Identification and developmental expression of ... - Semantic Scholar
Mechanisms of Development 99 (2000) 173±176
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Gene expression pattern
Identi®cation and developmental expression of three Distal-less homeobox containing genes in the ascidian Ciona intestinalis Anna Caracciolo 1, Anna Di Gregorio 1,2, Francesco Aniello, Roberto Di Lauro, Margherita Branno* Laboratory of Biochemistry and Molecular Biology, Stazione Zoologica ªAnton Dohrnº, Villa Comunale, 80121 Naples, Italy Received 26 July 2000; received in revised form 7 September 2000; accepted 11 September 2000
Abstract Several homeobox-containing genes related to Drosophila Distal-less (Dll) have been isolated from a wide variety of organisms and have been shown to function as developmental regulators. While in Drosophila only one Dll gene has been described so far, in Vertebrates many components of the Dlx multigenic family have been characterized. This suggests that, during the evolution of the Chordate phylum, the Dlx genes arose from an ancestral Dll/Dlx gene via gene duplication. We have previously reported the isolation of two Dll-related homeoboxes from the protochordate Ciona intestinalis, and described their clustered arrangement (Gene 156 (1995) 253). Here we present the detailed genomic organization and spatial-temporal expression of these two genes, Ci-Dll-A and Ci-Dll-B, and describe the isolation and characterization of another member of the ascidian family of Dll-related genes, which we tentatively named Ci-Dll-C. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ascidian; Ciona intestinalis; Homeobox-containing genes; Development; Myoplasm; Atrial siphon; Distal-less
1. Results 1.1. Structure of the Ciona Dll cluster and its similarity to vertebrate Dlx clusters With a screen for homeobox-containing genes in Ciona intestinalis, we isolated two genes, Ci-Dll-A and Ci-Dll-B, clustered on a 4 kb genomic insert. As shown in Fig. 1A, CiDll-A and Ci-Dll-B are organized in an inverted convergent (tail-to-tail) orientation; a comparison of genomic and cDNA sequences indicates that the distance between the two ORFs is ~1.5 kb. In this region, the two putative stop codons and polyadenylation signals have been identi®ed. Similar genomic arrangements of Dll-related homeoboxes have been described in vertebrate genomes: particularly, human DLX1 and DLX2 are found within a 20 kb genomic region (McGuinness et al., 1996) whilst DLX5 and DLX6 are contained in a 25 kb region (Nakamura et al., 1996). The same genomic arrangement is displayed by * Corresponding author. Tel.: 139-81-583-3275. E-mail address: [email protected] (M. Branno). 1 These authors contributed equally to this work. 2 Present address: Department of Molecular and Cell Biology, Division of Genetics and Development, 401 Barker Hall, University of California at Berkeley, CA 94720-3204, USA.
their mouse and zebra®sh cognates, Dlx1, Dlx2 and Dlx5, Dlx6 (e.g. Simeone et al., 1994; Zerucha et al., 2000). 1.2. Expression pattern of Ci-Dll-A The Ci-Dll-A cDNA clone predicts a protein of 453 amino acids. Northern blot analysis indicates that during Ciona embryogenesis a Ci-Dll-A transcript of about 1.6 kb is ®rst detectable at mid-tailbud stage and its levels are increased at the late tailbud and larval stages (Fig. 1B). Whole-mount in situ hybridization was performed on staged Ciona embryos (Fig. 2). Ci-Dll-A expression is ®rst detected at tailbud stage in a few ectodermal cells of the trunk (Fig. 2A). In hatched larvae, Ci-Dll-A is expressed in the bilateral primordia of the atrial siphon (Fig. 2B). As shown in Fig. 2C, in sagittal sections the atrial primordia consist of two pockets of cuboidal and columnar cells in the epidermis of the body wall, which will become fused, during metamorphosis, to form the single atrial siphon of the adult (Katz, 1983). 1.3. Expression pattern of Ci-Dll-B The isolated cDNA clone of Ci-Dll-B did not contain the entire coding region lacking the 5 0 UTR and the putative ATG and predicts a 606 bp open reading frame. Ci-Dll-B transcript (about 2.5 kb), monitored during embryogenesis
