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Lateral line, nervous system, and maternal expression of Frizzled 7a during zebrafish embryogenesis
Mechanisms of Development 115 (2002) 107–111 www.elsevier.com/locate/modo
Gene expression pattern
Lateral line, nervous system, and maternal expression of Frizzled 7a during zebrafish embryogenesis Saulius Sumanas 1, Hyon J. Kim, Spencer B. Hermanson, Stephen C. Ekker* Department of Genetics, Cell and Developmental Biology, University of Minnesota, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA Received 31 October 2001; received in revised form 22 February 2002; accepted 11 March 2002
Abstract We have isolated and mapped a new wnt receptor frizzled family member, zebrafish frizzled 7a. Fz7a and a previously reported zebrafish fz7 (El-Messaoudi and Renucci, 2001) make an orthologous gene pair, however, they display distinct expression patterns. Fz7a shows strong maternal as well as zygotic expression. Fz7a transcript is enriched dorsally starting with the shield stage. At the end of gastrulation, Fz7a is abundantly expressed within anterior neuroectoderm and expressed more weakly within lateral mesoderm. Fz7a is detected during somitogenesis within the central nervous system, somatic and posterior lateral mesoderm. At 24 hpf, fz7a is expressed in migrating lateral line primordium. At 48 hpf, fz7a is detected in the ear, pectoral fin bud, and within neuromasts, which had originated from the lateral line primordium. Radiation hybrid mapping using panel LN54 (Hukriede et al., 1999) places fz7a on linkage group 6, linked to the marker fi11h08 (distance 0.00cR, LOD score 14.1). To prove that fz7 and fz7a are indeed different genes we mapped fz7 as well using the same LN54 panel. Fz7 mapped to linkage group 9 with a LOD of 12.5, 27.31 cR from wnt 10a in between markers IBD2759 and fb50e04. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Wnt; Frizzled; Zebrafish; Expression; Maternal; Nervous system; Lateral line; Neuromast
1. Results Wnt signaling has been implicated in many patterning processes in a vertebrate embryo including the maternal control of dorsoventral patterning (Heasman, 1997; Nasevicius et al., 1998; Liu et al., 1999; Sumanas et al., 2000). Wnt proteins are known to transmit signals via the Frizzled seven-pass transmembrane receptor family (Bhanot et al., 1996). A maternally expressed frizzled family member, Xenopus frizzled-7, has been shown to be required for the dorsoventral axis induction in Xenopus laevis (Sumanas et al., 2000; Sumanas and Ekker, 2001). In zebrafish, Frizzled signaling has been implicated in axis induction as well (Nasevicius et al., 1998); the specific ortholog of Xfz7 previously has not been isolated from zebrafish. The recently reported zebrafish frizzled-7 gene is not expressed maternally (El-Messaoudi and Renucci, 2001), and consequently it is not a suitable candidate to function in the maternal axis induction pathway. We detected a zebrafish EST that is highly homologous to the Frizzled-7 protein subfamily. Using the available * Corresponding author. Tel.: 11-612-656-4509; fax: 11-612-626-7031. E-mail address: [email protected] (S.C. Ekker). 1 Present address: Discovery Genomics, Inc., 614 McKinley Pl. NE, Minneapolis, MN 55413, USA.
