of the deduced amino acid sequences of Cx43 of X. tropicalis and X. laevis. Amino acid differences are shaded. Putative transmembrane regions are boxed. A X. tropicalis genomic library in l-FixII was screened with a X. laevis Cx43 probe (see Section 2). Two overlapping clones were isolated. Sequencing revealed that both clones contain the entire protein coding region as well as putative promoter, 5 0 -UTR, intron and 3 0 -UTR sequences. The Genbank accesion number of the X. tropicalis nucleotide sequence is AY043270. (B) A single copy of Cx43 is present in X. tropicalis. Restriction sites in exon 2 and intron sequences of XtCx43 are depicted, and the region used as a probe is indicated. Total genomic DNA, digested with either PstI, BglII or XmnI, is visualized on an ethidium bromide stained agarose gel, and results of the derived probed Southern blot are depicted on the bottom right. Size of the signals (in kb) is shown on the right. To establish the copy number of the Cx43 gene in X. tropicalis genomic DNA was cut, blotted onto filter, and subsequently hybridized with a PstI-BglII fragment, encompassing ,1080 bp of the second exon. After PstI and BglII digestion bands of 5.1 and 7.8 kb, respectively, were detected, XmnI digestion resulted in a 1.0 kb and a 600 bp fragment, both also of expected size.
condensation (Fig. 4D,E). During limb development in the mouse, Cx43 is highly expressed in the apical epidermal ridge (AER) and mesenchyme in a proximal–distal gradient (Ruangvoravat and Lo, 1992) while later on it is expressed in and around precartilage condensations and presumptive perichondrium (Ruangvoravat and Lo, 1992; Yancey et al.,
Table 1 Amino acid identity of XtCx43 to Cx43 from other species Species
% Identity
Genbank accesion number
Xenopus laevis Gallus gallus Homo sapiens Mus musculus Rattus norvegicus Bos taurus Danio rerio Devario aequipinnatus
98.9 86.1 85.9 85.9 85.9 85.9 73.6 71.6
X17243 M29003 M65188 X61576 X06656 J05535 AF035481 AF067407
Fig. 3. Cx43 is expressed in the anterior lens epithelium. Transverse sections showing confined Cx43 expression in the anterior lens epithelium at stage 32 (A, arrows indicate inner ring, arrowhead indicates dorsal part of outer ring), 37 (B) and 40 (C). Cx43 expression in the anterior epithelium of isolated stage 52/53 lenses (D), inset: sense control (E). Enlargement of D, displaying Cx43 expression in individual epithelial cells. (A–C) v, ventral; d, dorsal. (E) l, lateral; a, anterior.
M.A.G. van der Heyden, L. Roeleveld / Mechanisms of Development 108 (2001) 217–220
219
dization was performed as described by Molenaar et al. (1998) using the entire X. tropicalis coding region to generate riboprobes for whole mount in situ hybridization on albino X. laevis embryos. Hybridizations were confirmed using X. laevis Cx43 probes (results not shown). References
Fig. 4. Cx43 expression in developing fore- (A) and hindlimbs (B). Cx43 is expressed intensely and widely in the early limb bud, becoming restricted during mesoderm condensation, later outlining the precartilage and perichondrium, forelimbs stages 52–55, hindlimbs stages 53–56. Enlargements of B, showing details of Cx43 expression during cartilage formation. Stage 53 (C), stage 54 (D) and stage 56 (E). Brown dots are pigment cells.