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00474-3
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A. Caracciolo et al. / Mechanisms of Development 99 (2000) 173±176
Fig. 1. Schematic representation of the genomic region containing the two Ci-Dll genes (A) and Northern blot analyses of the three Ci-Dll genes (B,C). (A) The subcloned of 4015 bp is represented in detail, showing that Ci-Dll-A and Ci-Dll-B are in an inverted convergent (tail to tail) arrangement. The coding sequences are boxed and include the homeobox (black boxes). The bold line indicate the introns and the intergenic region. The polyadenylation signal and the two putative stop codon are indicated with polyA and TGA respectively. (B,C) Poly(A) 1 RNAs (10 mg) were electrophoresed on a 1.3% agarose-3% formaldehyde gel and blotted onto Nylon ®lters (Hybond-N, Amersham, UK). Blots were hybridized using as probes the entire cDNA for Ci-Dll-A, Ci-Dll-B and Ci-Dll-C. As loading control was used a C. intestinalis calmodulin (Ci-CaM) probe (Di Gregorio et al., 1998). Poly(A) 1 was from: eggs (E), 16-stage cell (16), 64-stage cell (64), gastrula (G), neurula (N), early- (eT), middle- (mT), late-tailbud (lT) and swimming larva (SW). Size makers are on the right. The cDNA sequences have been deposited to the EMBL Data library under the accession numbers: Ci-Dll-A AJ278696; Ci-Dll-B AJ278697; Ci-Dll-C AJ278698.
by Northern blot analysis (Fig. 1B), is ®rst detected in unfertilized eggs, thus indicating a maternal storage; its levels then increase at 64-cell stage, peaking at gastrula stage, and decreasing rapidly soon after neurulation, becoming faint in early tailbud embryos and not detectable in later stages. The expression pattern of Ci-Dll-B is similar to that observed for the posterior end mark (pem) genes characterized in the ascidian Ciona savignyi (Yoshida et al. 1996; Satou and Satoh, 1997; Satou, 1999). Immediately after ooplasmic segregation, the Ci-Dll-B transcript appears localized to the embryonic region known as myoplasm (Fig. 2D). In the 8-cell-stage embryo, the distribution of Ci-Dll-B transcript was restricted to the posterior region of B4.1 cells (data not shown). Positive hybridization signals were detected at gastrula stage, not only in the progeny of the B4.1 cells, (B7.6; Fig. 2E) but also in nearly all the dorsal blastomeres, which include epidermal and neuroectodermal
precursors (Fig. 2F). Finally, early-tailbud embryos show positive signals in two cells of the endodermal strand, while middle-tailbud embryos show only one positive endodermal cell (Fig. 2G,H). 1.4. Expression pattern of Ci-Dll-C Ci-Dll-C was isolated screening a larva cDNA library and encodes a protein of 288 amino acids. Ci-Dll-C expression can be ®rst detected by Northern blot analysis at the gastrula stage and its levels increase at larval stage (Fig. 1C). In hatched larvae, the only site where this transcript is detected by whole-mount in situ hybridization is the adhesive organ, which is located at the anterior end of the body (Fig. 2I). This organ is used by ascidian larvae at the beginning of metamorphosis to attach to a solid substrate (Cloney, 1977).
A. Caracciolo et al. / Mechanisms of Development 99 (2000) 173±176
175
Fig. 2. Spatial expression of the three Distal-less transcripts. (A±C) Results with Ci-Dll-A antisense RNA; (D±H) results with Ci-Dll-B antisense RNA; (I) result with Ci-Dll-C antisense RNA. (A) An early tailbud embryo viewed from the lateral side. (B) A larva stage from dorsal view. (C) Sagittal section of the trunk. (D) Unfertilized egg. (E,F) An embryo at gastrula stage viewed from the ventral and dorsal side respectively. (G) An early tailbud embryo viewed from the lateral side. (H) A middle tailbud embryo viewed from the lateral side. (I) A trunk of larva stage from dorsal view. as, atrial siphon; ec, ectodermal cell; es, endodermal strand; my, myoplasm; p, palps; pas, primordia atrial siphon.
