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forkhead transcription factor FoxF1
Gene Expression Patterns 5 (2004) 29–34 www.elsevier.com/locate/modgep
Sequence and expression of the rainbow trout winged helix/forkhead transcription factor FoxF1 Yoshie Hidaka, Shigeyasu Tanaka, Masakazu Suzuki* Department of Biology, Faculty of Science, Shizuoka University, Ohya 836, Shizuoka, Shizuoka 422-8529, Japan Received 16 March 2004; received in revised form 24 June 2004; accepted 25 June 2004 Available online 5 August 2004
Abstract FoxF1 is a member of the winged helix/forkhead transcription factor gene family. We have cloned the cDNA encoding a rainbow trout FoxF1 homologue, and examined its developmental gene expression pattern. By 7 days postfertilization (dpf at 14 8C), FoxF1 is expressed throughout the alimentary tract in the mesenchymal cells adjacent to the endodermal epithelium, with intense signals on the dorsal side of the oral cavity and in the primitive stomach. As ontogeny proceeds, expression is down-regulated in the oral cavity and esophagus, but persists in the pharynx, stomach, and intestine. Hybridization signals are also detected in the developing liver, and in the mesenchyme layer around the notochord. From 18 dpf onwards, dramatic changes occur in gene expression in the branchial region. As the gill filaments elongate from the branchial arches, FoxF1 begins to be expressed along the central cell cord running through each gill filament, and then switches over its rodlike expression to a repetitive pattern, alternating on either side along the proximal part of the filament. A signal is further localized to the primitive pillar cells as they form gill lamellae. In addition to illustrating the conserved FoxF1 expression pattern in the developing digestive tract and liver, the results indicate a close association of FoxF1 with the formation of the fish gills. q 2004 Elsevier B.V. All rights reserved. Keywords: FoxF1; cDNA cloning; Development; Gills; Alimentary tract; Notochord; Bile duct; Hepatic stellate cells; Muscle layer; Mesenchyme; Rainbow trout; Fish; Gill filaments; Gill lamellae; Pillar cells; Capillary; Whole mount in situ hybridization; Alternate expression pattern
1. Results and discussion In mammals, transcription factors of the winged helix/forkhead (Fox) family are involved in many aspects of embryonic development and in the maintenance of the differentiated cell states of adult tissues (Carlsson and Mahlapuu, 2002; Kaufmann and Knochel, 1996; Lehmann et al., 2003). During embryonic development, FoxF1 (HFH8/FREAC-1) is expressed mostly in the mesenchyme of various organs, such as the digestive, respiratory, and urinary tracts (Clevidence et al., 1994; Kalinichenko et al., 2003a; Mahlapuu et al., 1998; Ormestad et al., 2004; Peterson et al., 1997). Foxf1 null embryos die at midgestation, showing defects in mesodermal differentiation and cell adhesion (Mahlapuu et al., 2001a), while haploinsufficiency
* Corresponding author. Tel.: C81-54-238-4769; fax: C81-54-2380986. E-mail address: [email protected] (M. Suzuki). 1567-133X/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2004.06.012
causes malformations of the foregut and lung (Kalinichenko et al., 2001; Mahlapuu et al., 2001b), and defects in the gall bladder (Kalinichenko et al., 2002). Because FoxF (biniou) is found in Drosophila (Zaffran et al., 2001), one can expect the FoxF1 gene to be conserved in the lower classes of vertebrates, such as amphibians and fish, which possess gills as respiratory organs. Accordingly, Xenopus FoxF1 (XFD13) gene has been isolated and shown to be expressed in head-derived neural crest cells, the dorsolateral plate, and other tissues during embryogenesis (Koster et al., 1999). However, the FoxF1 expression has not been fully characterized in the alimentary tract and its accessory organs at later stages. By screening for winged helix family members expressed in the ultimobranchial gland of rainbow trout (Oncorhynchus mykiss), we have isolated the rainbow trout orthologue of FoxF1 (Figs. 1,2). The FoxF1 sequence contains an open reading frame of 2416 nucleotides, encoding a 375 amino acid protein (Fig. 1). The predicted amino acid sequence of this protein has been highly
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Y. Hidaka et al. / Gene Expression Patterns 5 (2004) 29–34
Fig. 1. Nucleotide and amino acid sequences of the trout FoxF1 (DDBJ accession number AB164480). The winged helix domain and the polyadenylation signal are double-underlined and underlined, respectively.