sequence and polymerase chain reaction (PCR) we isolated a complete open reading frame of frizzled-7 homolog from fish gastrula library. The deduced protein sequence is 87% identical to the recently reported zebrafish frizzled-7 amino acid sequence (El-Messaoudi and Renucci, 2001), therefore we named our gene zebrafish frizzled-7a (fz7a). Frizzled 7a is a likely ortholog of other vertebrate Frizzled-7 proteins based on high homology to other Frizzled-7 subfamily members (Fig. 1; 81 and 79% amino acid sequence identity to Xenopus fz7 and mouse fz7, correspondingly) as well as similarities in expression patterns (Borello et al., 1999; Wheeler and Hoppler, 1999; Sumanas et al., 2000). High sequence similarity between zebrafish fz7 and fz7a suggests that the fz7 locus was duplicated in the course of evolution of Teleost fish. Although identical within conserved cysteine rich domain and transmembrane segments, fz7 and fz7a sequences are divergent in their signal sequences, the linker region before the first transmembrane domain, and untranslated regions (Fig. 1, and data not shown). Nucleotide sequence homology between fz7 and fz7a is 76% within the open reading frame, only 46% within 5 0 UTR, and 50% within 3 0 UTR regions (data not shown). Radiation hybrid mapping using panel LN54 (Hukriede et al., 1999) places fz7a on linkage group 6, linked to the marker fi11h08 (distance 0.00cR, LOD score 14.1). To
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00084-9
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Fig. 1. Zebrafish Frizzled 7a sequence alignment with its fish paralog fz7 and mouse homolog mfz7. Arrow marks the predicted signal sequence cleavage site, the ten conserved cysteines within the cysteine-rich domain are depicted in blue, and the seven putative transmembrane domains are marked with a line over them. Similar amino acids are shown in gray background. Sequence alignment and features predictions were performed using GeneWorks 2.5 software. Fz7a cDNA sequence was deposited to GenBank under accession number AF437316.
Fig. 2. Zebrafish frizzled 7a expression pattern during development. (A) Northern blotting of fz7a RNA. A single ,3.5 kb transcript was detected at all stages analyzed. Note the strong maternal expression (one cell and 256 cell stages). (B–N) In situ analysis of fz7a expression. (B) Shield stage. Animal view, dorsal is to the right. Fz7a transcript is enriched dorsally and partially excluded from the ventral side. (C) Seventy-five percent epiboly. Animal view, dorsal is to the right. Note the dorsal enrichment of fz7a transcript. (D) Tailbud stage. Dorsal view, anterior is up. Note the abundant expression of fz7a within anterior neuroectoderm, and fz7a expression within the presomitic and lateral mesoderm. tb, tailbud. (E) 10-somite stage, anterior part of an embryo, yolk removed. Dorsal view, anterior is up. Note the four visible stripes of fz7a RNA within the developing central nervous system: midbrain (mb), and rhombomeres 1, 3, and 4 of the hindbrain (hb). Arrowhead marks the midbrain–hindbrain boundary. Fz7a localization has been confirmed by double staining with pax2.1 and krox20 probes (Pfeffer et al., 1998; Oxtoby and Jowett, 1993; data not shown). (F) 5-somite stage, yolk removed. Dorsal view, anterior is up. Fz7a is expressed within midbrain (mb), hindbrain (hb, rhombomeres 1, 3, and 4), somites (s), and posterior lateral mesoderm surrounding tailbud (tb). (G) 10-somite stage, yolk removed. Dorsal view, anterior is up. Fz7a is expressed within midbrain (mb), hindbrain (hb, rhombomeres 1, 3, and 4), somites (s), and posterior lateral mesoderm surrounding tailbud (tb). (H) 20-somite stage. Fz7a is expressed within forebrain (fb), midbrain (mb), hindbrain (hb), somites (s) and tailbud mesenchyme (tb). (I) Same as (H), higher magnification view of the head region. (J) Tailbud of a 20-somite stage embryo, ventral view. Fz7a is expressed bilaterally within tailbud mesenchyme. (K–L) Twenty-six hours post fertilization. Fz7a is expressed in the brain region (mhb, midbrain–hindbrain boundary; hb, hindbrain), migrating lateral line primordium (black arrowhead), ventral (white arrowhead) and tailbud mesenchyme (tb, two longitudinal stripes observed from the ventral side, data not shown). (M–N) Forty-eight hours post fertilization. Fz7a is expressed in neuromasts (sample ones are marked with arrowheads), ear (e) and pectoral fin bud (fin).