1.5. Conclusion While only one Dll gene has been isolated from Drosophila (Cohen and Yurgens, 1989) and from amphioxus (Holland et al., 1996) and several Dlx genes have been isolated from Vertebrates (Liu et al., 1997), we have identi®ed three Dll-related genes in Ciona, which leads to the formulation of two main alternative hypotheses. First, the situation in Ciona represents the ancestral condition of primitive Chordates and, if so, divergent Dllrelated genes may also be present in the amphioxus genome. Second, the situation in Ciona is the result of a lineagespeci®c duplication event occurred after the divergence of Ascidians from the main chordate branch leading to Cephalochordates and Vertebrates.
RNAs isolated from hatched-larvae, using as a probe a genomic fragment corresponding to the Ci-Dll-A homeobox. A Ci-Dll-B cDNA clone was isolated screening a C. intestinalis cDNA library prepared from poly(A) 1 RNAs extracted from unfertilized eggs, using as a probe a genomic fragment encompassing the homeobox. A Ci-Dll-C cDNA clone was isolated from the C. intestinalis hatched-larvae cDNA library described above, using the CiHB1 degenerate oligo probe, as described in Di Gregorio et al. (1995). 2.2. Northern blot analysis Total RNA was extracted from embryos at various stages of development according to Chomczynski and Sacchi (1987) and poly(A) 1 RNAs were puri®ed by oligo(dT)cellulose chromatography (Sambrook et al., 1989).
2. Materials and methods
2.3. In situ hybridizations
2.1. Isolation of Ciona Dll-related genes
The cDNA inserts, cloned in the pBluescript SK(2) vector, were linearized and used as templates for the synthesis of DIG-11-UTP labelled sense or antisense RNA probes using a Digoxigenin RNA labeling kit (Boehringer
A positive Ci-Dll-A cDNA clone was isolated screening a C. intestinalis cDNA library prepared from poly-(A) 1
176
A. Caracciolo et al. / Mechanisms of Development 99 (2000) 173±176
Mannheim) according to the supplier's instructions. Wholemount in situ hybridizations on C. intestinalis staged embryos were carried out as described by Caracciolo et al. (1997). References Caracciolo, A., Gesualdo, I., Branno, M., Aniello, F., Di Lauro, R., Palumbo, A., 1997. Speci®c cellular localization of tyrosinase mRNA during Ciona intestinalis larval development. Dev. Growth Differ. 39, 437±444. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thyocianate-phenol-chloroform extraction. Anal. Biochem. 162, 156±159. Cloney, R.A., 1977. Larval adhesive organs and metamorphosis in ascidians. Cell Tissue Res. 183, 423±444. Cohen, S.M., Yurgens, G., 1989. Proximal-distal pattern formation in Drosophila: cell autonomous requirement for Distal-less gene activity in limb development. EMBO J. 8, 2045±2055. Di Gregorio, A., Spagnuolo, A., Ristoratore, F., Pischetola, M., Aniello, F., Branno, M., Cariello, L., Di Lauro, R., 1995. Cloning of ascidian homeobox genes provides evidence for a primordial chordate cluster. Gene 156, 253±257. Di Gregorio, A., Villani, M.G., Locascio, A., Ristoratore, F., Aniello, F., Branno, M., 1998. Developmental regulation and tissue-speci®c localization of calmodulin mRNA in the protochordate Ciona intestinalis. Dev. Growth Differ. 40, 387±394. Holland, N.D., Panganiban, G., Henyey, E.L., Holland, L.Z., 1996. Sequence and developmental expression of AmphiDll, an amphioxus Distal-less gene transcribed in the ectoderm, epidermis and nervous system: insights into evolution of craniate forebrain and neural crest. Development 122, 2911±2920.