1992). Similar expression patterns were found in chick (Dealy et al., 1994). Initial limb development in mouse and chicken (Becker et al., 1999; Makarenkova and Patel, 1999) and mouse skeleton formation (Lecanda et al., 2000) are affected by down regulation of Cx43. Comparison of the expression patterns of Cx43 in Xenopus with those in mammals and chicken reveals clear similarities in the developing lens, neural crest and limbs. We expect that the promoter of the Cx43 gene that we have isolated from X. tropicalis will be useful to investigate the mechanisms of transcriptional regulation in these tissues in transgenic embryos. 2. Methods 2.1. Cloning of X. tropicalis Cx43 A X. tropicalis genomic library ( generous gift from Dr L. Zimmerman and Dr R. Grainger) was screened at high stringency with a 800-bp probe encompassing the transmembrane domains 3 and 4, the C-terminal coding region and part of the 3 0 -UTR from X. laevis (sense primer: 5 0 CTTCGCACCTACATCATCAGCAT-3 0 ; antisense primer: 5 0 -ATTCTCCCTCGCCATATCAAAGTA-3 0 , accession number X17243) according to standard procedures. DNA was sequenced with the ABI Prism 377 DNA sequencer (Perkin Elmer, Foster City, CA, USA). 2.2. Embryo manipulation and in situ hybridization Methods of egg collection, fertilization and embryo culture were as described by Gao et al. (1994). In situ hybri-
Amaya, E., Offield, M.F., Grainger, R.M., 1998. Frog genetics: Xenopus tropicalis jumps into the future. Trends Genet. 14, 253–255. Becker, D.L., McGonnell, I., Makarenkova, H.P., Patel, K., Tickle, C., Lorimer, J., Green, C.R., 1999. Roles for a1 connexin in morphogenesis of chick embryos revealed using a novel antisense approach. Dev. Genet. 24, 33–42. Beyer, E.C., Kistler, J., Paul, D.L., Goodenough, D.A., 1989. Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J. Cell Biol. 108, 595–605. Bruzzone, R., White, T.W., Paul, D.L., 1996. Connections with connexins: the molecular basis of direct intercellular signaling. Eur. J. Biochem. 238, 1–27. Dealy, C.N., Beyer, E.C., Kosher, R.A., 1994. Expression patterns of mRNA for the gap junction proteins connexin43 and connexin42 suggest their involvement in chick limb morphogenesis and specification of the arterial vasculature. Dev. Dyn. 199, 156–167. Gao, Y., Spray, D.C., 1998. Structural changes in lenses of mice lacking the gap junction protein connexin43. Invest. Ophthalmol. Vis. Sci. 39, 1198–1209. Gao, X.M., Kuiken, G.A., Baarends, W.M., Koster, J.G., Destre´ e, O.H.J., 1994. Characterization of a functional promoter for the Xenopus Wnt-1 gene in vivo. Oncogene 9, 573–581. Gimlich, R.L., Kumar, N.M., Gilula, N.B., 1988. Sequence and developmental expression of mRNA coding for a gap junction protein in Xenopus. J. Cell Biol. 107, 1065–1073. Gimlich, R.L., Kumar, N.M., Gilula, N.B., 1990. Differential regulation of the levels of three gap junction mRNAs in Xenopus embryos. J. Cell Biol. 110, 597–605. Goodenough, D.A., Goliger, J.A., Paul, D.L., 1996. Connexins, connexons, and intercellular communication. Annu. Rev. Biochem. 65, 475–502. van der Heyden, M.A.G., Rook, M.B., Hermans, M.M.P., Rijksen, G., Boonstra, J., Defize, L.H.K., Destre´ e, O.H.J., 1998. Identification of connexin43 as a functional target for Wnt signalling. J. Cell Sci. 111, 1741–1749. Lecanda, F., Warlow, P.M., Sheikh, S., Furlan, F., Steinberg, T.H., Civitelli, R., 2000. Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction. J. Cell Biol. 151, 931–943. Makarenkova, H., Patel, K., 1999. Gap junction signalling mediated through connexin-43 is required for chick limb development. Dev. Biol. 207, 380–392. Molenaar, M., Roose, J., Peterson, J., Venanzi, S., Clevers, H., Destre´ e, O., 1998. Differential expression of the HMG box transcription factors Xtcf-3 and Xlef-1 during early Xenopus development. Mech. Dev. 75, 151–154. Musil, L.S., Beyer, E.C., Goodenough, D.A., 1990. Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation. J. Membr. Biol. 116, 163–175. Nadarajah, B., Makarenkova, H., Becker, D.L., Evans, W.H., Parnavelas, J.G., 1998. Basic FGF increases communication between cells of the developing neocortex. J. Neurosci. 18, 7881–7890. Ruangvoravat, C.P., Lo, C.W., 1992. Connexin 43 expression in the mouse embryo: localization of transcripts within developmentally significant domains. Dev. Dyn. 194, 261–281. Schiller, P.C., Roos, B.A., Howard, G.A., 1997. Parathyroid hormone up-