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prove that fz7 and fz7a are indeed different genes we mapped fz7 as well using the same LN54 panel. Fz7 mapped to linkage group 9 with a LOD of 12.5, 27.31 cR from wnt 10a in between markers IBD2759 and fb50e04. Thus fz7 and fz7a map to different linkage groups. We used Northern blotting to characterize the temporal expression pattern of fz7a. Fz7a mRNA migrates as a single approximately 3.5 kb size band, which is detected in all stages analyzed (Fig. 2A). Maternal expression is particularly strong (one cell and 256 cell stages, Fig. 2A). In situ expression analysis showed the dorsal enrichment of fz7a RNA at the shield stage (Fig. 2B). Such dorsal enrichment of fz7a is seen until the end of gastrulation (Fig. 2C). At the tailbud stage, fz7a is strongly expressed within anterior neuroectoderm and within the presomitic and lateral mesoderm along the whole body axis (Fig. 2D). By the 5-somite stage, the anterior domain splits into four distinct bands of expression within the brain region, corresponding to midbrain and rhombomeres 1, 3, and 4 within the hindbrain (Fig. 2E–G). Fz7a expression is also apparent in developing somites and posterior lateral mesoderm at this stage (Fig. 2F), which continues throughout somitogenesis (Fig. 2G). By the 20-somite stage, fz7a is detected in specific regions throughout the central nervous system (forebrain, midbrain, and hindbrain, Fig. 2H, I), in the somites where it is particularly abundant in the posterior region of an embryo (Fig. 2H), and tailbud mesenchyme (Fig. 2H, J). At 26 hpf, fz7a expression is found within the developing brain (Fig. 2K). Fz7a is also expressed within lateral line primordium (Fig. 2K, L), which migrates caudally along the midbody line (Metcalfe, 1985). Fz7a RNA is also detected in two longitudinal stripes within ventral mesenchyme along the posterior part of the body, particularly concentrated in the tailbud mesenchyme (Fig. 2K, L). At 48 hpf, fz7a RNA is localized to neuromast precursors that had separated from the migrating lateral line primordium (Fig. 2M, N; Metcalfe, 1985; Raible and Kruse, 2000). In addition to neuromasts, fz7a is localized to the ear and pectoral fin bud regions (Fig. 2M). Although fz7 and fz7a share a very strong sequence similarity, there are important differences in their expression patterns suggesting that the two zebrafish paralogues may have adopted different developmental roles in the course of evolution. Fz7a has a strong maternal expression while fz7 is expressed only zygotically (El-Messaoudi and Renucci, 2001). This expression pattern of fz7a makes it a candidate receptor for a maternal Wnt axis induction pathway since Frizzled signaling has been implicated in zebrafish axis induction (Nasevicius et al., 1998) and frizzled-7 subfamily members have been shown to be required for dorso-ventral axis induction in amphibia (Sumanas et al., 2000). Interestingly, two frizzled-7 orthologous pairs have also been reported in Xenopus laevis (Wheeler and Hoppler, 1999; Djiane et al., 2000; Medina et al., 2000; Sumanas et al., 2000) where they play a redundant maternal role in the dorso-ventral axis induction pathway (Sumanas and Ekker, 2001).
The zygotic expression pattern of fz7a partially overlaps with expression of other frizzled-7 subfamily members. Similarly to Xfz7 (Medina et al., 2000; Sumanas et al., 2000), zebrafish fz7a is dorsally enriched during gastrulation. At the end of gastrulation, fz7A possesses strong expression within anterior neuroectoderm, a pattern similar to zebrafish fz7 and Xenopus fz7 (Wheeler and Hoppler, 1999; Sumanas et al., 2000; El-Messaoudi and Renucci, 2001). Like zebrafish fz7, fz7a is detected within the developing central nervous system and somites, which is also similar to mouse fz7 expression pattern (Borello et al., 1999). Unlike Xfz7, we did not detect fz7a expression in the pronephros or branchial arches. We found fz7a, however, to be expressed within the lateral line primordium and neuromasts. This is the first report of expression of a Wnt signaling pathway molecule within these structures. Our data illustrates that, although the expression pattern of different frizzled-7 subfamily members partially overlaps, there are important differences in their expression pattern. 2. Materials and methods We utilized ESTs from the IMAGE consortium which spanned the complete open reading frame of fz7a (data not shown). Additional sequence information was obtained by performing PCR from an epiboly stage library. Utilizing EST sequence information, we isolated the fz7a open reading frame by PCR and subcloned into the expression vector T3TS (SpeI site; Hyatt and Ekker, 1999). To synthesize DIG-labeled probe for in situ hybridization, fz7a(2)T3TS antisense clone was digested with BstEII and transcribed with T3 RNA polymerase (Boehringer Mannheim). Hybridization in situ was performed as described (Jowett, 1999). In situ hybridization using DIG-labeled 780bp 3 0 UTR fragment of fz7a immediately downstream of the translation termination site confirmed the observed expression pattern (data not shown). For Northern blotting, RNA from ten embryos frozen at each stage was purified and loaded on a denaturing agarose gel. Hybridization was performed as described (Hopwood et al., 1989). A Fz7a-T3TS/SpeI fragment was labeled with 32P and used as a probe. Ethidium bromide staining of ribosomal RNA was used as a loading control. References Bhanot, P., Brink, M., Samos, C.H., Hseih, J.-C., Wang, Y., Macke, J.P., Nathans, J., Nusse, R., 1996. A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 382, 225–230. Borello, U., Buffa, V., Sonnino, C., Melchionna, R., Vivarelli, E., Cossu, G., 1999. Differential expression of the Wnt putative receptors Frizzled during mouse somitogenesis. Mech. Dev. 89, 173–177. Djiane, A., Riou, J., Umbhauer, M., Boucaut, J., Shi, D., 2000. Role of frizzled 7 in the regulation of convergent extension movements during gastrulation in Xenopus laevis. Development 127, 3091–3100.