conserved in the winged helix DNA binding domain (Fig. 2A). By molecular dissection analysis of human FOXF1, a lung-cell-specific activation domain and a general activation domain have been identified in an internal portion of 35 amino acid residues located downstream of the winged helix domain (Mahlapuu et al., 1998), and in the final 28 carboxy (C)-terminal amino acids (Hellqvist et al., 1996), respectively (Fig. 2A). These domains also show high similarity to the corresponding regions of the trout FoxF1: i.e. 74% identity in the former domain, and 72% identity in the latter domain. Temporal and spatial expression of FoxF1 during trout development was investigated using whole mount in situ hybridization histochemistry. At 7 days postfertilization (dpf at 14 8C), expression was already seen all along the alimentary tract, with prominent signals in the dorsal area of the oral cavity and in the primitive stomach (Fig. 3A). Throughout the alimentary tract, hybridization signals were detected in the mesenchyme adjacent to the endodermal epithelium, whereas the epithelium itself was not stained (Fig. 3B). FoxF1 expression was maintained from the oral cavity through the stomach to the anal canal, up until 12 dpf (Fig. 3C,D,G,I). In the pharynx, transcripts were restricted to the central part of the mesenchyme, next to the inner
epithelium (Fig. 3H), and hybridization signals were gradually intensified nearest to the epithelium (Fig. 3C,H). From 15 dpf onwards, expression appeared weaker in the oral cavity and esophagus, but remained strong in the pharynx, stomach, and intestine (Fig. 3J–N,P–R). In the stomach and intestine, expression was confined to the muscle layer (Fig. 3Q,R). FoxF1 was further observed in the developing liver at 18 dpf (Fig. 3K), and hybridization signals were localized to the bile duct mesenchyme (Fig. 3S) at 24 dpf (3 days after hatching). It also appeared that hepatic stellate cells had positive signals (Fig. 3T), as in the mouse liver (Kalinichenko et al., 2003b). In the murine embryo, FoxF1 is detected in the mesenchymal cells of various organs, such as the digestive tract, liver, lung, and kidney (Mahlapuu et al., 1998; Kalinichenko et al., 2003a). Therefore, the above results indicate that FoxF1 expression is basically conserved in many of the embryonic organs common to mammals and fish. Additionally, transcripts were seen in the mesenchyme around the notochord at 9 dpf (Fig. 3E) and at 12 dpf (Fig. 3F). On the other hand, a unique expression pattern occurred in the developing gills. At 15 dpf, FoxF1 were detected in the mesenchymal cells between the endodermal epithelium and differentiating gill cartilage (Fig. 4A,B), and continued
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Fig. 2. Characterization and classification of rainbow trout FoxF1. (A) Comparison of amino acid sequences between rainbow trout FoxF1 (AB164480) and human FOXF1 (AF085343). These sequences have been aligned using Clustal W (Thompson et al., 1994). The winged helix DNA binding domain of the trout FoxF1 is nearly identical with that of human FOXF1. The lung-cell-specific activation domain and the general activation domain of human FOXF1 are underlined in green and red, respectively. (B) Phylogenetic tree of vertebrate Fox proteins, showing the relationships between rainbow trout FoxF1, Xenopus FoxF1 (AJ242680), mouse Foxf1 (U42556), human FOXF1, mouse Foxf2 (Y12293), human FOXF2 (AF084939), and zebrafish FoxA1 (AF052250), FoxB1 (AF052246), and FoxG1 (AF067204). This tree has been generated by the neighbour joining method (Saitou and Nei, 1987). The length of each branch is proportional to the mean number of differences per residue. The topology shows that the trout Fox protein, encoded by the isolated cDNA clone, belongs to the FoxF1 subfamily. As compared over the full length, the trout FoxF1 has 73–77% amino acid identity to Xenopus and mammalian counterparts.