Katz, M.J., 1983. Comparative anatomy of the Tunicate tadpole. Biol. Bull. 164, 1±27. Liu, J.K., Ghattas, I., Liu, S., Chen, S., Rubenstein, J.L.R., 1997. Dlx gene encode DNA-binding proteins that are expressed in an overlapping and sequential pattern during basal ganglia differentiation. Dev. Dyn. 210, 498±512. McGuinness, T., Porteus, M.H., Smiga, S., Bulfone, A., Kingsley, C., Qiu, M., Liu, J.K., Long, J.E., Xu, D., Rubenstein, J.L., 1996. Sequence, organization, and transcription of the Dlx-1 and Dlx-2 locus. Genomics 35, 473±485. Nakamura, S., Stock, D.W., Wydner, K., Zhao, Z., Minowada, J., Lawrence, J.B., Weiss, K.M., Ruddle, F.H., 1996. Genomic analysis of a new mammalian Distal-less gene: Dlx7. Genomics 38, 314±324. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Satou, Y., 1999. Posterior end mark 3 (pem-3), an ascidian maternally expressed gene with localized mRNA encodes a protein with caenorhabditis elegans MEX-3-like KH domains. Dev. Biol. 212, 337±350. Satou, Y., Satoh, N., 1997. Posterior end mark 2 (pem-2), pem-4, pem-5, and pem-6: maternal gene localized mRNA in the ascidian embryo. Dev. Biol. 192, 467±481. Simeone, A., Acampora, D., Pannese, M., D'Esposito, M., Stornaiuolo, A., Gulisano, M., Kastury, K., Druck, T., Huebner, K., Boncinelli, E., 1994. Cloning and characterization of two new members of the vertebrate Dlx gene family. Proc. Natl. Acad. Sci. USA 91, 2250±2254. Yoshida, S., Marikawa, Y., Satoh, N., 1996. Posterior end mark, a novel maternal gene encoding a localized factor in ascidian embryo. Development 122, 2005±2012. Zerucha, T., Stuhmer, T., Hatch, G., Park, B.K., Long, Q., Yu, G., Gambarotta, A., Schultz, J.R., Rubenstein, J.L., Ekker, M., 2000. A highly conserved enhancer in the Dlx5/Dlx6 intergenic region is the site of cross-regulatory interactions between Dlx genes in the embryonic forebrain. J. Neurosci. 15, 709±721.
www.elsevier.com/locate/modo
Gene expression pattern
Identi®cation and developmental expression of three Distal-less homeobox containing genes in the ascidian Ciona intestinalis Anna Caracciolo 1, Anna Di Gregorio 1,2, Francesco Aniello, Roberto Di Lauro, Margherita Branno* Laboratory of Biochemistry and Molecular Biology, Stazione Zoologica ªAnton Dohrnº, Villa Comunale, 80121 Naples, Italy Received 26 July 2000; received in revised form 7 September 2000; accepted 11 September 2000
Abstract Several homeobox-containing genes related to Drosophila Distal-less (Dll) have been isolated from a wide variety of organisms and have been shown to function as developmental regulators. While in Drosophila only one Dll gene has been described so far, in Vertebrates many components of the Dlx multigenic family have been characterized. This suggests that, during the evolution of the Chordate phylum, the Dlx genes arose from an ancestral Dll/Dlx gene via gene duplication. We have previously reported the isolation of two Dll-related homeoboxes from the protochordate Ciona intestinalis, and described their clustered arrangement (Gene 156 (1995) 253). Here we present the detailed genomic organization and spatial-temporal expression of these two genes, Ci-Dll-A and Ci-Dll-B, and describe the isolation and characterization of another member of the ascidian family of Dll-related genes, which we tentatively named Ci-Dll-C. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ascidian; Ciona intestinalis; Homeobox-containing genes; Development; Myoplasm; Atrial siphon; Distal-less
1. Results 1.1. Structure of the Ciona Dll cluster and its similarity to vertebrate Dlx clusters With a screen for homeobox-containing genes in Ciona intestinalis, we isolated two genes, Ci-Dll-A and Ci-Dll-B, clustered on a 4 kb genomic insert. As shown in Fig. 1A, CiDll-A and Ci-Dll-B are organized in an inverted convergent (tail-to-tail) orientation; a comparison of genomic and cDNA sequences indicates that the distance between the two ORFs is ~1.5 kb. In this region, the two putative stop codons and polyadenylation signals have been identi®ed. Similar genomic arrangements of Dll-related homeoboxes have been described in vertebrate genomes: particularly, human DLX1 and DLX2 are found within a 20 kb genomic region (McGuinness et al., 1996) whilst DLX5 and DLX6 are contained in a 25 kb region (Nakamura et al., 1996). The same genomic arrangement is displayed by * Corresponding author. Tel.: 139-81-583-3275. E-mail address: [email protected] (M. Branno). 1 These authors contributed equally to this work. 2 Present address: Department of Molecular and Cell Biology, Division of Genetics and Development, 401 Barker Hall, University of California at Berkeley, CA 94720-3204, USA.
