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4, a POU III gene in amphioxus
Mechanisms of Development 116 (2002) 231–234 www.elsevier.com/locate/modo
Gene expression pattern
Cloning and developmental expression of AmphiBrn1/2/4, a POU III gene in amphioxus Simona Candiani a, Patrizio Castagnola b, Diana Oliveri a, Mario Pestarino a,* a
Dipartimento di Biologia Sperimentale, Ambientale e Applicata, Universita` di Genova, viale Benedetto XV 5, 16132 Genova, Italy b Istituto Nazionale per la Ricerca sul Cancro, largo R. Benzi 10, 16132 Genova, Italy Received 27 March 2002; received in revised form 1 May 2002; accepted 1 May 2002
Abstract The large family encoding POU transcription factors has been described in several species. In particular, class III POU genes regulate critical steps of vertebrate and invertebrate neurogenesis. A novel amphioxus class III POU gene, AmphiBrn1/2/4, has been isolated and its spatio-temporal expression has been reported. AmphiBrn1/2/4 is first expressed in the dorsal epiblast, then throughout the neural plate except for a gap at level of the anterior region of the cerebral vesicle. Transcripts are also detected in the primordium of gill slits, pharynx and left Hatschek’s diverticulum. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Homeodomain; POU-III class; AmphiBrn1/2/4; Developmental expression; Neural tube; Endodermic structures; Amphioxus; Branchiostoma floridae
1. Results and discussion POU proteins act as transcription factors during development, and in particular, those of class III play fundamental role in neural development (reviewed in Ryan and Rosenfeld, 1997; Schonemann et al., 1995). In invertebrates, POU III proteins are expressed both in nervous and non-nervous structures (Chavez et al., 1999; Fukuta et al., 1993; Bu¨rglin and Ruvkun, 2001). The only evidence of class III POU proteins in vertebrate non-nervous structures was demonstrated in zebrafish (Hauptmann and Gerster, 2000). To analyse POU genes potentially involved in the control of amphioxus neurogenesis, we have cloned a homologue of class III POU genes, AmphiBrn1/2/4, in Branchiostoma floridae. A fragment of AmphiBrn1/2/4 was amplified by polymerase chain reaction (PCR) and was used to isolate a cDNA clone from 26-h embryos cDNA library. Sequence analysis revealed an open reading frame with high sequence similarity to the known class III POU genes. At the same time, Northern blot analysis performed with total RNA from amphioxus adult identified a single major band of 2.7 kb (Fig. 1C), and in accordance with the data from the corresponding vertebrate genes, AmphiBrn1/2/4 is not only present during development but also in adults. The predicted * Corresponding author. Tel.: 139-010-35-38043; fax: 139-010-3538047. E-mail address: [email protected] (M. Pestarino).
AmphiBrn1/2/4 protein is 410 amino acids long including a POU domain (Fig. 1A) and is .90% identical to those of vertebrate class III POU genes. AmphiBrn1/2/4 POU domain is highly related to that of rat-Brn1, rat-Brn2, ratBrn4, Xl-POU3B, APH1 and Dm-cf1a (94, 95, 92, 95, 93 and 94% amino acid identity, respectively) (Fig. 1B). Spatio-temporal distribution of AmphiBrn1/2/4 was analysed by whole mount in situ hybridisation. Firstly, AmphiBrn1/2/4 is detected in dorsal epiblast at the midgastrula stage (Fig. 2A,B). At the onset of neurulation, transcripts become strong posteriorly and span to the anterior region of the neural plate (Fig. 2C,D). Soon after hatching, AmphiBrn1/2/4 expression is strong at level of two transverse patches located in the most anterior region of the neural plate, separated by a gap of expression (Fig. 2E,F). This expression is similar to that shown in the cerebral vesicle by AmphiOtx (Williams and Holland, 1996). In late neurula, AmphiBrn1/2/4 is restricted to the roof of the anterior region of the cerebral vesicle, whereas it is absent in the respective floor (Fig. 2G). Two large nerve cell bodies expressing AmphiBrn1/2/4 are visible just posterior to the cerebral vesicle (Fig. 2I), and belong to the primary motor centre (PMC) (Lacalli, 1996). In late neurula, left Hatschek’s diverticulum, anterodorsal pharyngeal endoderm and a small group of ventral endoderm cells which become the first gill slit, are labelled (Fig. 2H,L,M). Later on, expression is still detected in endoderm (Fig. 2N,O,P,R)
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00146-6
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S. Candiani et al. / Mechanisms of Development 116 (2002) 231–234
Fig. 1. (A) The deduced amino acid sequence of the AmphiBrn1/2/4 is shown. The conserved POU specific domain is underlined and the POU homeodomain is indicated by a dotted line. (B) Sequence alignment of POU domains of AmphiBrn1/2/4 and a selection of vertebrate and invertebrate class III POU proteins. The black background indicates full conserved residues in all sequences. (C) Northern blot analysis performed with total RNA from adult of B. floridae. A single band of 2.7 kb is indicated by an arrow. The position of RNA ladders is shown on the left.