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M.A.G. van der Heyden, L. Roeleveld / Mechanisms of Development 108 (2001) 217–220
regulation of connexin 43 gene expression in osteoblasts depends on cell phenotype. J. Bone Miner. Res. 12, 2005–2013. Yancey, S.B., Biswal, S., Revel, J.-P., 1992. Spatial and temporal patterns of distribution of the gap junction protein connexin43 during mouse gastrulation and organogenesis. Development 114, 203–212. Yang, D.I., Louis, C.F., 2000. Molecular cloning of ovine connexin44 and
temporal expression of gap junction proteins in a lens cell culture. Invest. Ophthalmol. Vis. Sci. 41, 2658–2664. Yoshizaki, G., Patin˜ o, R., 1995. Molecular cloning, tissue distribution, and hormonal control in the ovary of Cx41 mRNA, a novel Xenopus connexin gene transcript. Mol. Reprod. Dev. 36, 7–15.
Gene expression pattern
Connexin43 expression during Xenopus development Marcel A.G. van der Heyden a,b, Liesbeth Roeleveld b, Josi Peterson b, Olivier H.J. Destre´e b,* a
Department of Medical Physiology, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands b Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands Received 6 June 2001; received in revised form 11 July 2001; accepted 18 July 2001
Abstract The spatio-temporal expression of connexin43 in Xenopus laevis embryos was studied by in situ hybridization. Cx43 expression is first detected at stage 25 in the developing eye. In stage 32, expression was found in the margin of the lens placode, the cement gland, notochord, and in stage 37 in the branchial arches. Early limb buds show strong expression of Cx43 distally while later on expression is confined to sites of precartilage condensation. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Connexin; Xenopus laevis; Xenopus tropicalis; Embryo; Lens; Limb; Notochord; Neural crest; Branchial arch; Cartilage
1. Results In mammals, Cx43 is one of more than 15 cloned connexin genes (Bruzzone et al., 1996; Goodenough et al., 1996). Cx43 transcription is responsive to developmentally important signals like Wnt, PTH and FGF (van der Heyden et al., 1998; Nadarajah et al., 1998; Schiller et al., 1997). Only four connexins were described so far in amphibians, i.e. Cx30, Cx38, Cx41 and Cx43 for Xenopus laevis (Gimlich et al., 1988, 1990; Yoshizaki and Patin˜o, 1995). In contrast to mammals, Cx43 becomes expressed relatively late during development in X. laevis (Gimlich et al., 1990) and is expressed in many adult tissues (Gimlich et al., 1990; Yoshizaki and Patin˜o, 1995). Detailed expression patterns of Cx43 during Xenopus development have not been described. Recently, Xenopus tropicalis was introduced as a system for amphibian genetics (Amaya et al., 1998). We isolated the Cx43 gene from X. tropicalis; the coding region shows 95% identity with that of X. laevis Cx43 at the nucleotide level and 99% at the amino acid level (Fig. 1A). Protein similarity is high with human, mouse, rat, bovine, chicken and fish (Table 1). Southern blot hybridizations demonstrate that in X. tropicalis Cx43 is a single copy gene (Fig. 1B). 1.1. Cx43 is expressed in the developing lens, notochord, cement gland and limbs Cx43 expression was first detected by in situ hybridiza* Corresponding author. Tel.: 131-30-2121975; fax: 131-30-2516464. E-mail address: [email protected] (O.H.J. Destre´e).