to be expressed thereafter (Fig. 4C–E). In addition, expression began in the emerging gill filaments of 18 dpf embryos (Fig. 4C). As the primitive gill filaments elongated, hybridization signals became intensified and distributed along each filament with the highest levels at its distal tip (Fig. 4D,E). As the gill filaments developed further, FoxF1 changed its expression pattern within the proximal part of the filaments, and occurred alternately on either side of each filament (Fig. 4D,E). Because these characteristics of FoxF1 expression were evident in the gills at 24 dpf (Fig. 4E,F), the alevins of this stage were sectioned for detailed observation. Interestingly, the repetitive alternating expression pattern was already initiated just behind the leading edge of each gill filament, and FoxF1 was expressed in the primitive pillar cells
growing from the central cell cord within the gill filament, which are fated to form gill lamellae with the epithelial cells (Fig. 4G). The gill lamellae are the most important respiratory units of the gill system (Wilson and Laurent, 2002), and are considered to be involved in the selective conversion and production of hormonal signals (Olson, 1998). Within each lamella, the pillar cells—modified endothelial cells unique to fish gills—construct wellorganized capillary networks that are essential for efficient gas exchange (Laurent, 1984). Although, the gills serve important physiological functions, little is known about the molecular mechanisms underlying their organogenesis. To our knowledge, this is the first report to reveal the transcription factor closely associated with the development of gill filaments and gill lamellae.
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Fig. 3. Expression of FoxF1 in the trout embryos and alevins, by whole mount in situ hybridization. (A,D) Lateral views of 7 dpf (A) and 9 dpf embryos (D) show intense signals throughout the alimentary tract. (B,C) Parasagittal sections through the branchial region of 7 dpf (B) and 9 dpf embryos (C) reveal signals in the mesenchymal cells. ep, endodermal epithelium. (E,F) In transverse sections, signals are observed in the mesenchymal cells around the notochord at 9 dpf (E) and at 12 dpf (F). (G) Lateral view of 12 dpf embryo indicates intense signals along the alimentary tract. (H) Coronal section through the branchial region of a 12 dpf embryo reveals signals in the mesenchymal cells adjacent to the inner epithelium. (I) In a transverse section of a 12 dpf embryo, signals are seen in the mesenchymal cells surrounding the endodermal epithelium. (J,K,L) Lateral views of 15 dpf (J), 18 dpf (K), and 21 dpf embryos (L) show strong expression in the branchial arches (arrow) and stomach (arrowhead). (M,N) Ventral views of 21 dpf (hatching) (M) and 24 dpf (3 days after hatching) alevins (N) show strong expression in the gills (arrows) and stomach (arrowhead). (O) Control alevin hybridized with the sense probe exhibits no signal. (P–T) Parasagittal section of 24 dpf alevin shows that signals in the mesenchymal cells fade away from the stomach towards the esophagus (es) (P). Strong signals are localized to the muscle cells of the stomach (arrows) (Q) and intestine (arrows) (R), to the mesenchymal cells around the bile ducts (arrows) (S), and to presumed hepatic stellate cells (arrows) (T). (A–D,G,H,J–T) Anterior is left. (A–G,I–L,P–T) Dorsal is up. (H) Central is up. bd, bile duct; br, branchial region; ct, connective tissue; ep, endodermal epithelium; he, heart; li, liver; nc, notochord; oc, oral cavity; pf, pectoral fin; ph, pharynx; st, stomach. The scale bar to (A,D,G,J–O)Z 500 mm; the bar to (B,C,E,F,I,P–T)Z10 mm; the bar to (H)Z50 mm.
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Fig. 4. Expression of FoxF1 in the developing gill of rainbow trout, by whole mount in situ hybridization. (A) The gill, dissected out of the hybridized 15 dpf embryo, shows strong signals alongside the inner edge (arrows). (B) Parasagittal section of the branchial region of a 15 dpf embryo reveals signals in the mesenchymal cells (arrows) between the endodermal epithelium (ep) and the differentiating cartilage (ca). (C–E) The gills of 18 dpf embryo (C), and 21 dpf (D) and 24 dpf (E) alevins, show additional expression in the gill filaments (arrowheads). (F) Magnified view of the gill of 24 dpf alevin clearly exhibits transcripts at the leading edge (double arrowheads) of each gill filament, followed by a repetitive alternating expression pattern (arrowheads). (G) Parasagittal section of the gill of 24 dpf embryo localizes signals to the most distal cells (double arrowheads) of the central cell cord (cc) within the gill filament, and to the primitive pillar cells (arrowheads) growing outwards and obliquely to the axis of the filament. Weak expression initiates near the leading edge of the gill filament (white arrowheads), and then the signal intensity increases as the pillar cells develop. Bulges (white arrows) arise from the proximal part of the filament, leading to the formation of the gill lamellae. Anterior is left; dorsal is up. ba, branchial arch. The scale bar to (A,C,D,E)Z100 mm; the bar to (B,G)Z 10 mm; the bar to (F)Z50 mm.