S. Sumanas et al. / Mechanisms of Development 115 (2002) 107–111 El-Messaoudi, S., Renucci, A., 2001. Expression pattern of the frizzled 7 gene during zebrafish embryonic development. Mech. Dev. 102, 231– 234. Heasman, J., 1997. Patterning the Xenopus blastula. Development 124, 4179–4191. Hopwood, N.D., Pluck, A., Gurdon, J.B., 1989. MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. EMBO J. 8, 3409–3417. Hukriede, N.A., Joly, L., Tsang, M., Miles, J., Tellis, P., Epstein, J.A., Barbazuk, W.B., Li, F.N., Paw, B., Postlethwait, J.H., Hudson, T.J., Zon, L.I., McPherson, J.D., Chevrette, M., Dawid, I.B., Johnson, S.L., Ekker, M., 1999. Radiation hybrid mapping of the zebrafish genome. Proc. Natl Acad. Sci. USA 96, 9745–9750. Hyatt, T.M., Ekker, S.C., 1999. Vectors and techniques for ectopic gene expression in zebrafish. Methods Cell Biol. 59, 117–126. Jowett, T., 1999. Analysis of protein and gene expression. Methods Cell Biol. 59, 63–85. Liu, P., Wakamiya, M., Shea, M.J., Albrecht, U., Behringer, R.R., Bradley, A., 1999. Requirement for Wnt3 in vertebrate axis formation. Nat. Genet. 22, 361–365. Medina, A., Reintsch, W., Steinbeisser, H., 2000. Xenopus frizzled 7 can act in canonical and non-canonical Wnt signaling pathways: implications on early patterning and morphogenesis. Mech. Dev. 92, 227–237. Metcalfe, W.K., 1985. Sensory neuron growth cones comigrate with poster-
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ior lateral line primordial cells in zebrafish. J. Comp. Neurol. 238, 218– 224. Nasevicius, A., Hyatt, T., Kim, H., Guttman, J., Walsh, E., Sumanas, S., Wang, Y., Ekker, S.C., 1998. Evidence for a frizzled-mediated wnt pathway required for zebrafish dorsal mesoderm formation. Development 125, 4283–4292. Oxtoby, E., Jowett, T., 1993. Cloning of the zebrafish krox-20 gene (krx20) and its expression during hindbrain development. Nucleic Acids Res. 21, 1087–1095. Pfeffer, P.L., Gerster, T., Lun, K., Brand, M., Busslinger, M., 1998. Characterization of three novel members of the zebrafish Pax2/5/8 family: dependency of Pax5 and Pax8 expression on the Pax2.1 (noi) function. Development 125, 3063–3074. Raible, D.W., Kruse, G.J., 2000. Organization of the lateral line system in embryonic zebrafish. J. Comp. Neurol. 421, 189–198. Sumanas, S., Ekker, S.C., 2001. Xenopus frizzled-7 morphant displays defects in dorsoventral patterning and convergent extension movements during gastrulation. Genesis 30, 119–122. Sumanas, S., Strege, P., Heasman, J., Ekker, S.C., 2000. The putative wnt receptor Xenopus frizzled-7 functions upstream of beta- catenin in vertebrate dorsoventral mesoderm patterning. Development 127, 1981–1990. Wheeler, G.N., Hoppler, S., 1999. Two novel Xenopus frizzled genes expressed in developing heart and brain. Mech. Dev. 86, 203–207.