tion in the developing eye of stage 25 embryos (Fig. 2A,B). Expression levels in the eye increase, showing a double ring pattern at stage 32 (Fig. 2C,D). Sectioning reveals that Cx43 expression is confined to the anterior and lateral lens epithelium (Fig. 3A–C), and at low levels to the ciliary margin (Fig. 3A, arrowheads). The peripheral ring of expression in the eye (Fig. 2D, arrowheads) is hardly visible in sections, but is located at the border between head mesenchyme and retina (Fig. 3A). Expression is also found in the notochord (Fig. 2C,F), posterior part of the cement gland (Fig. 2D,E) and branchial arches (Fig. 2F,G). Expression in the notochord posteriorizes (compare Fig. 2C,F). Abundant expression of Cx43 in the notochord is also found in chick (Dealy et al., 1994) but not in the mouse. Staining in the branchial arches seen in whole mounts (Fig. 2G) is hardly visible in sections and presumably represents expression in derivatives of the neural crest as found in mammals (Ruangvoravat and Lo, 1992). Staining in the brain and otic vesicle is very weak and could not be ascertained in sections when compared to negative sense controls. In lenses isolated from stage 52/53 embryos, Cx43 is expressed in anterior and stronger in lateral epithelial cells (Fig. 3D,E). In the mouse, Cx43 is expressed in the lens placode (Yancey et al., 1992) and during fetal stages in the anterior and lateral lens epithelium (Beyer et al., 1989; Yancey et al., 1992; Gao and Spray, 1998). Similar expression patterns in the lens were found in chick (Musil et al., 1990) and sheep (Yang and Louis, 2000). Strong expression of Cx43 is found in the distal part of early limb buds (Fig. 4A,B). Expression becomes restricted to the developing digits and to regions of precartilage
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(01)00490-7
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Fig. 2. Cx43 is expressed in the eye, cement gland, notochord and branchial arches. Cx43 is expressed as a ring in the eye of stage 25 embryos (A,B arrow). Cx43 expression in the eye (C,D, arrow indicate inner ring, arrowhead indicate outer ring), part of the notochord (nt) (C) and in the posterior part of the cement gland (cg) (C,E) in stage 32 embryos. Cx43 expression in the lens region of the eye (F,G), in the posterior notochord (F) and in the branchial arches (arrowheads) (G) of stage 37 embryos.
Fig. 1. (A) Comparison of the deduced amino acid sequences of Cx43 of X. tropicalis and X. laevis. Amino acid differences are shaded. Putative transmembrane regions are boxed. A X. tropicalis genomic library in l-FixII was screened with a X. laevis Cx43 probe (see Section 2). Two overlapping clones were isolated. Sequencing revealed that both clones contain the entire protein coding region as well as putative promoter, 5 0 -UTR, intron and 3 0 -UTR sequences. The Genbank accesion number of the X. tropicalis nucleotide sequence is AY043270. (B) A single copy of Cx43 is present in X. tropicalis. Restriction sites in exon 2 and intron sequences of XtCx43 are depicted, and the region used as a probe is indicated. Total genomic DNA, digested with either PstI, BglII or XmnI, is visualized on an ethidium bromide stained agarose gel, and results of the derived probed Southern blot are depicted on the bottom right. Size of the signals (in kb) is shown on the right. To establish the copy number of the Cx43 gene in X. tropicalis genomic DNA was cut, blotted onto filter, and subsequently hybridized with a PstI-BglII fragment, encompassing ,1080 bp of the second exon. After PstI and BglII digestion bands of 5.1 and 7.8 kb, respectively, were detected, XmnI digestion resulted in a 1.0 kb and a 600 bp fragment, both also of expected size.