as well as in dorsal and ventral neurons (Fig. 2N,O,P), probably corresponding to the ventral and dorsal motor neurons expressing amphioxus islet (Jackman et al., 2000) and AmphiMnx (Ferrier et al., 2001), respectively. From 15day old larva until metamorphosis, AmphiBrn1/2/4 mRNA occurs exclusively in the neural tube (Fig. 2S,T). In conclusion, AmphiBrn1/2/4 is a further addition to the neural marker genes illustrating homologies between amphioxus and vertebrate neural tubes, particularly in the most anterior region. Furthermore, AmphiBrn1/2/4 expres-
sion pattern shows interesting similarities to the expression of some motor neuron markers, such as amphioxus islet and AmphiMnx.
2. Materials and methods 2.1. Obtaining and culturing embryos Adults of amphioxus B. floridae were collected in Tampa
S. Candiani et al. / Mechanisms of Development 116 (2002) 231–234
233
Fig. 2. Expression of AmphiBrn1/2/4 in amphioxus embryos and larvae as analysed by in situ hybridisation. Anterior is to left and dorsal is up in all whole mounts except for (I). Cross-sections (counterstained pink) are viewed from the anterior end of animal. (A) Lateral view and (B) cross-section of gastrula. The blastopore opens to the right. Staining is at level of the dorsal epiblast (presumptive neuroectoderm). (C) Side view of early neurula with strong posterior expression that spans anteriorly in the neural plate. (D) Cross-section through level d–d 0 in (C) showing AmphiBrn1/2/4 transcripts in the neural plate but not in the overgrowing dorsal epidermis (arrows). (E) Side view of neurula (somites are out of focus). The most anterior part of the neural plate is strongly labelled, but a gap of expression (arrow) is visible just behind it, with its posterior boundary corresponding to the anterior limit of the first somite. A faint signal is visible at level of a part of the anterior endoderm (arrowheads). (F) Same neurula in (E) viewed slightly rotated to the left side along the anterior–posterior axis. The gap of expression (arrow) shown in (E) is now better visible limited by two strong labelled patches. An irregular segmental arrangement is visible from the first somite to the posterior part of the neural plate. (G, H) Late neurula in lateral view. Neural expression is strongest in the ventral part of the posterior cerebral vesicle where the infundibular organ is located. In contrast, the anterior cerebral vesicle shows a weak expression only in the roof of the most rostral part, then a gap of expression is followed by reappearance of AmphiBrn1/2/4 in spinal cord. In (H) transcripts are present in the dorsal pharynx endoderm (arrowhead) and the rudiment of the primary gill slit (arrow). (I) Dorsal view of late neurula showing two labelled large cells bodies (arrows) corresponding to the ‘giant cells’ of the PMC. (L) Cross-section through l–l 0 in (H) showing intense expression in the ventral infundibular cells and the left Hatschek’s diverticulum. (M) Crosssection through m–m 0 in (H), strong expression is located in the neural tube and the rudiment of first gill slit (arrow), as well as at level of some cells of the dorsal pharynx endoderm (arrowheads). (N) Side view of the anterior half of 30-h larva with rudiments of first and second gill slits; the expression pattern is identical to that shown in late neurula, but additional expression is also visible at level of the rudiment of second gill slit (tandem arrows). (O) Cross-section through level o–o 0 in (N), AmphiBrn1/2/4 is detectable in ventral cells of the infundibular organ and in the left Hatschek’s diverticulum, now fused with ectoderm to form the preoral ciliated pit (arrow). (P) Cross-section through level p–p 0 in (N), transcripts are found in the opening first gill slit (arrow). (Q) Side view of the anterior end of 5-day larva showing transcripts in some cells behind the frontal eye (arrowhead) and in the preoral pit (arrow). (R) Anterior third of 7-day larva, expression is visible in the endostyle (arrow) and preoral pit (tandem arrows), whereas the neural expression appears down regulated along the anterior–posterior axis in the neural tube, even if strong labelling persists in the infundibular organ (arrowhead). (S) AmphiBrn1/2/4 occurs exclusively in the neural tube of 15-day larva (side view). (T) Cross-section through level t–t 0 in (S) showing a labelled ventrolateral neuron. Abbreviations: b, blastopore; cv, cerebral vesicle; ld, left Hatschek’s diverticulum; n, notochord; ps, pigment spot. Scale bars 20 mm in (N–T), 25 mm in (B,D,L,M) and 50 mm in (A,C,E–I).