Gene expression pattern
Lateral line, nervous system, and maternal expression of Frizzled 7a during zebrafish embryogenesis Saulius Sumanas 1, Hyon J. Kim, Spencer B. Hermanson, Stephen C. Ekker* Department of Genetics, Cell and Developmental Biology, University of Minnesota, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA Received 31 October 2001; received in revised form 22 February 2002; accepted 11 March 2002
Abstract We have isolated and mapped a new wnt receptor frizzled family member, zebrafish frizzled 7a. Fz7a and a previously reported zebrafish fz7 (El-Messaoudi and Renucci, 2001) make an orthologous gene pair, however, they display distinct expression patterns. Fz7a shows strong maternal as well as zygotic expression. Fz7a transcript is enriched dorsally starting with the shield stage. At the end of gastrulation, Fz7a is abundantly expressed within anterior neuroectoderm and expressed more weakly within lateral mesoderm. Fz7a is detected during somitogenesis within the central nervous system, somatic and posterior lateral mesoderm. At 24 hpf, fz7a is expressed in migrating lateral line primordium. At 48 hpf, fz7a is detected in the ear, pectoral fin bud, and within neuromasts, which had originated from the lateral line primordium. Radiation hybrid mapping using panel LN54 (Hukriede et al., 1999) places fz7a on linkage group 6, linked to the marker fi11h08 (distance 0.00cR, LOD score 14.1). To prove that fz7 and fz7a are indeed different genes we mapped fz7 as well using the same LN54 panel. Fz7 mapped to linkage group 9 with a LOD of 12.5, 27.31 cR from wnt 10a in between markers IBD2759 and fb50e04. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Wnt; Frizzled; Zebrafish; Expression; Maternal; Nervous system; Lateral line; Neuromast
1. Results Wnt signaling has been implicated in many patterning processes in a vertebrate embryo including the maternal control of dorsoventral patterning (Heasman, 1997; Nasevicius et al., 1998; Liu et al., 1999; Sumanas et al., 2000). Wnt proteins are known to transmit signals via the Frizzled seven-pass transmembrane receptor family (Bhanot et al., 1996). A maternally expressed frizzled family member, Xenopus frizzled-7, has been shown to be required for the dorsoventral axis induction in Xenopus laevis (Sumanas et al., 2000; Sumanas and Ekker, 2001). In zebrafish, Frizzled signaling has been implicated in axis induction as well (Nasevicius et al., 1998); the specific ortholog of Xfz7 previously has not been isolated from zebrafish. The recently reported zebrafish frizzled-7 gene is not expressed maternally (El-Messaoudi and Renucci, 2001), and consequently it is not a suitable candidate to function in the maternal axis induction pathway. We detected a zebrafish EST that is highly homologous to the Frizzled-7 protein subfamily. Using the available * Corresponding author. Tel.: 11-612-656-4509; fax: 11-612-626-7031. E-mail address: [email protected] (S.C. Ekker). 1 Present address: Discovery Genomics, Inc., 614 McKinley Pl. NE, Minneapolis, MN 55413, USA.
