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Analysis of cCx39 expression pattern during chick development
Developmental Brain Research 148 (2004) 179 – 183 www.elsevier.com/locate/devbrainres
Research report
Analysis of cCx39 expression pattern during chick development Annalisa Nicotra a, Federico Cicirata a,*, Salvador Martinez b a
Dipartimento di Scienze Fisiologiche, Universita` di Catania, V.le A. Doria 6, 95125 Catania, Italy b Instituto de Neurociencias UMH-CSIC San Juan de Alicante 03550-Spain Accepted 11 November 2003
Abstract The present study reports the expression pattern of connexin39 (cCx39) in chick embryos at different stages of central nervous system development. We examined the expression between HH17 and HH40 developmental stages of chicken embryos by in situ hybridization (ISH) technique. Connexin39 was first expressed at HH17. It stained neuroepithelial cells in the optic (OV) and telencephalic (TEL) vesicles, plus in the superficial mesenchyme of the two rostral branchial arches (maxilar and mandibular). These cells probably originated from the neural crest. This expression pattern changed drastically between stages HH17 and HH23, while it showed relatively little modifications from HH23 to HH29. At these times, connexin39 was expressed in three regions: the telencephalic vesicle, the diencephalon and the isthmus. At later stages, HH35 and HH40, connexin39 was mainly expressed in the ventricular epithelium and three cell layers of the stratum griseum and fibrosum superficialis (SGFS) in the optic tectum, as well as in granular and nuclear cells in the cerebellum. In conclusion, the expression pattern of connexin39 in embryonic nervous system is dynamic. This pattern is different from, and in some aspects complementary to, those showed by other connexins during brain development. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Pattern formation, compartments, and boundaries Keywords: Connexin; Development; Cellular communication; Nervous system; Chicken embryo
1. Introduction Gap junctions (GJs) are cell membrane structures that mediate direct cell– cell communication. A Gap junction channel is formed by two hemichannels called connexons, each composed of six connexin subunits [7]. Gap junction functional states can be regulated by several factors such as membrane voltage, pH, phosphorylation and biochemical signals. Because of this rich potential for the regulation of junctional conductance, and directional and molecular gating, gap junction communication plays a crucial role in normal tissue physiology. However, the most exciting role for gap junction communication is in the regulation of the information flow taking place during embryonic development. The involvement of Gap Junctions in the patterning and development of vertebrate and invertebrate embryos [9] was demonstrated by disturbing communication or connexin expression using blocking antibodies [2], dominant negative connexins [13], knockout mice [20] and antisense deoxy* Corresponding author. Tel.: +39-095333841; fax: +39-095330645. E-mail address: [email protected] (F. Cicirata). 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2003.11.009
oligonucleotide [2]. These experimental models always produced important developmental defects. Moreover, different gap junctional permeability has been demonstrated at the level of intersegmental boundaries in the developing chick rhomboencephalon [11]. The identification of new connexins and the analysis of their expression in different areas and developmental stages, is a basic tool for a better understanding of the role played by gap junctional communication in embryonic development. Here we describe the expression pattern during development of connexin39 (cCx39), a novel connexin gene recently cloned in chicken [12] (GenBank Accession Number: 509410).
2. Materials and methods 2.1. Digoxigenin labeled RNA and DNA probe Genomic DNA was isolated from chicken tissue by established procedures [16] and a region of 775 bp including the intracytoplasmatic loop of cCx39 gene was amplified by
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PCR using specific primers. The PCR product was cloned into a pCRII vector (Invitrogen-Life Technologies) and sequenced from both ends with vector-specific primers in order to confirm the sequence. One nanogram of this plasmidic DNA was then used as a template in a PCR reaction performed with 70 AM Digoxigenin-11-dUTP to amplify an internal region of 317 bp. This labeled fragment was purified with QIAquick PCR purification kit (Qiagen) and used as a probe in in situ hybridization (ISH) procedures at a concentration of 1 Ag/ml, previously denatured at 70 jC for 10 min. 2.2. Chicken embryos Fertilized eggs were incubated at 37 jC until the desired developmental stage was reached. Embryos col-
lected at stages HH17, HH23, HH25, HH27 and HH29 were fixed in 4% paraformaldeyde in PBS by immersion O/N, while embryos at HH35 and HH40 were first perfused with the same fixative solution and then postfixed O/N. 2.3. In situ hybridization Whole mount in situ hybridizations were performed following the protocol described by Shimamura et al. [17]. The embryos were then embedded in wax and cut in 8-Am sections. In situ hybridizations on 30-Am cryostat sections were performed as previously described [18], while those on 8-Am paraffin sections were performed as reported in the literature [10]. The hybridization temperature was 45 jC.