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S. Candiani et al. / Mechanisms of Development 116 (2002) 231–234
Bay, FL. Gametes from electrically stimulated adults were fertilised, and embryos and larvae were cultured at 238C in laboratory culture (Holland and Holland, 1993).
Pierce, J.M. Lawrence and R. Martinez Jr. (Tampa, FL), R. Cancedda (Genoa, Italy). This research was supported by University of Genova grants to S.C. and M.P.
2.2. Isolation of a AmphiBrn1/2/4 cDNA Using degenerate primers (5 0 -GACGGATCCAC(A/C/ G)AC(A/C/G)AT(A/C/T)TG(C/T)(A/C/G)GNTT(C/T)GA3 0 and 5 0 -GACGAATTCGTGNC(G/T)NC(G/T)(G/A)TT(G/A)TT(G/A)CA(G/A)AACC-3 0 , where N ¼ A/C/G/T), a 306 bp fragment was amplified from a cDNA library in Lambda Zap II made from 26-h B. floridae embryos (kindly provided by L. Holland). The PCR protocol was: 2 min, 948C; five cycles of 1 min, 948; 1 min; 458C; 1 min, 728C; 35 cycles of 1 min, 948C; 1 min, 558C; 1 min, 728C; one cycle of 7 min, 728C. The PCR product was cloned into pCR2.1 vector (InVitrogen), sequenced and used to screen a cDNA library at high stringency from 26-h embryos of B. floridae (Max-Planck-Institute, Berlin). Two cDNA clones of approximate sizes 2.5 and 2.6 kb (clone identification numbers MPIMGp53132O15 and MPMGp53139B17, respectively) hybridised strongly and when sequenced were found to be derived from the same gene. The longest AmphiBrn1/2/4 cDNA clone (39B17) (GenBank accession number: AY078995) was used for all experimental procedures. 2.3. Northern blot analysis Total RNAs (20 mg) from B. floridae adults prepared with Trizol (Gibco), were electrophoresed and blotted onto Hybond N membranes (Amersham Pharmacia). The blot was hybridised at high stringency with 32P-labelled AmphiBrn1/2/4 cDNA clone as probe. 2.4. Whole mount in situ hybridisation and histology Antisense digoxigenin (DIG)-labelled RNA probes were prepared against an AmphiBrn1/2/4 cDNA clone. In situ hybridisation and histological sections were according to Holland et al. (1996) and Holland and Holland (1996). Acknowledgements We thank L. Holland and N. Holland (La Jolla, CA), S.K.