sequence and polymerase chain reaction (PCR) we isolated a complete open reading frame of frizzled-7 homolog from fish gastrula library. The deduced protein sequence is 87% identical to the recently reported zebrafish frizzled-7 amino acid sequence (El-Messaoudi and Renucci, 2001), therefore we named our gene zebrafish frizzled-7a (fz7a). Frizzled 7a is a likely ortholog of other vertebrate Frizzled-7 proteins based on high homology to other Frizzled-7 subfamily members (Fig. 1; 81 and 79% amino acid sequence identity to Xenopus fz7 and mouse fz7, correspondingly) as well as similarities in expression patterns (Borello et al., 1999; Wheeler and Hoppler, 1999; Sumanas et al., 2000). High sequence similarity between zebrafish fz7 and fz7a suggests that the fz7 locus was duplicated in the course of evolution of Teleost fish. Although identical within conserved cysteine rich domain and transmembrane segments, fz7 and fz7a sequences are divergent in their signal sequences, the linker region before the first transmembrane domain, and untranslated regions (Fig. 1, and data not shown). Nucleotide sequence homology between fz7 and fz7a is 76% within the open reading frame, only 46% within 5 0 UTR, and 50% within 3 0 UTR regions (data not shown). Radiation hybrid mapping using panel LN54 (Hukriede et al., 1999) places fz7a on linkage group 6, linked to the marker fi11h08 (distance 0.00cR, LOD score 14.1). To
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Fig. 1. Zebrafish Frizzled 7a sequence alignment with its fish paralog fz7 and mouse homolog mfz7. Arrow marks the predicted signal sequence cleavage site, the ten conserved cysteines within the cysteine-rich domain are depicted in blue, and the seven putative transmembrane domains are marked with a line over them. Similar amino acids are shown in gray background. Sequence alignment and features predictions were performed using GeneWorks 2.5 software. Fz7a cDNA sequence was deposited to GenBank under accession number AF437316.
Fig. 2. Zebrafish frizzled 7a expression pattern during development. (A) Northern blotting of fz7a RNA. A single ,3.5 kb transcript was detected at all stages analyzed. Note the strong maternal expression (one cell and 256 cell stages). (B–N) In situ analysis of fz7a expression. (B) Shield stage. Animal view, dorsal is to the right. Fz7a transcript is enriched dorsally and partially excluded from the ventral side. (C) Seventy-five percent epiboly. Animal view, dorsal is to the right. Note the dorsal enrichment of fz7a transcript. (D) Tailbud stage. Dorsal view, anterior is up. Note the abundant expression of fz7a within anterior neuroectoderm, and fz7a expression within the presomitic and lateral mesoderm. tb, tailbud. (E) 10-somite stage, anterior part of an embryo, yolk removed. Dorsal view, anterior is up. Note the four visible stripes of fz7a RNA within the developing central nervous system: midbrain (mb), and rhombomeres 1, 3, and 4 of the hindbrain (hb). Arrowhead marks the midbrain–hindbrain boundary. Fz7a localization has been confirmed by double staining with pax2.1 and krox20 probes (Pfeffer et al., 1998; Oxtoby and Jowett, 1993; data not shown). (F) 5-somite stage, yolk removed. Dorsal view, anterior is up. Fz7a is expressed within midbrain (mb), hindbrain (hb, rhombomeres 1, 3, and 4), somites (s), and posterior lateral mesoderm surrounding tailbud (tb). (G) 10-somite stage, yolk removed. Dorsal view, anterior is up. Fz7a is expressed within midbrain (mb), hindbrain (hb, rhombomeres 1, 3, and 4), somites (s), and posterior lateral mesoderm surrounding tailbud (tb). (H) 20-somite stage. Fz7a is expressed within forebrain (fb), midbrain (mb), hindbrain (hb), somites (s) and tailbud mesenchyme (tb). (I) Same as (H), higher magnification view of the head region. (J) Tailbud of a 20-somite stage embryo, ventral view. Fz7a is expressed bilaterally within tailbud mesenchyme. (K–L) Twenty-six hours post fertilization. Fz7a is expressed in the brain region (mhb, midbrain–hindbrain boundary; hb, hindbrain), migrating lateral line primordium (black arrowhead), ventral (white arrowhead) and tailbud mesenchyme (tb, two longitudinal stripes observed from the ventral side, data not shown). (M–N) Forty-eight hours post fertilization. Fz7a is expressed in neuromasts (sample ones are marked with arrowheads), ear (e) and pectoral fin bud (fin).