Fig. 1. Expression of cCx39 in early (HH17 – HH29) and late stages (HH35 – HH40) of chick embryos. (A) Lateral view of whole embryo at HH17, indicating the section planes shown as microphotographs in A1, A2 and A3. The anterior part of the embryo and the heart show a strong signal. The labeled cells in neural crest (NC), optic vesicle (OV), foregut (FG), telencephalic neuroepithelium (Tel) and heart (H) are indicated by arrowheads in A1, A2 and A3. (B) Lateral view of embryonic brain at HH23. A strong labeling is present in the caudal part of telencephalic vesicle (TEL), in the pretectum (Pt) and the isthmus (Is). (C) Lateral view of embryonic brain at HH25. The telencephalic vesicle is almost completely labeled. The pretectum and isthmus are also positive. (D) Lateral view of embryonic brain at HH27, indicating the sections shown as microphotographs in D1, D2 and D3. Telencephalon, part of diencephalon and pretectum are the positive regions. Details of cCx39 expressing cells are shown in D1, D2 and D3. (E) Lateral view of embryonic brain at HH29. cCx39 expression covers the whole telencephalic vesicle. The pineal anlage, indicated by an arrowhead, is labeled strongly positive.
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Expression was shown by colorimetric reaction with antiDIG-AP and NBT/BCIP substrates.
3. Results cCx39 transcription was analyzed in chick embryos of stages HH17-40 by in situ hybridization (ISH) with DNA probes. The nervous system was analyzed in whole mount at 5 developmental stages from HH17 to HH29, and in histological sections at later stages of development, HH35 and HH40. 3.1. Expression of cCx39 at early stages of development (HH17 to HH29) At HH17, when the neurulation process is completed [6], cCx39 DNA hybridization stained neuroepithelial cells in the optic (OV) and telencephalic (TEL) vesicles (Fig. 1A and A1). Some cells expressing cCx39 were found in the superficial mesenchyme of the two rostral branchial arches
181
(maxilar and mandibular), (Fig. 1A1 and A2). The localization of these cells suggested a neural crest origin [8]. Strongly positive cells were also shown in the roof of the foregut, ventrally to the prosencephalon. This region of transition between the anterior endoderm epithelium (foregut) and the buco-pharyngea membrane (Fig. 1A2), also known as the mesendoderm and has been characterized as an important organizer region for the normal development of the prosencephalon [21]. In the heart tube, cCx39 is expressed in the luminal epithelium (Fig. 1A3) which will develop into the endocardium [3]. This expression pattern changed drastically between stages HH17 and HH23, while it showed relatively little modifications in the next analyzed stages: HH25, HH27 and HH29. At these times, the expression of cCx39 was detected in three regions: the telencephalic vesicle, the diencephalon and the isthmus. (1) At HH23, the telencephalic vesicle was principally positive in its caudal pole. From this caudal domain, the expression progressively advanced towards the rostral telencephalic pole between stages HH25 and HH29, when almost the whole telence-
Fig. 2. (A) Sagittal section of HH35 brain embryo showing the optic tectum. The ventricular [ventricular epithelium (VE)] and the external layer [superficial cell layer (Scl)] express cCx39. (B) Sagittal section of HH40 brain embryo showing the optic tectum. Four layers indicated by arrowheads are strongly labeled [stratum griseum and fibrosum superficialis (SGFS); stratum griseum centrale (SGC)]. (C) Sagittal section of HH35 brain embryo showing the cerebellum. Arrowheads show cells expressing cCx39 in external layer [external granular layer (egl)] and cerebellar anlage. (D) Sagittal section of HH40 brain embryo showing the cerebellum. The external and internal [internal granular layer (igl)] granular layers are strongly labeled. The lower arrowheads indicate cerebellar nuclei (cn) cells expressing cCx39. (D1) Inset of Panel D shown at higher magnification highlights the labeled cells of egl and igl in the cerebellum.