References Bu¨ rglin, T., Ruvkun, G., 2001. Regulation of ectodermal and excretory function by the C. elegans POU homeobox gene ceh-6. Development 128, 779–790. Chavez, M., Landry, C., Loret, S., Muller, M., Figueroa, J., Peers, B., Rentier-Delrue, F., Rousseau, G.G., Krauskopf, M., Martial, J.A., 1999. APH-1, a POU homeobox gene expressed in the salt gland of the crustacean Artemia franciscana. Mech. Dev. 87, 207–212. Ferrier, D.E.K., Brooke, N.M., Panopoulou, G., Holland, P.W.H., 2001. The Mnx homeobox gene class defined by HB9, MNR2 and amphioxus AmphiMnx. Dev. Genes Evol. 211, 103–107. Fukuta, M., Matsuno, K., Hui, C., Nagata, T., Takiya, S., Xu, P., Ueno, K., Suzuki, Y., 1993. Molecular cloning of POU domain-containing factor involved in the regulation of the Bombyx Sericin-1 Gene. J. Biol. Chem. 268, 9471–9475. Hauptmann, G., Gerster, T., 2000. Combinatorial expression of zebrafish Brn-1- and Brn-2-related POU genes in the embryonic brain, pronephric primordium, and pharyngeal arches. Dev. Dyn. 218, 345–358. Holland, L.Z., Holland, P.W.H., Holland, N.D., 1996. Revealing homologies between body parts of distantly related animals by in situ hybridisation to developmental genes: amphioxus versus vertebrates. In: Ferraris, J.D., Palumbi, S.R. (Eds.). Molecular Zoology: Advances, Strategies, and Protocols, Wiley-Liss, New York, NY, pp. 267– 282. Holland, N.D., Holland, L.Z., 1996. Embryos and larvae of invertebrate deuterostomes. In: Stern, C.D., Holland, P.W.H. (Eds.). Essential Developmental Biology: a Practical Approach, IRL Press, Oxford, pp. 21–32. Jackman, W.R., Langeland, J.A., Kimmel, C.B., 2000. Islet reveals segmentation in the amphioxus hindbrain homolog. Dev. Biol. 220, 16–26. Lacalli, T.C., 1996. Frontal eye circuitry, rostral sensory pathways and brain organization in amphioxus larvae: evidence from 3D reconstructions. Philos. Trans. R. Soc. Lond. B Biol. Sci. 351, 243–263. Ryan, A.K., Rosenfeld, M.G., 1997. POU domain family values: flexibility, partnerships, and developmental codes. Genes Dev. 11, 1207– 1225. Schonemann, M.D., Ryan, A.K., McEvilly, R.J., O’Connell, S.M., Arias, C.A., Kalla, K.A., Li, P., Sawchenko, P.E., Rosenfeld, M.G., 1995. Development and survival of the endocrine hypothalamus and posterior pituitary gland requires the neuronal POU domain factor Brn-2. Genes Dev. 9, 3122–3135. Williams, N.A., Holland, P.W.H., 1996. Old head on young shoulders. Nature 383, 490.
Gene expression pattern
Cloning and developmental expression of AmphiBrn1/2/4, a POU III gene in amphioxus Simona Candiani a, Patrizio Castagnola b, Diana Oliveri a, Mario Pestarino a,* a
Dipartimento di Biologia Sperimentale, Ambientale e Applicata, Universita` di Genova, viale Benedetto XV 5, 16132 Genova, Italy b Istituto Nazionale per la Ricerca sul Cancro, largo R. Benzi 10, 16132 Genova, Italy Received 27 March 2002; received in revised form 1 May 2002; accepted 1 May 2002
Abstract The large family encoding POU transcription factors has been described in several species. In particular, class III POU genes regulate critical steps of vertebrate and invertebrate neurogenesis. A novel amphioxus class III POU gene, AmphiBrn1/2/4, has been isolated and its spatio-temporal expression has been reported. AmphiBrn1/2/4 is first expressed in the dorsal epiblast, then throughout the neural plate except for a gap at level of the anterior region of the cerebral vesicle. Transcripts are also detected in the primordium of gill slits, pharynx and left Hatschek’s diverticulum. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Homeodomain; POU-III class; AmphiBrn1/2/4; Developmental expression; Neural tube; Endodermic structures; Amphioxus; Branchiostoma floridae
1. Results and discussion POU proteins act as transcription factors during development, and in particular, those of class III play fundamental role in neural development (reviewed in Ryan and Rosenfeld, 1997; Schonemann et al., 1995). In invertebrates, POU III proteins are expressed both in nervous and non-nervous structures (Chavez et al., 1999; Fukuta et al., 1993; Bu¨rglin and Ruvkun, 2001). The only evidence of class III POU proteins in vertebrate non-nervous structures was demonstrated in zebrafish (Hauptmann and Gerster, 2000). To analyse POU genes potentially involved in the control of amphioxus neurogenesis, we have cloned a homologue of class III POU genes, AmphiBrn1/2/4, in Branchiostoma floridae. A fragment of AmphiBrn1/2/4 was amplified by polymerase chain reaction (PCR) and was used to isolate a cDNA clone from 26-h embryos cDNA library. Sequence analysis revealed an open reading frame with high sequence similarity to the known class III POU genes. At the same time, Northern blot analysis performed with total RNA from amphioxus adult identified a single major band of 2.7 kb (Fig. 1C), and in accordance with the data from the corresponding vertebrate genes, AmphiBrn1/2/4 is not only present during development but also in adults. The predicted * Corresponding author. Tel.: 139-010-35-38043; fax: 139-010-3538047. E-mail address: [email protected] (M. Pestarino).