condensation (Fig. 4D,E). During limb development in the mouse, Cx43 is highly expressed in the apical epidermal ridge (AER) and mesenchyme in a proximal–distal gradient (Ruangvoravat and Lo, 1992) while later on it is expressed in and around precartilage condensations and presumptive perichondrium (Ruangvoravat and Lo, 1992; Yancey et al.,
Table 1 Amino acid identity of XtCx43 to Cx43 from other species Species
% Identity
Genbank accesion number
Xenopus laevis Gallus gallus Homo sapiens Mus musculus Rattus norvegicus Bos taurus Danio rerio Devario aequipinnatus
98.9 86.1 85.9 85.9 85.9 85.9 73.6 71.6
X17243 M29003 M65188 X61576 X06656 J05535 AF035481 AF067407
Fig. 3. Cx43 is expressed in the anterior lens epithelium. Transverse sections showing confined Cx43 expression in the anterior lens epithelium at stage 32 (A, arrows indicate inner ring, arrowhead indicates dorsal part of outer ring), 37 (B) and 40 (C). Cx43 expression in the anterior epithelium of isolated stage 52/53 lenses (D), inset: sense control (E). Enlargement of D, displaying Cx43 expression in individual epithelial cells. (A–C) v, ventral; d, dorsal. (E) l, lateral; a, anterior.
M.A.G. van der Heyden, L. Roeleveld / Mechanisms of Development 108 (2001) 217–220
219
dization was performed as described by Molenaar et al. (1998) using the entire X. tropicalis coding region to generate riboprobes for whole mount in situ hybridization on albino X. laevis embryos. Hybridizations were confirmed using X. laevis Cx43 probes (results not shown). References
Fig. 4. Cx43 expression in developing fore- (A) and hindlimbs (B). Cx43 is expressed intensely and widely in the early limb bud, becoming restricted during mesoderm condensation, later outlining the precartilage and perichondrium, forelimbs stages 52–55, hindlimbs stages 53–56. Enlargements of B, showing details of Cx43 expression during cartilage formation. Stage 53 (C), stage 54 (D) and stage 56 (E). Brown dots are pigment cells.
1992). Similar expression patterns were found in chick (Dealy et al., 1994). Initial limb development in mouse and chicken (Becker et al., 1999; Makarenkova and Patel, 1999) and mouse skeleton formation (Lecanda et al., 2000) are affected by down regulation of Cx43. Comparison of the expression patterns of Cx43 in Xenopus with those in mammals and chicken reveals clear similarities in the developing lens, neural crest and limbs. We expect that the promoter of the Cx43 gene that we have isolated from X. tropicalis will be useful to investigate the mechanisms of transcriptional regulation in these tissues in transgenic embryos. 2. Methods 2.1. Cloning of X. tropicalis Cx43 A X. tropicalis genomic library ( generous gift from Dr L. Zimmerman and Dr R. Grainger) was screened at high stringency with a 800-bp probe encompassing the transmembrane domains 3 and 4, the C-terminal coding region and part of the 3 0 -UTR from X. laevis (sense primer: 5 0 CTTCGCACCTACATCATCAGCAT-3 0 ; antisense primer: 5 0 -ATTCTCCCTCGCCATATCAAAGTA-3 0 , accession number X17243) according to standard procedures. DNA was sequenced with the ABI Prism 377 DNA sequencer (Perkin Elmer, Foster City, CA, USA). 2.2. Embryo manipulation and in situ hybridization Methods of egg collection, fertilization and embryo culture were as described by Gao et al. (1994). In situ hybri-