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phalic vesicle was positive (Fig. 1B, C, D, E). (2) In the diencephalon, the transversal domain of the pretectum appeared positive at stage HH23. Thereafter, the expression decreased during subsequent stages to almost disappear at HH29 (Fig. 1B, C, D, E; [15]). At this stage (HH29), only the pineal anlage, in the roof of the dorsal thalamus, appeared positive in the diencephalon (Fig. 1E). (3) In the isthmus, expression was clearly observable at stage HH23 and progressively disappeared between stages HH25 and HH27 (Fig. 1B, C, D). Histological section of neural tube at HH27 showed that expression domains were formed by cells frequently distributed in a radial pattern, from the ventricular to superficial areas of the brain wall in the diencephalon and telencephalon (Fig. 1D1, D2, D3). We did not observe this pattern of positive cells either at older developmental stages or in other brain areas. Such distribution suggested the expression of cCx39 in specific cells during the radial migration from the germinative to the mantle layers. 3.2. Expression of cCx39 in late stages of development (HH35 and HH40) At later stages, HH35 and HH40, the cCx39 was mainly expressed in two brain regions: the optic tectum and the cerebellum. The optic tectum showed abundant positive cells in different layers. At HH35, the expression was localized in the ventricular epithelium and a superficial cell layer (Fig. 2A). At HH40, the staining concerned the ventricular epithelium plus the stratum griseum centrale (SGC) and two cell layers of the stratum griseum and fibrosum superficialis (SGFS) (Fig. 2B). The laminar expression of cCx39 in the optic tectum during the interval HH35 –HH40, in which the layers differentiated, suggests a possible involvement of cCx39 in this process. The cerebellum expressed cCx39 in both granular cells in the cortex and nuclear cells. At HH35, most granular cells migrating along the external granular layer (egl) were stained (Fig. 2C). The germinal area of these cells, the caudal rhombic lip [22], did not show cCx39 expression. This evidence suggests that the gene cCx39 is activated in migrating granular cells some time after their generation. Moreover, some cells were also positive in the central domain of the cerebellar anlage (Fig. 2C) corresponding to cerebellar nuclei (cn) progenitors and cortical neurons [4]. At HH40, granule cells of both external and internal layers expressed cCx39 (Fig. 2D and D1). The expression of Cx39 was strong in the external granular layer (egl) and relatively weak in the internal granular layer (igl). Because granular progenitors are located in the external layer and mature granular cells in the inner granular layer, we hypothesize that the intensity of cCx39 expression decreases in granular cells with their cellular maturation. Most neurons of cerebellar nuclei (cn) also expressed cCx39 (Fig. 2D).
4. Discussion It is well known that gap junctions play a key role in several aspects of embryogenesis and that during this period most, if not all cells, are coupled by GJ channels [9]. Because the composition of connexins that oligomerize to form the GJ channels is a critical factor for controlling the permeability to signaling molecules, it is important to identify which connexins are expressed in different regions of the developing brain. In fact, it has been commonly hypothesized that the development of the embryonic brain in regions composed of cells morphologically and functionally homologues or mysteriously integrated, is due to morphogenic factors spreading from cell to cell, from primary organizers to surrounding areas through GJs. Thus, the identification of which connexins are expressed where in the developing brain is an important contribution to understanding one basic mechanism regulating embryogenesis. This line of investigation has not yet been extensively approached. In fact, despite the various connexins expressed in adult brain, only two studies have previously addressed the problem, investigating the spatio-temporal expression pattern of connexin43 [14] and connexin36 [5] in developing mouse brain by in situ hybridization analysis. Their expression pattern was compared to that of connexin39, which was studied in chicken, using the table elaborated by Wessels and Markwald [19] for the comparison of the different developmental times of the mouse and chicken. These connexins were studied along different time intervals, so that it was possible to compare their expression at only two times investigated in all studies, E10.5 in mouse, which corresponds to HH17 in chicken, and E12.5 in mouse, which corresponds to HH26 –HH29 in chicken. At the former time (E10.5 in mouse—HH17 in chicken), cCx39 is expressed in all parts of the telencephalic vesicles. The Cx36 domain extends from the most anterior territory of the brain up to the pretectum area. Cx43 transcripts were found at high level throughout caudal regions of the neural tube, while at the rostral end the hybridization signal was restricted predominantly to the dorsal aspect and to the midbrain/hindbrain junction, a region where Cx39 is expressed later. At the latter time (E12.5 in mouse—HH26 –HH29 in chicken), Cx36 is expressed in the boundary between dorsal and ventral telencephalon, in the Zona Limitans Intrathalamica, in the dorsal telencephalon, in the ventral part of the isthmus and in the pretectum. This expression pattern differs from that of cCx39 which is expressed in all parts of the telencephalic vesicles, through a caudal –rostral progression, the entire isthmus and pretectum. Cx43 is expressed above all in the dorsal – lateral telencephalon and in the floor of diencephalons, but it overlaps the Cx39 expression in the isthmus and in the epiphysis. In conclusion, these data show that the development of different brain regions is associated with the expression of different connexins. Thus, the existence of a connexin-
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specific pattern for developing brain areas strongly supports the hypothesis that they play specific roles in the control of embryonic regionalization. In particular, because cCx39 is expressed early in organizing regions as the isthmus and the mesoendoderm, we hypothesize that gap junction channels formed by cCx39 could be concerned in the appearance of morphogenetic molecules generated by these organizing regions.
