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Identification and developmental expression of cyclin-dependent kinase 4 gene in Xenopus laevis
Mechanisms of Development 70 (1998) 197–200
Gene expression pattern
Identification and developmental expression of cyclin-dependent kinase 4 gene in Xenopus laevis Ce´line Goisset, Jean-Claude Boucaut, De-Li Shi* Groupe de Biologie Expe´rimentale, Laboratoire de Biologie Mole´culaire et Cellulaire du De´veloppement, CNRS URA 1135, Universite´ P. et M. Curie, 9 quai Saint-Bernard, 75005 Paris, France Received 30 September 1997; revised version received 30 October 1997; accepted 5 November 1997
Abstract Cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases which play a pivotal role in the eucaryote cell cycle regulation. We have identified the Xenopus homologue of mammalian CDK4 (XCDK4). The protein sequence of XCDK4 has 78 and 77% overall identity to human and mouse CDK4, respectively. Northern blot analysis revealed a single transcript of approximately 4.5 kb present at various stages. XCDK4 transcripts show very dynamic expression during early development. The level of expression is higher during cleavage and gastrulation. In situ hybridization analysis revealed that the transcripts are enriched in the dorsal mesoderm at the beginning of gastrulation, then extend to both the lateral and ventral mesoderm. At the end of gastrulation, XCDK4 transcripts are mainly distributed in the blastoporal region and in the anterior neural fold. During neurulation they become restricted to optic vesicles and to neural crest cells organizing the branchial arches. As development proceeds, XCDK4 transcripts are highly expressed in the branchial arches. At late tail-bud stages, XCDK4 transcripts are also detected in ventral hematopoietic precursor cells. Therefore, this analysis clearly shows that XCDK4 has a regionalized expression during Xenopus embryogenesis. 1998 Elsevier Science Ireland Ltd. Keywords: Cyclin-dependent kinase; CDK4; Cell cycle; Mesoderm; Hematopoietic cells; Neural crest cells; Branchial arches; Optic vesicles; Xenopus
1. Results Several members of the cyclin-dependent kinase family are involved in embryonic cell cycle regulation during early Xenopus development. The role of cdc2 (CDK1) and CDK2 in regulating the cell cycle as well as mid-blastula transition in Xenopus has been described (Guadagno and Newport, 1992; Hartley et al., 1996, 1997). We have isolated a fulllength cDNA encoding a novel Xenopus CDK. The deduced protein sequence has characteristic features expected of members of the CDK family (Fig. 1). It has 78 and 77% overall identity to human (Hanks, 1987) and mouse (Matsushime et al., 1992) CDK4, respectively. It also shows 72%
* Corresponding author. Tel.: +33 1 44272772; fax: +33 1 44273445; e-mail: [email protected]. fr
0925-4773/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0925-4773 (97 )0 0184-6
overall identity to human CDK6. Identities to other members of the CDK family are less important. In particular, comparison with the known Xenopus CDK2 (Paris et al., 1991) shows 48% overall identity. Therefore this clone most likely represents an additional member of the CDK family in Xenopus. Because of its highest identity to human CDK4, this clone is designated XCDK4. RNase protection analysis of the temporal expression of XCDK4 shows that it is expressed as a maternally-derived RNA. High level of expression is detected during cleavage and gastrula stages. The expression level is slightly lower during neurulation and tail-bud stages, and decreases sharply at larval stage (Fig. 2A). Northern blot analysis indicates that a single transcript of approximately 4.5 kb is expressed at various stages of development (Fig. 2B). Members of the CDK family were shown to have distinct tissue functions. For example, CDK5 is expressed in
198
C. Goisset et al. / Mechanisms of Development 70 (1998) 197–200
Fig. 1. Protein sequence alignment between Xenopus, human and mouse CDK4. Dots indicate divergent residues and dashes indicate the absence of amino acid residues at the corresponding position. The GxPxxxxREx motif present in all CDKs is underlined.