their mouse and zebra®sh cognates, Dlx1, Dlx2 and Dlx5, Dlx6 (e.g. Simeone et al., 1994; Zerucha et al., 2000). 1.2. Expression pattern of Ci-Dll-A The Ci-Dll-A cDNA clone predicts a protein of 453 amino acids. Northern blot analysis indicates that during Ciona embryogenesis a Ci-Dll-A transcript of about 1.6 kb is ®rst detectable at mid-tailbud stage and its levels are increased at the late tailbud and larval stages (Fig. 1B). Whole-mount in situ hybridization was performed on staged Ciona embryos (Fig. 2). Ci-Dll-A expression is ®rst detected at tailbud stage in a few ectodermal cells of the trunk (Fig. 2A). In hatched larvae, Ci-Dll-A is expressed in the bilateral primordia of the atrial siphon (Fig. 2B). As shown in Fig. 2C, in sagittal sections the atrial primordia consist of two pockets of cuboidal and columnar cells in the epidermis of the body wall, which will become fused, during metamorphosis, to form the single atrial siphon of the adult (Katz, 1983). 1.3. Expression pattern of Ci-Dll-B The isolated cDNA clone of Ci-Dll-B did not contain the entire coding region lacking the 5 0 UTR and the putative ATG and predicts a 606 bp open reading frame. Ci-Dll-B transcript (about 2.5 kb), monitored during embryogenesis
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00474-3
174
A. Caracciolo et al. / Mechanisms of Development 99 (2000) 173±176
Fig. 1. Schematic representation of the genomic region containing the two Ci-Dll genes (A) and Northern blot analyses of the three Ci-Dll genes (B,C). (A) The subcloned of 4015 bp is represented in detail, showing that Ci-Dll-A and Ci-Dll-B are in an inverted convergent (tail to tail) arrangement. The coding sequences are boxed and include the homeobox (black boxes). The bold line indicate the introns and the intergenic region. The polyadenylation signal and the two putative stop codon are indicated with polyA and TGA respectively. (B,C) Poly(A) 1 RNAs (10 mg) were electrophoresed on a 1.3% agarose-3% formaldehyde gel and blotted onto Nylon ®lters (Hybond-N, Amersham, UK). Blots were hybridized using as probes the entire cDNA for Ci-Dll-A, Ci-Dll-B and Ci-Dll-C. As loading control was used a C. intestinalis calmodulin (Ci-CaM) probe (Di Gregorio et al., 1998). Poly(A) 1 was from: eggs (E), 16-stage cell (16), 64-stage cell (64), gastrula (G), neurula (N), early- (eT), middle- (mT), late-tailbud (lT) and swimming larva (SW). Size makers are on the right. The cDNA sequences have been deposited to the EMBL Data library under the accession numbers: Ci-Dll-A AJ278696; Ci-Dll-B AJ278697; Ci-Dll-C AJ278698.