References [2] D.L. Becker, I. McGonnell, H.P. Makarenkova, K. Patel, C. Tickle, J. Lorimer, C.R. Green, Roles for alpha 1 connexin in morphogenesis of chick embryos revealed using a novel antisense approach, Dev. Genet. 24 (1999) 33 – 42. [3] D.L. Brutsaert, G.W. De Keulenaer, P. Fransen, P. Mohan, G.L. Kaluza, L.J. Andries, J.L. Rouleau, S.U. Sys, The cardiac endothelium: functional morphology, development, and physiology, Prog. Cardiovasc. Dis. 39 (1996) 239 – 262. [4] D. Goldowitz, K. Hamre, The cells and molecules that make a cerebellum, Trends Neurosci. 21 (1998) 375 – 382. [5] M. Gulisano, R. Parenti, F. Spinella, F. Cicirata, Cx36 is dynamically expressed during early development of mouse brain and nervous system, NeuroReport 11 (2000) 3823 – 3828. [6] V. Hamburger, H. Hamilton, A series of normal stages in the development of the chick embryo, J. Morphol. 88 (1951) 49 – 92. [7] N.M. Kumar, N.B. Gilula, The gap junction communication channel, Cell 84 (1996) 381 – 388. [8] N.M. Le Douarin, E. Dupin, C. Ziller, Genetic and epigenetic control in neural crest development, Curr. Opin. Genet. Dev. 4 (1994) 685 – 695. [9] M. Levin, Isolation and community: a review of the role of GapJunctional communication in embryonic patterning, J. Membr. Biol. 185 (2001) 177 – 192. [10] R. Mahmood, I. Mason, in: P. Sharpe, I. Mason (Eds.), Methods in Molecular Biology/Molecular Embryology, Humana Press, Totowa, NJ, 1999.
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[11] S. Martı´nez, E. Geijo, M.V. Sanchez-Vives, L. Puelles, R. Gallego, Reduced junctional permeability at interrhombomeric boundaries, Development 116 (1992) 1069 – 1076. [12] A. Nicotra, D. Cicero, M. Gulisano, S. Martinez, F. Cicirata, Cloning and expression pattern of connexin39, a new member of the gap junction gene family, FENS Abstr. 1, FENS Forum 2003, Paris, France (A003.16). [13] D.L. Paul, K. Yu, R. Bruzzone, R.L. Gimlich, D.A. Goodenough, Expression of a dominant negative inhibitor of intercellular communication in the early Xenopus embryo causes delamination and extrusion of cells, Development 121 (1995) 371 – 381. [14] C.P. Ruangvoravat, C.W. Lo, Connexin 43 expression in the mouse embryo: localization of transcripts within developmentally significant domains, Dev. Dyn. 194 (1992) 261 – 281. [15] J.L. Rubenstein, S. Martinez, K. Shimamura, L. Puelles, The embryonic vertebrate forebrain: the prosomeric model, Science 266 (1994) 578 – 580. [16] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, North Harbor, 1989. [17] K. Shimamura, S. Hirano, A.P. McMahon, M. Takeichi, Wnt-1 dependent regulation of local E-cadherin and aN-catenin expression in the embryonic mouse brain, Development 120 (1994) 2225 – 2234. [18] N. Spassky, K. Heydon, A. Mangatal, A. Jankovski, C. Olivier, F. Queraud-Lesaux, C. Goujet-Zalc, J.L. Thomas, B. Zalc, Sonic hedgehog-dependent emergence of oligodendrocytes in the telencephalon: evidence for a source of oligodendrocytes in the olfactory bulb that is independent of PDGFRa signalling, Development 128 (2001) 4993 – 5004. [19] A. Wessels, R. Markwald, in: Developmental Biology Protocols, vol. II, Humana Press, Totowa, NJ, 2000, pp. 239 – 259. [20] T.W. White, D.L. Paul, Genetic diseases and gene knockouts reveal diverse connexin functions, Annu. Rev. Physiol. 61 (1999) 283 – 310. [21] R.J. Wingate, The rhombic lip and early cerebellar development, Curr. Opin. Neurobiol. 11 (2001) 82 – 88. [22] S. Withington, R. Beddington, J. Cooke, Foregut endoderm is required at head process stages for anteriormost neural patterning in chick, Development 128 (2001) 309 – 320.