Amaya, E., Offield, M.F., Grainger, R.M., 1998. Frog genetics: Xenopus tropicalis jumps into the future. Trends Genet. 14, 253–255. Becker, D.L., McGonnell, I., Makarenkova, H.P., Patel, K., Tickle, C., Lorimer, J., Green, C.R., 1999. Roles for a1 connexin in morphogenesis of chick embryos revealed using a novel antisense approach. Dev. Genet. 24, 33–42. Beyer, E.C., Kistler, J., Paul, D.L., Goodenough, D.A., 1989. Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J. Cell Biol. 108, 595–605. Bruzzone, R., White, T.W., Paul, D.L., 1996. Connections with connexins: the molecular basis of direct intercellular signaling. Eur. J. Biochem. 238, 1–27. Dealy, C.N., Beyer, E.C., Kosher, R.A., 1994. Expression patterns of mRNA for the gap junction proteins connexin43 and connexin42 suggest their involvement in chick limb morphogenesis and specification of the arterial vasculature. Dev. Dyn. 199, 156–167. Gao, Y., Spray, D.C., 1998. Structural changes in lenses of mice lacking the gap junction protein connexin43. Invest. Ophthalmol. Vis. Sci. 39, 1198–1209. Gao, X.M., Kuiken, G.A., Baarends, W.M., Koster, J.G., Destre´ e, O.H.J., 1994. Characterization of a functional promoter for the Xenopus Wnt-1 gene in vivo. Oncogene 9, 573–581. Gimlich, R.L., Kumar, N.M., Gilula, N.B., 1988. Sequence and developmental expression of mRNA coding for a gap junction protein in Xenopus. J. Cell Biol. 107, 1065–1073. Gimlich, R.L., Kumar, N.M., Gilula, N.B., 1990. Differential regulation of the levels of three gap junction mRNAs in Xenopus embryos. J. Cell Biol. 110, 597–605. Goodenough, D.A., Goliger, J.A., Paul, D.L., 1996. Connexins, connexons, and intercellular communication. Annu. Rev. Biochem. 65, 475–502. van der Heyden, M.A.G., Rook, M.B., Hermans, M.M.P., Rijksen, G., Boonstra, J., Defize, L.H.K., Destre´ e, O.H.J., 1998. Identification of connexin43 as a functional target for Wnt signalling. J. Cell Sci. 111, 1741–1749. Lecanda, F., Warlow, P.M., Sheikh, S., Furlan, F., Steinberg, T.H., Civitelli, R., 2000. Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction. J. Cell Biol. 151, 931–943. Makarenkova, H., Patel, K., 1999. Gap junction signalling mediated through connexin-43 is required for chick limb development. Dev. Biol. 207, 380–392. Molenaar, M., Roose, J., Peterson, J., Venanzi, S., Clevers, H., Destre´ e, O., 1998. Differential expression of the HMG box transcription factors Xtcf-3 and Xlef-1 during early Xenopus development. Mech. Dev. 75, 151–154. Musil, L.S., Beyer, E.C., Goodenough, D.A., 1990. Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation. J. Membr. Biol. 116, 163–175. Nadarajah, B., Makarenkova, H., Becker, D.L., Evans, W.H., Parnavelas, J.G., 1998. Basic FGF increases communication between cells of the developing neocortex. J. Neurosci. 18, 7881–7890. Ruangvoravat, C.P., Lo, C.W., 1992. Connexin 43 expression in the mouse embryo: localization of transcripts within developmentally significant domains. Dev. Dyn. 194, 261–281. Schiller, P.C., Roos, B.A., Howard, G.A., 1997. Parathyroid hormone up-
220
M.A.G. van der Heyden, L. Roeleveld / Mechanisms of Development 108 (2001) 217–220
regulation of connexin 43 gene expression in osteoblasts depends on cell phenotype. J. Bone Miner. Res. 12, 2005–2013. Yancey, S.B., Biswal, S., Revel, J.-P., 1992. Spatial and temporal patterns of distribution of the gap junction protein connexin43 during mouse gastrulation and organogenesis. Development 114, 203–212. Yang, D.I., Louis, C.F., 2000. Molecular cloning of ovine connexin44 and
temporal expression of gap junction proteins in a lens cell culture. Invest. Ophthalmol. Vis. Sci. 41, 2658–2664. Yoshizaki, G., Patin˜ o, R., 1995. Molecular cloning, tissue distribution, and hormonal control in the ovary of Cx41 mRNA, a novel Xenopus connexin gene transcript. Mol. Reprod. Dev. 36, 7–15.