by Northern blot analysis (Fig. 1B), is ®rst detected in unfertilized eggs, thus indicating a maternal storage; its levels then increase at 64-cell stage, peaking at gastrula stage, and decreasing rapidly soon after neurulation, becoming faint in early tailbud embryos and not detectable in later stages. The expression pattern of Ci-Dll-B is similar to that observed for the posterior end mark (pem) genes characterized in the ascidian Ciona savignyi (Yoshida et al. 1996; Satou and Satoh, 1997; Satou, 1999). Immediately after ooplasmic segregation, the Ci-Dll-B transcript appears localized to the embryonic region known as myoplasm (Fig. 2D). In the 8-cell-stage embryo, the distribution of Ci-Dll-B transcript was restricted to the posterior region of B4.1 cells (data not shown). Positive hybridization signals were detected at gastrula stage, not only in the progeny of the B4.1 cells, (B7.6; Fig. 2E) but also in nearly all the dorsal blastomeres, which include epidermal and neuroectodermal
precursors (Fig. 2F). Finally, early-tailbud embryos show positive signals in two cells of the endodermal strand, while middle-tailbud embryos show only one positive endodermal cell (Fig. 2G,H). 1.4. Expression pattern of Ci-Dll-C Ci-Dll-C was isolated screening a larva cDNA library and encodes a protein of 288 amino acids. Ci-Dll-C expression can be ®rst detected by Northern blot analysis at the gastrula stage and its levels increase at larval stage (Fig. 1C). In hatched larvae, the only site where this transcript is detected by whole-mount in situ hybridization is the adhesive organ, which is located at the anterior end of the body (Fig. 2I). This organ is used by ascidian larvae at the beginning of metamorphosis to attach to a solid substrate (Cloney, 1977).
A. Caracciolo et al. / Mechanisms of Development 99 (2000) 173±176
175
Fig. 2. Spatial expression of the three Distal-less transcripts. (A±C) Results with Ci-Dll-A antisense RNA; (D±H) results with Ci-Dll-B antisense RNA; (I) result with Ci-Dll-C antisense RNA. (A) An early tailbud embryo viewed from the lateral side. (B) A larva stage from dorsal view. (C) Sagittal section of the trunk. (D) Unfertilized egg. (E,F) An embryo at gastrula stage viewed from the ventral and dorsal side respectively. (G) An early tailbud embryo viewed from the lateral side. (H) A middle tailbud embryo viewed from the lateral side. (I) A trunk of larva stage from dorsal view. as, atrial siphon; ec, ectodermal cell; es, endodermal strand; my, myoplasm; p, palps; pas, primordia atrial siphon.
1.5. Conclusion While only one Dll gene has been isolated from Drosophila (Cohen and Yurgens, 1989) and from amphioxus (Holland et al., 1996) and several Dlx genes have been isolated from Vertebrates (Liu et al., 1997), we have identi®ed three Dll-related genes in Ciona, which leads to the formulation of two main alternative hypotheses. First, the situation in Ciona represents the ancestral condition of primitive Chordates and, if so, divergent Dllrelated genes may also be present in the amphioxus genome. Second, the situation in Ciona is the result of a lineagespeci®c duplication event occurred after the divergence of Ascidians from the main chordate branch leading to Cephalochordates and Vertebrates.
RNAs isolated from hatched-larvae, using as a probe a genomic fragment corresponding to the Ci-Dll-A homeobox. A Ci-Dll-B cDNA clone was isolated screening a C. intestinalis cDNA library prepared from poly(A) 1 RNAs extracted from unfertilized eggs, using as a probe a genomic fragment encompassing the homeobox. A Ci-Dll-C cDNA clone was isolated from the C. intestinalis hatched-larvae cDNA library described above, using the CiHB1 degenerate oligo probe, as described in Di Gregorio et al. (1995). 2.2. Northern blot analysis Total RNA was extracted from embryos at various stages of development according to Chomczynski and Sacchi (1987) and poly(A) 1 RNAs were puri®ed by oligo(dT)cellulose chromatography (Sambrook et al., 1989).