embryonic mouse nervous systems and plays a critical role during neuronal differentiation (Tsai et al., 1993; Nikolic et al., 1996). In situ hybridization analysis has revealed a restricted expression of XCDK4 during early development. The expression of XCDK4 transcripts is first enriched in the dorsal mesoderm at the beginning of gastrulation (Fig. 3A), and then extends to the lateral and ventral regions by midgastrula stages (Fig. 3B). At the end of gastrulation, the transcripts are expressed around the blastopore and in the dorsoanterior region (Fig. 3C,D). During neurulation expression of XCDK4 is essentially detected in the posterior mesoderm, and at the level of anterior neural folds which will form the optic vesicles and anterior neural crest. No expression is detected in the trunk neural fold or the neural groove (Fig. 3D–F). As development proceeds, XCDK4 transcripts become enriched in the optic vesicles and in the migrating neural crest cells that will give rise to the branchial arches (Fig. 3G,H), while their expression in the posterior mesoderm becomes barely detectable. Higher level of expression persists in the optic vesicles and branchial arches during tail-bud stages (Fig. 3I–K). Interestingly, at the end of the tail-bud stage, XCDK4 transcripts are expressed in the ventral region of the embryo, beginning on both sides of the heart and extending into the vicinity of the proctodeum (Fig. 3L). These ventral cells most likely
correspond to hematopoietic precursors. Our results therefore reveal a restricted expression pattern of XCDK4 in the early embryo.
Fig. 2. Temporal expression of XCDK4 transcripts. (A) RNase protection assay. Total RNAs were extracted from indicated stages and hybridized with a XCDK4-specific probe. An EF-1a probe was used as a loading control. (B) Northern blot analysis showing the presence of a 4.5 kb transcript at various stages.
C. Goisset et al. / Mechanisms of Development 70 (1998) 197–200
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Fig. 3. Whole-mount in situ hybridization analysis of the tissue distribution of XCDK4 transcripts. (A) Stage 10.5; vegetal view. (B) Stage 11; vegetal view. (C) Stage 11.5; dorsovegetal view. (D) Stage 12; dorsal view. (E,F,G,H) Stages 13, 15, 19 and 22; dorsal views. (I,J,K,L) Stages 23, 24, 25 and 32; lateral views, yp, yolk plug; np, neural plate; ov, optic vesicle; ba, branchial arches; h, heart.
2. Methods 2.1. Isolation of XCDK4 cDNA A partial cDNA encoding a novel Xenopus CDK was obtained initially by PCR using degenerate oligonucleotides corresponding to consensus motifs of serine/ threonine protein kinases. This PCR product was used to screen an oligo-d(T)-primed lZAP II larval stage cDNA library (gift from Dr. D. W. DeSimone; University of Virginia, Charlottesville), as previously described (Shi et al., 1992). One positive plaque was obtained and rescued as pBluescript phagemids (Stratagene) by in vivo excision
according to the manufacture’s protocol. The nucleotide sequence was determined using the Sequenase Version 2.0 kit (USB). 2.2. RNase protection assay and northern blot Obtaining of Xenopus eggs and extraction of RNA were as described previously (Launay et al., 1996). Embryonic stages were determined according to the table of Nieuwkoop and Faber (1967). The XCDK4 template is an EcoRI/Apa I fragment (nucleotide position 490–646). RNase protection and northern blot were performed as previously described (Shi et al., 1992; Launay et al., 1996).
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C. Goisset et al. / Mechanisms of Development 70 (1998) 197–200
2.3. In situ hybridization In situ hybridization was performed as described (Harland, 1991). Briefly, embryos were treated for 10 min by proteinase K (10 mg ml−1). Following refixation with 4% paraformaldehyde and prehybridization, hybridization was performed overnight at 60°C. The embryos were then treated with RNase A and RNase T1 at 37°C and washes were done sequentially with 2 X and 0.2 X SSC at 60°C, followed by washes in MAB (100 mM maleic acid, 150 mM NaCl, pH 7.5) at room temperature. The embryos were then incubated in MAB and 2% Boehringer Mannheim Blocking Reagent (BMB), followed by MAB, 2% BMB and 20% heat inactivated lamb serum. Incubation of embryos with anti-digoxigenin antibody coupled with alkaline phosphatase (Boehringer Mannheim) was performed for 4 h at room temperature. After extensive washes in MAB for 24 h, chromogenic reaction was done using BM purple as substrate (Boehringer Mannheim).
Acknowledgements The authors would like to acknowledge Dr. D. W. DeSimone (University of Virginia, Charlottesville) for gift of cDNA library. This work was supported by grants from the Centre National de la Recherche Scientifique (CNRS), the Association Franc¸aise de la Myopathie (AFM) and the Association pour la Recherche contre le Cancer (ARC).
References Guadagno, T.M., Newport, J.W., 1992. Cdk2 kinase is required for entry into mitosis as a positive regulator of Cdc-cyclin B kinase activity. Cell 84, 73–82.