2. Materials and methods Approximately 40,000 plaques from an ultimobranchial gland cDNA library of rainbow trout were screened using the forkhead domain of rainbow trout FoxN4. Embryos and alevins were reared at 14 8C. Developmental stages specified by dpf apparently correspond to those by Vernier (1836) in the following manner: 7 dpf (Pulsations of the heart begin. The otic vesicles are already present), stage 20;
9 dpf (Pigment formation begins on the eyes. Five pairs of primitive branchial arches are formed), stage 21; 12 dpf (five pairs of aortic arches are present), stage 24; 15 dpf (The head bends ventralwards. Cartilagenous rays are present in both the dorsal and anal fins), stage 27; 18 dpf (Cartilagenous rays are also evident in the caudal fin. Primitive gill filaments begin to grow), stage 29; 21 dpf (the alevins hatch), stage 30; and 24 dpf, stage 31, although several morphological features are different between these
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two series of stages. In situ hybridization for trout embryos and alevins was performed as described previously (Hidaka et al., 2004). Antisense digoxigenin probe was prepared using the Sac II–Sal I region (at nucleotides 436–1492 in Fig. 1) of the trout FoxF1 cDNA.
Acknowledgements We are grateful to the staff of Fuji Trout Hatchery and the Fuji Branch of Shizuoka Prefectural Fisheries Experimental Station for providing rainbow trout. We express our sincere gratitude to Prof. S. Hyodo, University of Tokyo, Japan, for his assistance, and to Dr B.I. Baker for critical reading of the manuscript. This work was supported by Grants-in Aid from the Ministry of Education, Science, Sports, and Culture of Japan, and in part by funds from the cooperative program (No. 9, 2003) provided by Ocean Research Institute, University of Tokyo.
References Carlsson, P., Mahlapuu, M., 2002. Forkhead transcription factors: key players in development and metabolism. Dev. Biol. 250, 1–23. Clevidence, D.E., Overdier, D.G., Peterson, R.S., Porcella, A., Ye, H., Paulson, E.K., Costa, R.H., 1994. Members of the HNF-3/fork head family of transcription factors exhibit distinct cellular expression patterns in lung and regulate the surfactant protein B promoter. Dev. Biol. 166, 195–209. Hellqvist, M., Mahlapuu, M., Samuelsson, L., Enerback, S., Carlsson, P., 1996. Differential activation of lung-specific genes by two forkhead proteins, FREAC-1 and FREAC-2. J. Biol. Chem. 271, 4482–4490. Hidaka, Y., Tanaka, S., Suzuki, M., 2004. Analysis of salmon calcitonin I in the ultimobranchial gland and gill filaments during development of rainbow trout, Oncorhynchus mykiss, by in situ hybridization and immunohistochemical staining. Zool. Sci. 21, 629–637. Kalinichenko, V.V., Lim, L., Beer-Stolts, D., Shin, B., Rausa, F.M., Clark, J., et al., 2001. Defects in pulmonary vasculature and perinatal lung hemorrhage in mice heterozygous null for the forkhead box f1 transcription factor. Dev. Biol. 235, 489–506. Kalinichenko, V.V., Zhou, Y., Bhattacharyya, D., Kim, W., Shin, B., Bambal, K., Costa, R.H., 2002. Haploinsufficiency of the mouse Forkhead Box f1 gene causes defects in gall bladder development. J. Biol. Chem. 277, 12369–12374. Kalinichenko, V.V., Gusarova, G.A., Shin, B., Bambal, K., Costa, R.H., 2003a. The forkhead box F1 transcription factor is expressed in brain
and head mesenchyme during mouse embryonic development. Gene Exp. Patterns 3, 153–158. Kalinichenko, V.V., Bhattacharyya, D., Zhou, Y., Gusarova, G.A., Kim, W., Shin, B., Costa, R.H., 2003b. Foxf1 C/K mice exhibit defective stellate cell activation and abnormal liver regeneration following CCl4 injury. Hepatology 37, 107–117. Kaufmann, E., Knochel, W., 1996. Five years on the wings of fork head. Mech. Dev. 57, 3–20. Koster, M., Dillinger, K., Knochel, W., 1999. Genomic structure and embryonic expression of the Xenopus winged helix factors XFD-13/13 0 . Mech. Dev. 88, 89–93. Laurent, P., 1984. Gill internal morphology, in: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology, vol. XA. Academic Press Inc, San Diego, pp. 73–183. Lehmann, O.J., Sowden, J.C., Carlsson, P., Jordan, T., Battacharya, S.S., 2003. Fox’s in development and disease. Trends Genet. 