Research report
Analysis of cCx39 expression pattern during chick development Annalisa Nicotra a, Federico Cicirata a,*, Salvador Martinez b a
Dipartimento di Scienze Fisiologiche, Universita` di Catania, V.le A. Doria 6, 95125 Catania, Italy b Instituto de Neurociencias UMH-CSIC San Juan de Alicante 03550-Spain Accepted 11 November 2003
Abstract The present study reports the expression pattern of connexin39 (cCx39) in chick embryos at different stages of central nervous system development. We examined the expression between HH17 and HH40 developmental stages of chicken embryos by in situ hybridization (ISH) technique. Connexin39 was first expressed at HH17. It stained neuroepithelial cells in the optic (OV) and telencephalic (TEL) vesicles, plus in the superficial mesenchyme of the two rostral branchial arches (maxilar and mandibular). These cells probably originated from the neural crest. This expression pattern changed drastically between stages HH17 and HH23, while it showed relatively little modifications from HH23 to HH29. At these times, connexin39 was expressed in three regions: the telencephalic vesicle, the diencephalon and the isthmus. At later stages, HH35 and HH40, connexin39 was mainly expressed in the ventricular epithelium and three cell layers of the stratum griseum and fibrosum superficialis (SGFS) in the optic tectum, as well as in granular and nuclear cells in the cerebellum. In conclusion, the expression pattern of connexin39 in embryonic nervous system is dynamic. This pattern is different from, and in some aspects complementary to, those showed by other connexins during brain development. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Pattern formation, compartments, and boundaries Keywords: Connexin; Development; Cellular communication; Nervous system; Chicken embryo
1. Introduction Gap junctions (GJs) are cell membrane structures that mediate direct cell– cell communication. A Gap junction channel is formed by two hemichannels called connexons, each composed of six connexin subunits [7]. Gap junction functional states can be regulated by several factors such as membrane voltage, pH, phosphorylation and biochemical signals. Because of this rich potential for the regulation of junctional conductance, and directional and molecular gating, gap junction communication plays a crucial role in normal tissue physiology. However, the most exciting role for gap junction communication is in the regulation of the information flow taking place during embryonic development. The involvement of Gap Junctions in the patterning and development of vertebrate and invertebrate embryos [9] was demonstrated by disturbing communication or connexin expression using blocking antibodies [2], dominant negative connexins [13], knockout mice [20] and antisense deoxy* Corresponding author. Tel.: +39-095333841; fax: +39-095330645. E-mail address: [email protected] (F. Cicirata). 0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2003.11.009
oligonucleotide [2]. These experimental models always produced important developmental defects. Moreover, different gap junctional permeability has been demonstrated at the level of intersegmental boundaries in the developing chick rhomboencephalon [11]. The identification of new connexins and the analysis of their expression in different areas and developmental stages, is a basic tool for a better understanding of the role played by gap junctional communication in embryonic development. Here we describe the expression pattern during development of connexin39 (cCx39), a novel connexin gene recently cloned in chicken [12] (GenBank Accession Number: 509410).
2. Materials and methods 2.1. Digoxigenin labeled RNA and DNA probe Genomic DNA was isolated from chicken tissue by established procedures [16] and a region of 775 bp including the intracytoplasmatic loop of cCx39 gene was amplified by
180
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PCR using specific primers. The PCR product was cloned into a pCRII vector (Invitrogen-Life Technologies) and sequenced from both ends with vector-specific primers in order to confirm the sequence. One nanogram of this plasmidic DNA was then used as a template in a PCR reaction performed with 70 AM Digoxigenin-11-dUTP to amplify an internal region of 317 bp. This labeled fragment was purified with QIAquick PCR purification kit (Qiagen) and used as a probe in in situ hybridization (ISH) procedures at a concentration of 1 Ag/ml, previously denatured at 70 jC for 10 min. 2.2. Chicken embryos Fertilized eggs were incubated at 37 jC until the desired developmental stage was reached. Embryos col-
lected at stages HH17, HH23, HH25, HH27 and HH29 were fixed in 4% paraformaldeyde in PBS by immersion O/N, while embryos at HH35 and HH40 were first perfused with the same fixative solution and then postfixed O/N. 2.3. In situ hybridization Whole mount in situ hybridizations were performed following the protocol described by Shimamura et al. [17]. The embryos were then embedded in wax and cut in 8-Am sections. In situ hybridizations on 30-Am cryostat sections were performed as previously described [18], while those on 8-Am paraffin sections were performed as reported in the literature [10]. The hybridization temperature was 45 jC.