AmphiBrn1/2/4 protein is 410 amino acids long including a POU domain (Fig. 1A) and is .90% identical to those of vertebrate class III POU genes. AmphiBrn1/2/4 POU domain is highly related to that of rat-Brn1, rat-Brn2, ratBrn4, Xl-POU3B, APH1 and Dm-cf1a (94, 95, 92, 95, 93 and 94% amino acid identity, respectively) (Fig. 1B). Spatio-temporal distribution of AmphiBrn1/2/4 was analysed by whole mount in situ hybridisation. Firstly, AmphiBrn1/2/4 is detected in dorsal epiblast at the midgastrula stage (Fig. 2A,B). At the onset of neurulation, transcripts become strong posteriorly and span to the anterior region of the neural plate (Fig. 2C,D). Soon after hatching, AmphiBrn1/2/4 expression is strong at level of two transverse patches located in the most anterior region of the neural plate, separated by a gap of expression (Fig. 2E,F). This expression is similar to that shown in the cerebral vesicle by AmphiOtx (Williams and Holland, 1996). In late neurula, AmphiBrn1/2/4 is restricted to the roof of the anterior region of the cerebral vesicle, whereas it is absent in the respective floor (Fig. 2G). Two large nerve cell bodies expressing AmphiBrn1/2/4 are visible just posterior to the cerebral vesicle (Fig. 2I), and belong to the primary motor centre (PMC) (Lacalli, 1996). In late neurula, left Hatschek’s diverticulum, anterodorsal pharyngeal endoderm and a small group of ventral endoderm cells which become the first gill slit, are labelled (Fig. 2H,L,M). Later on, expression is still detected in endoderm (Fig. 2N,O,P,R)
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00146-6
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S. Candiani et al. / Mechanisms of Development 116 (2002) 231–234
Fig. 1. (A) The deduced amino acid sequence of the AmphiBrn1/2/4 is shown. The conserved POU specific domain is underlined and the POU homeodomain is indicated by a dotted line. (B) Sequence alignment of POU domains of AmphiBrn1/2/4 and a selection of vertebrate and invertebrate class III POU proteins. The black background indicates full conserved residues in all sequences. (C) Northern blot analysis performed with total RNA from adult of B. floridae. A single band of 2.7 kb is indicated by an arrow. The position of RNA ladders is shown on the left.
as well as in dorsal and ventral neurons (Fig. 2N,O,P), probably corresponding to the ventral and dorsal motor neurons expressing amphioxus islet (Jackman et al., 2000) and AmphiMnx (Ferrier et al., 2001), respectively. From 15day old larva until metamorphosis, AmphiBrn1/2/4 mRNA occurs exclusively in the neural tube (Fig. 2S,T). In conclusion, AmphiBrn1/2/4 is a further addition to the neural marker genes illustrating homologies between amphioxus and vertebrate neural tubes, particularly in the most anterior region. Furthermore, AmphiBrn1/2/4 expres-
sion pattern shows interesting similarities to the expression of some motor neuron markers, such as amphioxus islet and AmphiMnx.