19, 339–344. Mahlapuu, M., Pelto-Huikko, M., Aitola, M., Enerback, S., Carlsson, P., 1998. FREAC-1 contains a cell-type-specific transcriptional activation domain and is expressed in epithelial–mesenchymal interfaces. Dev. Biol. 202, 183–195. Mahlapuu, M., Ormestad, M., Enerback, S., Carlsson, P., 2001a. The forkhead transcription factor Foxf1 is required for differentiation of extraembryonic and lateral plate mesoderm. Development 128, 155– 166. Mahlapuu, M., Enerback, S., Carlsson, P., 2001b. Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signaling, causes lung and foregut malformation. Development 128, 2397–2406. Olson, K.R., 1998. Hormone metabolism by the fish gill. Comp. Biochem. Physiol. 119A, 55–65. Ormestad, M., Astorga, J., Carlsson, P., 2004. Differences in the embryonic expression patterns of mouse Foxf1 and -2 match their distinct mutant phenotype. Dev. Dyn. 229, 328–333. Peterson, R., Lim, L., Ye, H., Zhou, H., Overdier, D., Costa, R.H., 1997. The winged helix transcriptional activator HFH-8 is expressed in the mesoderm of the primitive streak stage of mouse embryos and its cellular derivatives. Mech. Dev. 69, 53–69. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Vernier, J.-M., 1836. Table chronologique du developpement embryonnaire de la truite arc-en-ciel, Salmo gairdneri Rich. Annal. Embryol. Morph. 2, 495–520. Wilson, J.M., Laurent, P., 2002. Fish gill morphology: inside out. J. Exp. Zool. 293, 192–213. Zaffran, S., Kuchler, A., Lee, H.-H., Frasch, M., 2001. biniou (FoxF), a central component in a regulatory network controlling visceral mesoderm development and midgut morphogenesis in Drosophila. Genes Dev. 15, 2900–2915.
Sequence and expression of the rainbow trout winged helix/forkhead transcription factor FoxF1 Yoshie Hidaka, Shigeyasu Tanaka, Masakazu Suzuki* Department of Biology, Faculty of Science, Shizuoka University, Ohya 836, Shizuoka, Shizuoka 422-8529, Japan Received 16 March 2004; received in revised form 24 June 2004; accepted 25 June 2004 Available online 5 August 2004
Abstract FoxF1 is a member of the winged helix/forkhead transcription factor gene family. We have cloned the cDNA encoding a rainbow trout FoxF1 homologue, and examined its developmental gene expression pattern. By 7 days postfertilization (dpf at 14 8C), FoxF1 is expressed throughout the alimentary tract in the mesenchymal cells adjacent to the endodermal epithelium, with intense signals on the dorsal side of the oral cavity and in the primitive stomach. As ontogeny proceeds, expression is down-regulated in the oral cavity and esophagus, but persists in the pharynx, stomach, and intestine. Hybridization signals are also detected in the developing liver, and in the mesenchyme layer around the notochord. From 18 dpf onwards, dramatic changes occur in gene expression in the branchial region. As the gill filaments elongate from the branchial arches, FoxF1 begins to be expressed along the central cell cord running through each gill filament, and then switches over its rodlike expression to a repetitive pattern, alternating on either side along the proximal part of the filament. A signal is further localized to the primitive pillar cells as they form gill lamellae. In addition to illustrating the conserved FoxF1 expression pattern in the developing digestive tract and liver, the results indicate a close association of FoxF1 with the formation of the fish gills. q 2004 Elsevier B.V. All rights reserved. Keywords: FoxF1; cDNA cloning; Development; Gills; Alimentary tract; Notochord; Bile duct; Hepatic stellate cells; Muscle layer; Mesenchyme; Rainbow trout; Fish; Gill filaments; Gill lamellae; Pillar cells; Capillary; Whole mount in situ hybridization; Alternate expression pattern