Hanks, S.K., 1987. Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family. Proc. Natl. Acad. Sci. USA 84, 388–392. Harland, R.M., 1991. In situ hybridization: an improved whole-mount method for Xenopus embryos. Meth. Cell Biol. 36, 685–695. Hartley, R.S., Rempel, R.E., Maller, J.L., 1996. In vivo regulation of the embryonic cell cycle in Xenopus. Dev. Biol. 173, 408–419. Hartley, R.S., Sible, J.C., Lewellyn, A.L., Maller, J.L., 1997. A role for Cyclin E/Cdk2 in the timing of the midblastula transition in Xenopus embryos. Dev. Biol. 188, 312–321. Launay, C., Fromentoux, V., Shi, D. L, Boucaut, J.C., 1996. A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers. Development 122, 869–880. Matsushime, H., Ewen, M.E., Strom, D.K., Kato, J.Y., Hanks, S.K., Roussel, M.F., Sherr, C.J., 1992. Identification and properties of an atypical catalytic subunit (p34psk-j3/cdk4) for mammalian D type G1 cyclins. Cell 71, 323–334. Nieuwkoop, P.D. and Faber, J., 1967. Normal Table of Xenopus laevis (Daudin), North-Holland, Amsterdam. Nikolic, M., Dudek, H., Kwon, Y.T., Ramos, Y.F.M., Tsai, L.H., 1996. The cdk5/p35 kinase is essential for neurite outgrowth during neuronal differentiation. Genes Dev. 10, 816–825. Paris, J., Le, G.R., Couturier, A., Le, G.K., Omilli, F., Camonis, J., MacNeill, S., Philippe, M., 1991. Cloning by differential screening of Xenopus cDNA coding for a protein highly homologous to cdc2. Proc. Natl. Acad. Sci. USA 88, 1039–1043. Shi, D.L., Feige, J.J., Riou, J.F., DeSimone, D.W., Boucaut, J.C., 1992. Differential expression and regulation of two distinct fibroblast growth factor receptors during early development of the urodele amphibian Pleurodeles waltl. Development 116, 261–273. Tsai, L.H., Takahashi, T., Caviness, V.S. Jr., Harlow, E., 1993. Activity and expression pattern of cyclin-dependent kinase 5 in the embryonic mouse nervous system. Development 119, 1029–1040.
Gene expression pattern
Identification and developmental expression of cyclin-dependent kinase 4 gene in Xenopus laevis Ce´line Goisset, Jean-Claude Boucaut, De-Li Shi* Groupe de Biologie Expe´rimentale, Laboratoire de Biologie Mole´culaire et Cellulaire du De´veloppement, CNRS URA 1135, Universite´ P. et M. Curie, 9 quai Saint-Bernard, 75005 Paris, France Received 30 September 1997; revised version received 30 October 1997; accepted 5 November 1997
Abstract Cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases which play a pivotal role in the eucaryote cell cycle regulation. We have identified the Xenopus homologue of mammalian CDK4 (XCDK4). The protein sequence of XCDK4 has 78 and 77% overall identity to human and mouse CDK4, respectively. Northern blot analysis revealed a single transcript of approximately 4.5 kb present at various stages. XCDK4 transcripts show very dynamic expression during early development. The level of expression is higher during cleavage and gastrulation. In situ hybridization analysis revealed that the transcripts are enriched in the dorsal mesoderm at the beginning of gastrulation, then extend to both the lateral and ventral mesoderm. At the end of gastrulation, XCDK4 transcripts are mainly distributed in the blastoporal region and in the anterior neural fold. During neurulation they become restricted to optic vesicles and to neural crest cells organizing the branchial arches. As development proceeds, XCDK4 transcripts are highly expressed in the branchial arches. At late tail-bud stages, XCDK4 transcripts are also detected in ventral hematopoietic precursor cells. Therefore, this analysis clearly shows that XCDK4 has a regionalized expression during Xenopus embryogenesis. 1998 Elsevier Science Ireland Ltd. Keywords: Cyclin-dependent kinase; CDK4; Cell cycle; Mesoderm; Hematopoietic cells; Neural crest cells; Branchial arches; Optic vesicles; Xenopus
1. Results Several members of the cyclin-dependent kinase family are involved in embryonic cell cycle regulation during early Xenopus development. The role of cdc2 (CDK1) and CDK2 in regulating the cell cycle as well as mid-blastula transition in Xenopus has been described (Guadagno and Newport, 1992; Hartley et al., 1996, 1997). We have isolated a fulllength cDNA encoding a novel Xenopus CDK. The deduced protein sequence has characteristic features expected of members of the CDK family (Fig. 1). It has 78 and 77% overall identity to human (Hanks, 1987) and mouse (Matsushime et al., 1992) CDK4, respectively. It also shows 72%