2. Materials and methods 2.1. Obtaining and culturing embryos Adults of amphioxus B. floridae were collected in Tampa
S. Candiani et al. / Mechanisms of Development 116 (2002) 231–234
233
Fig. 2. Expression of AmphiBrn1/2/4 in amphioxus embryos and larvae as analysed by in situ hybridisation. Anterior is to left and dorsal is up in all whole mounts except for (I). Cross-sections (counterstained pink) are viewed from the anterior end of animal. (A) Lateral view and (B) cross-section of gastrula. The blastopore opens to the right. Staining is at level of the dorsal epiblast (presumptive neuroectoderm). (C) Side view of early neurula with strong posterior expression that spans anteriorly in the neural plate. (D) Cross-section through level d–d 0 in (C) showing AmphiBrn1/2/4 transcripts in the neural plate but not in the overgrowing dorsal epidermis (arrows). (E) Side view of neurula (somites are out of focus). The most anterior part of the neural plate is strongly labelled, but a gap of expression (arrow) is visible just behind it, with its posterior boundary corresponding to the anterior limit of the first somite. A faint signal is visible at level of a part of the anterior endoderm (arrowheads). (F) Same neurula in (E) viewed slightly rotated to the left side along the anterior–posterior axis. The gap of expression (arrow) shown in (E) is now better visible limited by two strong labelled patches. An irregular segmental arrangement is visible from the first somite to the posterior part of the neural plate. (G, H) Late neurula in lateral view. Neural expression is strongest in the ventral part of the posterior cerebral vesicle where the infundibular organ is located. In contrast, the anterior cerebral vesicle shows a weak expression only in the roof of the most rostral part, then a gap of expression is followed by reappearance of AmphiBrn1/2/4 in spinal cord. In (H) transcripts are present in the dorsal pharynx endoderm (arrowhead) and the rudiment of the primary gill slit (arrow). (I) Dorsal view of late neurula showing two labelled large cells bodies (arrows) corresponding to the ‘giant cells’ of the PMC. (L) Cross-section through l–l 0 in (H) showing intense expression in the ventral infundibular cells and the left Hatschek’s diverticulum. (M) Crosssection through m–m 0 in (H), strong expression is located in the neural tube and the rudiment of first gill slit (arrow), as well as at level of some cells of the dorsal pharynx endoderm (arrowheads). (N) Side view of the anterior half of 30-h larva with rudiments of first and second gill slits; the expression pattern is identical to that shown in late neurula, but additional expression is also visible at level of the rudiment of second gill slit (tandem arrows). (O) Cross-section through level o–o 0 in (N), AmphiBrn1/2/4 is detectable in ventral cells of the infundibular organ and in the left Hatschek’s diverticulum, now fused with ectoderm to form the preoral ciliated pit (arrow). (P) Cross-section through level p–p 0 in (N), transcripts are found in the opening first gill slit (arrow). (Q) Side view of the anterior end of 5-day larva showing transcripts in some cells behind the frontal eye (arrowhead) and in the preoral pit (arrow). (R) Anterior third of 7-day larva, expression is visible in the endostyle (arrow) and preoral pit (tandem arrows), whereas the neural expression appears down regulated along the anterior–posterior axis in the neural tube, even if strong labelling persists in the infundibular organ (arrowhead). (S) AmphiBrn1/2/4 occurs exclusively in the neural tube of 15-day larva (side view). (T) Cross-section through level t–t 0 in (S) showing a labelled ventrolateral neuron. Abbreviations: b, blastopore; cv, cerebral vesicle; ld, left Hatschek’s diverticulum; n, notochord; ps, pigment spot. Scale bars 20 mm in (N–T), 25 mm in (B,D,L,M) and 50 mm in (A,C,E–I).
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S. Candiani et al. / Mechanisms of Development 116 (2002) 231–234
Bay, FL. Gametes from electrically stimulated adults were fertilised, and embryos and larvae were cultured at 238C in laboratory culture (Holland and Holland, 1993).
Pierce, J.M. Lawrence and R. Martinez Jr. (Tampa, FL), R. Cancedda (Genoa, Italy). This research was supported by University of Genova grants to S.C. and M.P.