1. Results and discussion In mammals, transcription factors of the winged helix/forkhead (Fox) family are involved in many aspects of embryonic development and in the maintenance of the differentiated cell states of adult tissues (Carlsson and Mahlapuu, 2002; Kaufmann and Knochel, 1996; Lehmann et al., 2003). During embryonic development, FoxF1 (HFH8/FREAC-1) is expressed mostly in the mesenchyme of various organs, such as the digestive, respiratory, and urinary tracts (Clevidence et al., 1994; Kalinichenko et al., 2003a; Mahlapuu et al., 1998; Ormestad et al., 2004; Peterson et al., 1997). Foxf1 null embryos die at midgestation, showing defects in mesodermal differentiation and cell adhesion (Mahlapuu et al., 2001a), while haploinsufficiency
* Corresponding author. Tel.: C81-54-238-4769; fax: C81-54-2380986. E-mail address: [email protected] (M. Suzuki). 1567-133X/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2004.06.012
causes malformations of the foregut and lung (Kalinichenko et al., 2001; Mahlapuu et al., 2001b), and defects in the gall bladder (Kalinichenko et al., 2002). Because FoxF (biniou) is found in Drosophila (Zaffran et al., 2001), one can expect the FoxF1 gene to be conserved in the lower classes of vertebrates, such as amphibians and fish, which possess gills as respiratory organs. Accordingly, Xenopus FoxF1 (XFD13) gene has been isolated and shown to be expressed in head-derived neural crest cells, the dorsolateral plate, and other tissues during embryogenesis (Koster et al., 1999). However, the FoxF1 expression has not been fully characterized in the alimentary tract and its accessory organs at later stages. By screening for winged helix family members expressed in the ultimobranchial gland of rainbow trout (Oncorhynchus mykiss), we have isolated the rainbow trout orthologue of FoxF1 (Figs. 1,2). The FoxF1 sequence contains an open reading frame of 2416 nucleotides, encoding a 375 amino acid protein (Fig. 1). The predicted amino acid sequence of this protein has been highly
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Fig. 1. Nucleotide and amino acid sequences of the trout FoxF1 (DDBJ accession number AB164480). The winged helix domain and the polyadenylation signal are double-underlined and underlined, respectively.
conserved in the winged helix DNA binding domain (Fig. 2A). By molecular dissection analysis of human FOXF1, a lung-cell-specific activation domain and a general activation domain have been identified in an internal portion of 35 amino acid residues located downstream of the winged helix domain (Mahlapuu et al., 1998), and in the final 28 carboxy (C)-terminal amino acids (Hellqvist et al., 1996), respectively (Fig. 2A). These domains also show high similarity to the corresponding regions of the trout FoxF1: i.e. 74% identity in the former domain, and 72% identity in the latter domain. Temporal and spatial expression of FoxF1 during trout development was investigated using whole mount in situ hybridization histochemistry. At 7 days postfertilization (dpf at 14 8C), expression was already seen all along the alimentary tract, with prominent signals in the dorsal area of the oral cavity and in the primitive stomach (Fig. 3A). Throughout the alimentary tract, hybridization signals were detected in the mesenchyme adjacent to the endodermal epithelium, whereas the epithelium itself was not stained (Fig. 3B). FoxF1 expression was maintained from the oral cavity through the stomach to the anal canal, up until 12 dpf (Fig. 3C,D,G,I). In the pharynx, transcripts were restricted to the central part of the mesenchyme, next to the inner