* Corresponding author. Tel.: +33 1 44272772; fax: +33 1 44273445; e-mail: [email protected]. fr
0925-4773/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0925-4773 (97 )0 0184-6
overall identity to human CDK6. Identities to other members of the CDK family are less important. In particular, comparison with the known Xenopus CDK2 (Paris et al., 1991) shows 48% overall identity. Therefore this clone most likely represents an additional member of the CDK family in Xenopus. Because of its highest identity to human CDK4, this clone is designated XCDK4. RNase protection analysis of the temporal expression of XCDK4 shows that it is expressed as a maternally-derived RNA. High level of expression is detected during cleavage and gastrula stages. The expression level is slightly lower during neurulation and tail-bud stages, and decreases sharply at larval stage (Fig. 2A). Northern blot analysis indicates that a single transcript of approximately 4.5 kb is expressed at various stages of development (Fig. 2B). Members of the CDK family were shown to have distinct tissue functions. For example, CDK5 is expressed in
198
C. Goisset et al. / Mechanisms of Development 70 (1998) 197–200
Fig. 1. Protein sequence alignment between Xenopus, human and mouse CDK4. Dots indicate divergent residues and dashes indicate the absence of amino acid residues at the corresponding position. The GxPxxxxREx motif present in all CDKs is underlined.
embryonic mouse nervous systems and plays a critical role during neuronal differentiation (Tsai et al., 1993; Nikolic et al., 1996). In situ hybridization analysis has revealed a restricted expression of XCDK4 during early development. The expression of XCDK4 transcripts is first enriched in the dorsal mesoderm at the beginning of gastrulation (Fig. 3A), and then extends to the lateral and ventral regions by midgastrula stages (Fig. 3B). At the end of gastrulation, the transcripts are expressed around the blastopore and in the dorsoanterior region (Fig. 3C,D). During neurulation expression of XCDK4 is essentially detected in the posterior mesoderm, and at the level of anterior neural folds which will form the optic vesicles and anterior neural crest. No expression is detected in the trunk neural fold or the neural groove (Fig. 3D–F). As development proceeds, XCDK4 transcripts become enriched in the optic vesicles and in the migrating neural crest cells that will give rise to the branchial arches (Fig. 3G,H), while their expression in the posterior mesoderm becomes barely detectable. Higher level of expression persists in the optic vesicles and branchial arches during tail-bud stages (Fig. 3I–K). Interestingly, at the end of the tail-bud stage, XCDK4 transcripts are expressed in the ventral region of the embryo, beginning on both sides of the heart and extending into the vicinity of the proctodeum (Fig. 3L). These ventral cells most likely
correspond to hematopoietic precursors. Our results therefore reveal a restricted expression pattern of XCDK4 in the early embryo.
Fig. 2. Temporal expression of XCDK4 transcripts. (A) RNase protection assay. Total RNAs were extracted from indicated stages and hybridized with a XCDK4-specific probe. An EF-1a probe was used as a loading control. (B) Northern blot analysis showing the presence of a 4.5 kb transcript at various stages.
C. Goisset et al. / Mechanisms of Development 70 (1998) 197–200
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Fig. 3. Whole-mount in situ hybridization analysis of the tissue distribution of XCDK4 transcripts. (A) Stage 10.5; vegetal view. (B) Stage 11; vegetal view. (C) Stage 11.5; dorsovegetal view. (D) Stage 12; dorsal view. (E,F,G,H) Stages 13, 15, 19 and 22; dorsal views. (I,J,K,L) Stages 23, 24, 25 and 32; lateral views, yp, yolk plug; np, neural plate; ov, optic vesicle; ba, branchial arches; h, heart.