Fig. 1. Expression of cCx39 in early (HH17 – HH29) and late stages (HH35 – HH40) of chick embryos. (A) Lateral view of whole embryo at HH17, indicating the section planes shown as microphotographs in A1, A2 and A3. The anterior part of the embryo and the heart show a strong signal. The labeled cells in neural crest (NC), optic vesicle (OV), foregut (FG), telencephalic neuroepithelium (Tel) and heart (H) are indicated by arrowheads in A1, A2 and A3. (B) Lateral view of embryonic brain at HH23. A strong labeling is present in the caudal part of telencephalic vesicle (TEL), in the pretectum (Pt) and the isthmus (Is). (C) Lateral view of embryonic brain at HH25. The telencephalic vesicle is almost completely labeled. The pretectum and isthmus are also positive. (D) Lateral view of embryonic brain at HH27, indicating the sections shown as microphotographs in D1, D2 and D3. Telencephalon, part of diencephalon and pretectum are the positive regions. Details of cCx39 expressing cells are shown in D1, D2 and D3. (E) Lateral view of embryonic brain at HH29. cCx39 expression covers the whole telencephalic vesicle. The pineal anlage, indicated by an arrowhead, is labeled strongly positive.
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Expression was shown by colorimetric reaction with antiDIG-AP and NBT/BCIP substrates.
3. Results cCx39 transcription was analyzed in chick embryos of stages HH17-40 by in situ hybridization (ISH) with DNA probes. The nervous system was analyzed in whole mount at 5 developmental stages from HH17 to HH29, and in histological sections at later stages of development, HH35 and HH40. 3.1. Expression of cCx39 at early stages of development (HH17 to HH29) At HH17, when the neurulation process is completed [6], cCx39 DNA hybridization stained neuroepithelial cells in the optic (OV) and telencephalic (TEL) vesicles (Fig. 1A and A1). Some cells expressing cCx39 were found in the superficial mesenchyme of the two rostral branchial arches
181
(maxilar and mandibular), (Fig. 1A1 and A2). The localization of these cells suggested a neural crest origin [8]. Strongly positive cells were also shown in the roof of the foregut, ventrally to the prosencephalon. This region of transition between the anterior endoderm epithelium (foregut) and the buco-pharyngea membrane (Fig. 1A2), also known as the mesendoderm and has been characterized as an important organizer region for the normal development of the prosencephalon [21]. In the heart tube, cCx39 is expressed in the luminal epithelium (Fig. 1A3) which will develop into the endocardium [3]. This expression pattern changed drastically between stages HH17 and HH23, while it showed relatively little modifications in the next analyzed stages: HH25, HH27 and HH29. At these times, the expression of cCx39 was detected in three regions: the telencephalic vesicle, the diencephalon and the isthmus. (1) At HH23, the telencephalic vesicle was principally positive in its caudal pole. From this caudal domain, the expression progressively advanced towards the rostral telencephalic pole between stages HH25 and HH29, when almost the whole telence-
Fig. 2. (A) Sagittal section of HH35 brain embryo showing the optic tectum. The ventricular [ventricular epithelium (VE)] and the external layer [superficial cell layer (Scl)] express cCx39. (B) Sagittal section of HH40 brain embryo showing the optic tectum. Four layers indicated by arrowheads are strongly labeled [stratum griseum and fibrosum superficialis (SGFS); stratum griseum centrale (SGC)]. (C) Sagittal section of HH35 brain embryo showing the cerebellum. Arrowheads show cells expressing cCx39 in external layer [external granular layer (egl)] and cerebellar anlage. (D) Sagittal section of HH40 brain embryo showing the cerebellum. The external and internal [internal granular layer (igl)] granular layers are strongly labeled. The lower arrowheads indicate cerebellar nuclei (cn) cells expressing cCx39. (D1) Inset of Panel D shown at higher magnification highlights the labeled cells of egl and igl in the cerebellum.