epithelium (Fig. 3H), and hybridization signals were gradually intensified nearest to the epithelium (Fig. 3C,H). From 15 dpf onwards, expression appeared weaker in the oral cavity and esophagus, but remained strong in the pharynx, stomach, and intestine (Fig. 3J–N,P–R). In the stomach and intestine, expression was confined to the muscle layer (Fig. 3Q,R). FoxF1 was further observed in the developing liver at 18 dpf (Fig. 3K), and hybridization signals were localized to the bile duct mesenchyme (Fig. 3S) at 24 dpf (3 days after hatching). It also appeared that hepatic stellate cells had positive signals (Fig. 3T), as in the mouse liver (Kalinichenko et al., 2003b). In the murine embryo, FoxF1 is detected in the mesenchymal cells of various organs, such as the digestive tract, liver, lung, and kidney (Mahlapuu et al., 1998; Kalinichenko et al., 2003a). Therefore, the above results indicate that FoxF1 expression is basically conserved in many of the embryonic organs common to mammals and fish. Additionally, transcripts were seen in the mesenchyme around the notochord at 9 dpf (Fig. 3E) and at 12 dpf (Fig. 3F). On the other hand, a unique expression pattern occurred in the developing gills. At 15 dpf, FoxF1 were detected in the mesenchymal cells between the endodermal epithelium and differentiating gill cartilage (Fig. 4A,B), and continued
Y. Hidaka et al. / Gene Expression Patterns 5 (2004) 29–34
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Fig. 2. Characterization and classification of rainbow trout FoxF1. (A) Comparison of amino acid sequences between rainbow trout FoxF1 (AB164480) and human FOXF1 (AF085343). These sequences have been aligned using Clustal W (Thompson et al., 1994). The winged helix DNA binding domain of the trout FoxF1 is nearly identical with that of human FOXF1. The lung-cell-specific activation domain and the general activation domain of human FOXF1 are underlined in green and red, respectively. (B) Phylogenetic tree of vertebrate Fox proteins, showing the relationships between rainbow trout FoxF1, Xenopus FoxF1 (AJ242680), mouse Foxf1 (U42556), human FOXF1, mouse Foxf2 (Y12293), human FOXF2 (AF084939), and zebrafish FoxA1 (AF052250), FoxB1 (AF052246), and FoxG1 (AF067204). This tree has been generated by the neighbour joining method (Saitou and Nei, 1987). The length of each branch is proportional to the mean number of differences per residue. The topology shows that the trout Fox protein, encoded by the isolated cDNA clone, belongs to the FoxF1 subfamily. As compared over the full length, the trout FoxF1 has 73–77% amino acid identity to Xenopus and mammalian counterparts.
to be expressed thereafter (Fig. 4C–E). In addition, expression began in the emerging gill filaments of 18 dpf embryos (Fig. 4C). As the primitive gill filaments elongated, hybridization signals became intensified and distributed along each filament with the highest levels at its distal tip (Fig. 4D,E). As the gill filaments developed further, FoxF1 changed its expression pattern within the proximal part of the filaments, and occurred alternately on either side of each filament (Fig. 4D,E). Because these characteristics of FoxF1 expression were evident in the gills at 24 dpf (Fig. 4E,F), the alevins of this stage were sectioned for detailed observation. Interestingly, the repetitive alternating expression pattern was already initiated just behind the leading edge of each gill filament, and FoxF1 was expressed in the primitive pillar cells
growing from the central cell cord within the gill filament, which are fated to form gill lamellae with the epithelial cells (Fig. 4G). The gill lamellae are the most important respiratory units of the gill system (Wilson and Laurent, 2002), and are considered to be involved in the selective conversion and production of hormonal signals (Olson, 1998). Within each lamella, the pillar cells—modified endothelial cells unique to fish gills—construct wellorganized capillary networks that are essential for efficient gas exchange (Laurent, 1984). Although, the gills serve important physiological functions, little is known about the molecular mechanisms underlying their organogenesis. To our knowledge, this is the first report to reveal the transcription factor closely associated with the development of gill filaments and gill lamellae.