2.2. Isolation of a AmphiBrn1/2/4 cDNA Using degenerate primers (5 0 -GACGGATCCAC(A/C/ G)AC(A/C/G)AT(A/C/T)TG(C/T)(A/C/G)GNTT(C/T)GA3 0 and 5 0 -GACGAATTCGTGNC(G/T)NC(G/T)(G/A)TT(G/A)TT(G/A)CA(G/A)AACC-3 0 , where N ¼ A/C/G/T), a 306 bp fragment was amplified from a cDNA library in Lambda Zap II made from 26-h B. floridae embryos (kindly provided by L. Holland). The PCR protocol was: 2 min, 948C; five cycles of 1 min, 948; 1 min; 458C; 1 min, 728C; 35 cycles of 1 min, 948C; 1 min, 558C; 1 min, 728C; one cycle of 7 min, 728C. The PCR product was cloned into pCR2.1 vector (InVitrogen), sequenced and used to screen a cDNA library at high stringency from 26-h embryos of B. floridae (Max-Planck-Institute, Berlin). Two cDNA clones of approximate sizes 2.5 and 2.6 kb (clone identification numbers MPIMGp53132O15 and MPMGp53139B17, respectively) hybridised strongly and when sequenced were found to be derived from the same gene. The longest AmphiBrn1/2/4 cDNA clone (39B17) (GenBank accession number: AY078995) was used for all experimental procedures. 2.3. Northern blot analysis Total RNAs (20 mg) from B. floridae adults prepared with Trizol (Gibco), were electrophoresed and blotted onto Hybond N membranes (Amersham Pharmacia). The blot was hybridised at high stringency with 32P-labelled AmphiBrn1/2/4 cDNA clone as probe. 2.4. Whole mount in situ hybridisation and histology Antisense digoxigenin (DIG)-labelled RNA probes were prepared against an AmphiBrn1/2/4 cDNA clone. In situ hybridisation and histological sections were according to Holland et al. (1996) and Holland and Holland (1996). Acknowledgements We thank L. Holland and N. Holland (La Jolla, CA), S.K.
References Bu¨ rglin, T., Ruvkun, G., 2001. Regulation of ectodermal and excretory function by the C. elegans POU homeobox gene ceh-6. Development 128, 779–790. Chavez, M., Landry, C., Loret, S., Muller, M., Figueroa, J., Peers, B., Rentier-Delrue, F., Rousseau, G.G., Krauskopf, M., Martial, J.A., 1999. APH-1, a POU homeobox gene expressed in the salt gland of the crustacean Artemia franciscana. Mech. Dev. 87, 207–212. Ferrier, D.E.K., Brooke, N.M., Panopoulou, G., Holland, P.W.H., 2001. The Mnx homeobox gene class defined by HB9, MNR2 and amphioxus AmphiMnx. Dev. Genes Evol. 211, 103–107. Fukuta, M., Matsuno, K., Hui, C., Nagata, T., Takiya, S., Xu, P., Ueno, K., Suzuki, Y., 1993. Molecular cloning of POU domain-containing factor involved in the regulation of the Bombyx Sericin-1 Gene. J. Biol. Chem. 268, 9471–9475. Hauptmann, G., Gerster, T., 2000. Combinatorial expression of zebrafish Brn-1- and Brn-2-related POU genes in the embryonic brain, pronephric primordium, and pharyngeal arches. Dev. Dyn. 218, 345–358. Holland, L.Z., Holland, P.W.H., Holland, N.D., 1996. Revealing homologies between body parts of distantly related animals by in situ hybridisation to developmental genes: amphioxus versus vertebrates. In: Ferraris, J.D., Palumbi, S.R. (Eds.). Molecular Zoology: Advances, Strategies, and Protocols, Wiley-Liss, New York, NY, pp. 267– 282. Holland, N.D., Holland, L.Z., 1996. Embryos and larvae of invertebrate deuterostomes. In: Stern, C.D., Holland, P.W.H. (Eds.). Essential Developmental Biology: a Practical Approach, IRL Press, Oxford, pp. 21–32. Jackman, W.R., Langeland, J.A., Kimmel, C.B., 2000. Islet reveals segmentation in the amphioxus hindbrain homolog. Dev. Biol. 220, 16–26. Lacalli, T.C., 1996. Frontal eye circuitry, rostral sensory pathways and brain organization in amphioxus larvae: evidence from 3D reconstructions. Philos. Trans. R. Soc. Lond. B Biol. Sci. 351, 243–263. Ryan, A.K., Rosenfeld, M.G., 1997. POU domain family values: flexibility, partnerships, and developmental codes. Genes Dev. 11, 1207– 1225. Schonemann, M.D., Ryan, A.K., McEvilly, R.J., O’Connell, S.M., Arias, C.A., Kalla, K.A., Li, P., Sawchenko, P.E., Rosenfeld, M.G., 1995. Development and survival of the endocrine hypothalamus and posterior pituitary gland requires the neuronal POU domain factor Brn-2. Genes Dev. 9, 3122–3135. Williams, N.A., Holland, P.W.H., 1996. Old head on young shoulders. Nature 383, 490.