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phalic vesicle was positive (Fig. 1B, C, D, E). (2) In the diencephalon, the transversal domain of the pretectum appeared positive at stage HH23. Thereafter, the expression decreased during subsequent stages to almost disappear at HH29 (Fig. 1B, C, D, E; [15]). At this stage (HH29), only the pineal anlage, in the roof of the dorsal thalamus, appeared positive in the diencephalon (Fig. 1E). (3) In the isthmus, expression was clearly observable at stage HH23 and progressively disappeared between stages HH25 and HH27 (Fig. 1B, C, D). Histological section of neural tube at HH27 showed that expression domains were formed by cells frequently distributed in a radial pattern, from the ventricular to superficial areas of the brain wall in the diencephalon and telencephalon (Fig. 1D1, D2, D3). We did not observe this pattern of positive cells either at older developmental stages or in other brain areas. Such distribution suggested the expression of cCx39 in specific cells during the radial migration from the germinative to the mantle layers. 3.2. Expression of cCx39 in late stages of development (HH35 and HH40) At later stages, HH35 and HH40, the cCx39 was mainly expressed in two brain regions: the optic tectum and the cerebellum. The optic tectum showed abundant positive cells in different layers. At HH35, the expression was localized in the ventricular epithelium and a superficial cell layer (Fig. 2A). At HH40, the staining concerned the ventricular epithelium plus the stratum griseum centrale (SGC) and two cell layers of the stratum griseum and fibrosum superficialis (SGFS) (Fig. 2B). The laminar expression of cCx39 in the optic tectum during the interval HH35 –HH40, in which the layers differentiated, suggests a possible involvement of cCx39 in this process. The cerebellum expressed cCx39 in both granular cells in the cortex and nuclear cells. At HH35, most granular cells migrating along the external granular layer (egl) were stained (Fig. 2C). The germinal area of these cells, the caudal rhombic lip [22], did not show cCx39 expression. This evidence suggests that the gene cCx39 is activated in migrating granular cells some time after their generation. Moreover, some cells were also positive in the central domain of the cerebellar anlage (Fig. 2C) corresponding to cerebellar nuclei (cn) progenitors and cortical neurons [4]. At HH40, granule cells of both external and internal layers expressed cCx39 (Fig. 2D and D1). The expression of Cx39 was strong in the external granular layer (egl) and relatively weak in the internal granular layer (igl). Because granular progenitors are located in the external layer and mature granular cells in the inner granular layer, we hypothesize that the intensity of cCx39 expression decreases in granular cells with their cellular maturation. Most neurons of cerebellar nuclei (cn) also expressed cCx39 (Fig. 2D).
4. Discussion It is well known that gap junctions play a key role in several aspects of embryogenesis and that during this period most, if not all cells, are coupled by GJ channels [9]. Because the composition of connexins that oligomerize to form the GJ channels is a critical factor for controlling the permeability to signaling molecules, it is important to identify which connexins are expressed in different regions of the developing brain. In fact, it has been commonly hypothesized that the development of the embryonic brain in regions composed of cells morphologically and functionally homologues or mysteriously integrated, is due to morphogenic factors spreading from cell to cell, from primary organizers to surrounding areas through GJs. Thus, the identification of which connexins are expressed where in the developing brain is an important contribution to understanding one basic mechanism regulating embryogenesis. This line of investigation has not yet been extensively approached. In fact, despite the various connexins expressed in adult brain, only two studies have previously addressed the problem, investigating the spatio-temporal expression pattern of connexin43 [14] and connexin36 [5] in developing mouse brain by in situ hybridization analysis. Their expression pattern was compared to that of connexin39, which was studied in chicken, using the table elaborated by Wessels and Markwald [19] for the comparison of the different developmental times of the mouse and chicken. These connexins were studied along different time intervals, so that it was possible to compare their expression at only two times investigated in all studies, E10.5 in mouse, which corresponds to HH17 in chicken, and E12.5 in mouse, which corresponds to HH26 –HH29 in chicken. At the former time (E10.5 in mouse—HH17 in chicken), cCx39 is expressed in all parts of the telencephalic vesicles. The Cx36 domain extends from the most anterior territory of the brain up to the pretectum area. Cx43 transcripts were found at high level throughout caudal regions of the neural tube, while at the rostral end the hybridization signal was restricted predominantly to the dorsal aspect and to the midbrain/hindbrain junction, a region where Cx39 is expressed later. At the latter time (E12.5 in mouse—HH26 –HH29 in chicken), Cx36 is expressed in the boundary between dorsal and ventral telencephalon, in the Zona Limitans Intrathalamica, in the dorsal telencephalon, in the ventral part of the isthmus and in the pretectum. This expression pattern differs from that of cCx39 which is expressed in all parts of the telencephalic vesicles, through a caudal –rostral progression, the entire isthmus and pretectum. Cx43 is expressed above all in the dorsal – lateral telencephalon and in the floor of diencephalons, but it overlaps the Cx39 expression in the isthmus and in the epiphysis. In conclusion, these data show that the development of different brain regions is associated with the expression of different connexins. Thus, the existence of a connexin-
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specific pattern for developing brain areas strongly supports the hypothesis that they play specific roles in the control of embryonic regionalization. In particular, because cCx39 is expressed early in organizing regions as the isthmus and the mesoendoderm, we hypothesize that gap junction channels formed by cCx39 could be concerned in the appearance of morphogenetic molecules generated by these organizing regions.