2. Materials and methods
2.3. In situ hybridizations
2.1. Isolation of Ciona Dll-related genes
The cDNA inserts, cloned in the pBluescript SK(2) vector, were linearized and used as templates for the synthesis of DIG-11-UTP labelled sense or antisense RNA probes using a Digoxigenin RNA labeling kit (Boehringer
A positive Ci-Dll-A cDNA clone was isolated screening a C. intestinalis cDNA library prepared from poly-(A) 1
176
A. Caracciolo et al. / Mechanisms of Development 99 (2000) 173±176
Mannheim) according to the supplier's instructions. Wholemount in situ hybridizations on C. intestinalis staged embryos were carried out as described by Caracciolo et al. (1997). References Caracciolo, A., Gesualdo, I., Branno, M., Aniello, F., Di Lauro, R., Palumbo, A., 1997. Speci®c cellular localization of tyrosinase mRNA during Ciona intestinalis larval development. Dev. Growth Differ. 39, 437±444. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thyocianate-phenol-chloroform extraction. Anal. Biochem. 162, 156±159. Cloney, R.A., 1977. Larval adhesive organs and metamorphosis in ascidians. Cell Tissue Res. 183, 423±444. Cohen, S.M., Yurgens, G., 1989. Proximal-distal pattern formation in Drosophila: cell autonomous requirement for Distal-less gene activity in limb development. EMBO J. 8, 2045±2055. Di Gregorio, A., Spagnuolo, A., Ristoratore, F., Pischetola, M., Aniello, F., Branno, M., Cariello, L., Di Lauro, R., 1995. Cloning of ascidian homeobox genes provides evidence for a primordial chordate cluster. Gene 156, 253±257. Di Gregorio, A., Villani, M.G., Locascio, A., Ristoratore, F., Aniello, F., Branno, M., 1998. Developmental regulation and tissue-speci®c localization of calmodulin mRNA in the protochordate Ciona intestinalis. Dev. Growth Differ. 40, 387±394. Holland, N.D., Panganiban, G., Henyey, E.L., Holland, L.Z., 1996. Sequence and developmental expression of AmphiDll, an amphioxus Distal-less gene transcribed in the ectoderm, epidermis and nervous system: insights into evolution of craniate forebrain and neural crest. Development 122, 2911±2920.
Katz, M.J., 1983. Comparative anatomy of the Tunicate tadpole. Biol. Bull. 164, 1±27. Liu, J.K., Ghattas, I., Liu, S., Chen, S., Rubenstein, J.L.R., 1997. Dlx gene encode DNA-binding proteins that are expressed in an overlapping and sequential pattern during basal ganglia differentiation. Dev. Dyn. 210, 498±512. McGuinness, T., Porteus, M.H., Smiga, S., Bulfone, A., Kingsley, C., Qiu, M., Liu, J.K., Long, J.E., Xu, D., Rubenstein, J.L., 1996. Sequence, organization, and transcription of the Dlx-1 and Dlx-2 locus. Genomics 35, 473±485. Nakamura, S., Stock, D.W., Wydner, K., Zhao, Z., Minowada, J., Lawrence, J.B., Weiss, K.M., Ruddle, F.H., 1996. Genomic analysis of a new mammalian Distal-less gene: Dlx7. Genomics 38, 314±324. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Satou, Y., 1999. Posterior end mark 3 (pem-3), an ascidian maternally expressed gene with localized mRNA encodes a protein with caenorhabditis elegans MEX-3-like KH domains. Dev. Biol. 212, 337±350. Satou, Y., Satoh, N., 1997. Posterior end mark 2 (pem-2), pem-4, pem-5, and pem-6: maternal gene localized mRNA in the ascidian embryo. Dev. Biol. 192, 467±481. Simeone, A., Acampora, D., Pannese, M., D'Esposito, M., Stornaiuolo, A., Gulisano, M., Kastury, K., Druck, T., Huebner, K., Boncinelli, E., 1994. Cloning and characterization of two new members of the vertebrate Dlx gene family. Proc. Natl. Acad. Sci. USA 91, 2250±2254. Yoshida, S., Marikawa, Y., Satoh, N., 1996. Posterior end mark, a novel maternal gene encoding a localized factor in ascidian embryo. Development 122, 2005±2012. Zerucha, T., Stuhmer, T., Hatch, G., Park, B.K., Long, Q., Yu, G., Gambarotta, A., Schultz, J.R., Rubenstein, J.L., Ekker, M., 2000. A highly conserved enhancer in the Dlx5/Dlx6 intergenic region is the site of cross-regulatory interactions between Dlx genes in the embryonic forebrain. J. Neurosci. 15, 709±721.