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Fig. 3. Expression of FoxF1 in the trout embryos and alevins, by whole mount in situ hybridization. (A,D) Lateral views of 7 dpf (A) and 9 dpf embryos (D) show intense signals throughout the alimentary tract. (B,C) Parasagittal sections through the branchial region of 7 dpf (B) and 9 dpf embryos (C) reveal signals in the mesenchymal cells. ep, endodermal epithelium. (E,F) In transverse sections, signals are observed in the mesenchymal cells around the notochord at 9 dpf (E) and at 12 dpf (F). (G) Lateral view of 12 dpf embryo indicates intense signals along the alimentary tract. (H) Coronal section through the branchial region of a 12 dpf embryo reveals signals in the mesenchymal cells adjacent to the inner epithelium. (I) In a transverse section of a 12 dpf embryo, signals are seen in the mesenchymal cells surrounding the endodermal epithelium. (J,K,L) Lateral views of 15 dpf (J), 18 dpf (K), and 21 dpf embryos (L) show strong expression in the branchial arches (arrow) and stomach (arrowhead). (M,N) Ventral views of 21 dpf (hatching) (M) and 24 dpf (3 days after hatching) alevins (N) show strong expression in the gills (arrows) and stomach (arrowhead). (O) Control alevin hybridized with the sense probe exhibits no signal. (P–T) Parasagittal section of 24 dpf alevin shows that signals in the mesenchymal cells fade away from the stomach towards the esophagus (es) (P). Strong signals are localized to the muscle cells of the stomach (arrows) (Q) and intestine (arrows) (R), to the mesenchymal cells around the bile ducts (arrows) (S), and to presumed hepatic stellate cells (arrows) (T). (A–D,G,H,J–T) Anterior is left. (A–G,I–L,P–T) Dorsal is up. (H) Central is up. bd, bile duct; br, branchial region; ct, connective tissue; ep, endodermal epithelium; he, heart; li, liver; nc, notochord; oc, oral cavity; pf, pectoral fin; ph, pharynx; st, stomach. The scale bar to (A,D,G,J–O)Z 500 mm; the bar to (B,C,E,F,I,P–T)Z10 mm; the bar to (H)Z50 mm.
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Fig. 4. Expression of FoxF1 in the developing gill of rainbow trout, by whole mount in situ hybridization. (A) The gill, dissected out of the hybridized 15 dpf embryo, shows strong signals alongside the inner edge (arrows). (B) Parasagittal section of the branchial region of a 15 dpf embryo reveals signals in the mesenchymal cells (arrows) between the endodermal epithelium (ep) and the differentiating cartilage (ca). (C–E) The gills of 18 dpf embryo (C), and 21 dpf (D) and 24 dpf (E) alevins, show additional expression in the gill filaments (arrowheads). (F) Magnified view of the gill of 24 dpf alevin clearly exhibits transcripts at the leading edge (double arrowheads) of each gill filament, followed by a repetitive alternating expression pattern (arrowheads). (G) Parasagittal section of the gill of 24 dpf embryo localizes signals to the most distal cells (double arrowheads) of the central cell cord (cc) within the gill filament, and to the primitive pillar cells (arrowheads) growing outwards and obliquely to the axis of the filament. Weak expression initiates near the leading edge of the gill filament (white arrowheads), and then the signal intensity increases as the pillar cells develop. Bulges (white arrows) arise from the proximal part of the filament, leading to the formation of the gill lamellae. Anterior is left; dorsal is up. ba, branchial arch. The scale bar to (A,C,D,E)Z100 mm; the bar to (B,G)Z 10 mm; the bar to (F)Z50 mm.
2. Materials and methods Approximately 40,000 plaques from an ultimobranchial gland cDNA library of rainbow trout were screened using the forkhead domain of rainbow trout FoxN4. Embryos and alevins were reared at 14 8C. Developmental stages specified by dpf apparently correspond to those by Vernier (1836) in the following manner: 7 dpf (Pulsations of the heart begin. The otic vesicles are already present), stage 20;
9 dpf (Pigment formation begins on the eyes. Five pairs of primitive branchial arches are formed), stage 21; 12 dpf (five pairs of aortic arches are present), stage 24; 15 dpf (The head bends ventralwards. Cartilagenous rays are present in both the dorsal and anal fins), stage 27; 18 dpf (Cartilagenous rays are also evident in the caudal fin. Primitive gill filaments begin to grow), stage 29; 21 dpf (the alevins hatch), stage 30; and 24 dpf, stage 31, although several morphological features are different between these
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two series of stages. In situ hybridization for trout embryos and alevins was performed as described previously (Hidaka et al., 2004). Antisense digoxigenin probe was prepared using the Sac II–Sal I region (at nucleotides 436–1492 in Fig. 1) of the trout FoxF1 cDNA.
Acknowledgements We are grateful to the staff of Fuji Trout Hatchery and the Fuji Branch of Shizuoka Prefectural Fisheries Experimental Station for providing rainbow trout. We express our sincere gratitude to Prof. S. Hyodo, University of Tokyo, Japan, for his assistance, and to Dr B.I. Baker for critical reading of the manuscript. This work was supported by Grants-in Aid from the Ministry of Education, Science, Sports, and Culture of Japan, and in part by funds from the cooperative program (No. 9, 2003) provided by Ocean Research Institute, University of Tokyo.
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