References [2] D.L. Becker, I. McGonnell, H.P. Makarenkova, K. Patel, C. Tickle, J. Lorimer, C.R. Green, Roles for alpha 1 connexin in morphogenesis of chick embryos revealed using a novel antisense approach, Dev. Genet. 24 (1999) 33 – 42. [3] D.L. Brutsaert, G.W. De Keulenaer, P. Fransen, P. Mohan, G.L. Kaluza, L.J. Andries, J.L. Rouleau, S.U. Sys, The cardiac endothelium: functional morphology, development, and physiology, Prog. Cardiovasc. Dis. 39 (1996) 239 – 262. [4] D. Goldowitz, K. Hamre, The cells and molecules that make a cerebellum, Trends Neurosci. 21 (1998) 375 – 382. [5] M. Gulisano, R. Parenti, F. Spinella, F. Cicirata, Cx36 is dynamically expressed during early development of mouse brain and nervous system, NeuroReport 11 (2000) 3823 – 3828. [6] V. Hamburger, H. Hamilton, A series of normal stages in the development of the chick embryo, J. Morphol. 88 (1951) 49 – 92. [7] N.M. Kumar, N.B. Gilula, The gap junction communication channel, Cell 84 (1996) 381 – 388. [8] N.M. Le Douarin, E. Dupin, C. Ziller, Genetic and epigenetic control in neural crest development, Curr. Opin. Genet. Dev. 4 (1994) 685 – 695. [9] M. Levin, Isolation and community: a review of the role of GapJunctional communication in embryonic patterning, J. Membr. Biol. 185 (2001) 177 – 192. [10] R. Mahmood, I. Mason, in: P. Sharpe, I. Mason (Eds.), Methods in Molecular Biology/Molecular Embryology, Humana Press, Totowa, NJ, 1999.
183
[11] S. Martı´nez, E. Geijo, M.V. Sanchez-Vives, L. Puelles, R. Gallego, Reduced junctional permeability at interrhombomeric boundaries, Development 116 (1992) 1069 – 1076. [12] A. Nicotra, D. Cicero, M. Gulisano, S. Martinez, F. Cicirata, Cloning and expression pattern of connexin39, a new member of the gap junction gene family, FENS Abstr. 1, FENS Forum 2003, Paris, France (A003.16). [13] D.L. Paul, K. Yu, R. Bruzzone, R.L. Gimlich, D.A. Goodenough, Expression of a dominant negative inhibitor of intercellular communication in the early Xenopus embryo causes delamination and extrusion of cells, Development 121 (1995) 371 – 381. [14] C.P. Ruangvoravat, C.W. Lo, Connexin 43 expression in the mouse embryo: localization of transcripts within developmentally significant domains, Dev. Dyn. 194 (1992) 261 – 281. [15] J.L. Rubenstein, S. Martinez, K. Shimamura, L. Puelles, The embryonic vertebrate forebrain: the prosomeric model, Science 266 (1994) 578 – 580. [16] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, North Harbor, 1989. [17] K. Shimamura, S. Hirano, A.P. McMahon, M. Takeichi, Wnt-1 dependent regulation of local E-cadherin and aN-catenin expression in the embryonic mouse brain, Development 120 (1994) 2225 – 2234. [18] N. Spassky, K. Heydon, A. Mangatal, A. Jankovski, C. Olivier, F. Queraud-Lesaux, C. Goujet-Zalc, J.L. Thomas, B. Zalc, Sonic hedgehog-dependent emergence of oligodendrocytes in the telencephalon: evidence for a source of oligodendrocytes in the olfactory bulb that is independent of PDGFRa signalling, Development 128 (2001) 4993 – 5004. [19] A. Wessels, R. Markwald, in: Developmental Biology Protocols, vol. II, Humana Press, Totowa, NJ, 2000, pp. 239 – 259. [20] T.W. White, D.L. Paul, Genetic diseases and gene knockouts reveal diverse connexin functions, Annu. Rev. Physiol. 61 (1999) 283 – 310. [21] R.J. Wingate, The rhombic lip and early cerebellar development, Curr. Opin. Neurobiol. 11 (2001) 82 – 88. [22] S. Withington, R. Beddington, J. Cooke, Foregut endoderm is required at head process stages for anteriormost neural patterning in chick, Development 128 (2001) 309 – 320.