2. Methods 2.1. Isolation of XCDK4 cDNA A partial cDNA encoding a novel Xenopus CDK was obtained initially by PCR using degenerate oligonucleotides corresponding to consensus motifs of serine/ threonine protein kinases. This PCR product was used to screen an oligo-d(T)-primed lZAP II larval stage cDNA library (gift from Dr. D. W. DeSimone; University of Virginia, Charlottesville), as previously described (Shi et al., 1992). One positive plaque was obtained and rescued as pBluescript phagemids (Stratagene) by in vivo excision
according to the manufacture’s protocol. The nucleotide sequence was determined using the Sequenase Version 2.0 kit (USB). 2.2. RNase protection assay and northern blot Obtaining of Xenopus eggs and extraction of RNA were as described previously (Launay et al., 1996). Embryonic stages were determined according to the table of Nieuwkoop and Faber (1967). The XCDK4 template is an EcoRI/Apa I fragment (nucleotide position 490–646). RNase protection and northern blot were performed as previously described (Shi et al., 1992; Launay et al., 1996).
200
C. Goisset et al. / Mechanisms of Development 70 (1998) 197–200
2.3. In situ hybridization In situ hybridization was performed as described (Harland, 1991). Briefly, embryos were treated for 10 min by proteinase K (10 mg ml−1). Following refixation with 4% paraformaldehyde and prehybridization, hybridization was performed overnight at 60°C. The embryos were then treated with RNase A and RNase T1 at 37°C and washes were done sequentially with 2 X and 0.2 X SSC at 60°C, followed by washes in MAB (100 mM maleic acid, 150 mM NaCl, pH 7.5) at room temperature. The embryos were then incubated in MAB and 2% Boehringer Mannheim Blocking Reagent (BMB), followed by MAB, 2% BMB and 20% heat inactivated lamb serum. Incubation of embryos with anti-digoxigenin antibody coupled with alkaline phosphatase (Boehringer Mannheim) was performed for 4 h at room temperature. After extensive washes in MAB for 24 h, chromogenic reaction was done using BM purple as substrate (Boehringer Mannheim).
Acknowledgements The authors would like to acknowledge Dr. D. W. DeSimone (University of Virginia, Charlottesville) for gift of cDNA library. This work was supported by grants from the Centre National de la Recherche Scientifique (CNRS), the Association Franc¸aise de la Myopathie (AFM) and the Association pour la Recherche contre le Cancer (ARC).
References Guadagno, T.M., Newport, J.W., 1992. Cdk2 kinase is required for entry into mitosis as a positive regulator of Cdc-cyclin B kinase activity. Cell 84, 73–82.
Hanks, S.K., 1987. Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family. Proc. Natl. Acad. Sci. USA 84, 388–392. Harland, R.M., 1991. In situ hybridization: an improved whole-mount method for Xenopus embryos. Meth. Cell Biol. 36, 685–695. Hartley, R.S., Rempel, R.E., Maller, J.L., 1996. In vivo regulation of the embryonic cell cycle in Xenopus. Dev. Biol. 173, 408–419. Hartley, R.S., Sible, J.C., Lewellyn, A.L., Maller, J.L., 1997. A role for Cyclin E/Cdk2 in the timing of the midblastula transition in Xenopus embryos. Dev. Biol. 188, 312–321. Launay, C., Fromentoux, V., Shi, D. L, Boucaut, J.C., 1996. A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers. Development 122, 869–880. Matsushime, H., Ewen, M.E., Strom, D.K., Kato, J.Y., Hanks, S.K., Roussel, M.F., Sherr, C.J., 1992. Identification and properties of an atypical catalytic subunit (p34psk-j3/cdk4) for mammalian D type G1 cyclins. Cell 71, 323–334. Nieuwkoop, P.D. and Faber, J., 1967. Normal Table of Xenopus laevis (Daudin), North-Holland, Amsterdam. Nikolic, M., Dudek, H., Kwon, Y.T., Ramos, Y.F.M., Tsai, L.H., 1996. The cdk5/p35 kinase is essential for neurite outgrowth during neuronal differentiation. Genes Dev. 10, 816–825. Paris, J., Le, G.R., Couturier, A., Le, G.K., Omilli, F., Camonis, J., MacNeill, S., Philippe, M., 1991. Cloning by differential screening of Xenopus cDNA coding for a protein highly homologous to cdc2. Proc. Natl. Acad. Sci. USA 88, 1039–1043. Shi, D.L., Feige, J.J., Riou, J.F., DeSimone, D.W., Boucaut, J.C., 1992. Differential expression and regulation of two distinct fibroblast growth factor receptors during early development of the urodele amphibian Pleurodeles waltl. Development 116, 261–273. Tsai, L.H., Takahashi, T., Caviness, V.S. Jr., Harlow, E., 1993. Activity and expression pattern of cyclin-dependent kinase 5 in the embryonic mouse nervous system. Development 119, 1029–1040.