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prove that fz7 and fz7a are indeed different genes we mapped fz7 as well using the same LN54 panel. Fz7 mapped to linkage group 9 with a LOD of 12.5, 27.31 cR from wnt 10a in between markers IBD2759 and fb50e04. Thus fz7 and fz7a map to different linkage groups. We used Northern blotting to characterize the temporal expression pattern of fz7a. Fz7a mRNA migrates as a single approximately 3.5 kb size band, which is detected in all stages analyzed (Fig. 2A). Maternal expression is particularly strong (one cell and 256 cell stages, Fig. 2A). In situ expression analysis showed the dorsal enrichment of fz7a RNA at the shield stage (Fig. 2B). Such dorsal enrichment of fz7a is seen until the end of gastrulation (Fig. 2C). At the tailbud stage, fz7a is strongly expressed within anterior neuroectoderm and within the presomitic and lateral mesoderm along the whole body axis (Fig. 2D). By the 5-somite stage, the anterior domain splits into four distinct bands of expression within the brain region, corresponding to midbrain and rhombomeres 1, 3, and 4 within the hindbrain (Fig. 2E–G). Fz7a expression is also apparent in developing somites and posterior lateral mesoderm at this stage (Fig. 2F), which continues throughout somitogenesis (Fig. 2G). By the 20-somite stage, fz7a is detected in specific regions throughout the central nervous system (forebrain, midbrain, and hindbrain, Fig. 2H, I), in the somites where it is particularly abundant in the posterior region of an embryo (Fig. 2H), and tailbud mesenchyme (Fig. 2H, J). At 26 hpf, fz7a expression is found within the developing brain (Fig. 2K). Fz7a is also expressed within lateral line primordium (Fig. 2K, L), which migrates caudally along the midbody line (Metcalfe, 1985). Fz7a RNA is also detected in two longitudinal stripes within ventral mesenchyme along the posterior part of the body, particularly concentrated in the tailbud mesenchyme (Fig. 2K, L). At 48 hpf, fz7a RNA is localized to neuromast precursors that had separated from the migrating lateral line primordium (Fig. 2M, N; Metcalfe, 1985; Raible and Kruse, 2000). In addition to neuromasts, fz7a is localized to the ear and pectoral fin bud regions (Fig. 2M). Although fz7 and fz7a share a very strong sequence similarity, there are important differences in their expression patterns suggesting that the two zebrafish paralogues may have adopted different developmental roles in the course of evolution. Fz7a has a strong maternal expression while fz7 is expressed only zygotically (El-Messaoudi and Renucci, 2001). This expression pattern of fz7a makes it a candidate receptor for a maternal Wnt axis induction pathway since Frizzled signaling has been implicated in zebrafish axis induction (Nasevicius et al., 1998) and frizzled-7 subfamily members have been shown to be required for dorso-ventral axis induction in amphibia (Sumanas et al., 2000). Interestingly, two frizzled-7 orthologous pairs have also been reported in Xenopus laevis (Wheeler and Hoppler, 1999; Djiane et al., 2000; Medina et al., 2000; Sumanas et al., 2000) where they play a redundant maternal role in the dorso-ventral axis induction pathway (Sumanas and Ekker, 2001).
The zygotic expression pattern of fz7a partially overlaps with expression of other frizzled-7 subfamily members. Similarly to Xfz7 (Medina et al., 2000; Sumanas et al., 2000), zebrafish fz7a is dorsally enriched during gastrulation. At the end of gastrulation, fz7A possesses strong expression within anterior neuroectoderm, a pattern similar to zebrafish fz7 and Xenopus fz7 (Wheeler and Hoppler, 1999; Sumanas et al., 2000; El-Messaoudi and Renucci, 2001). Like zebrafish fz7, fz7a is detected within the developing central nervous system and somites, which is also similar to mouse fz7 expression pattern (Borello et al., 1999). Unlike Xfz7, we did not detect fz7a expression in the pronephros or branchial arches. We found fz7a, however, to be expressed within the lateral line primordium and neuromasts. This is the first report of expression of a Wnt signaling pathway molecule within these structures. Our data illustrates that, although the expression pattern of different frizzled-7 subfamily members partially overlaps, there are important differences in their expression pattern. 2. Materials and methods We utilized ESTs from the IMAGE consortium which spanned the complete open reading frame of fz7a (data not shown). Additional sequence information was obtained by performing PCR from an epiboly stage library. Utilizing EST sequence information, we isolated the fz7a open reading frame by PCR and subcloned into the expression vector T3TS (SpeI site; Hyatt and Ekker, 1999). To synthesize DIG-labeled probe for in situ hybridization, fz7a(2)T3TS antisense clone was digested with BstEII and transcribed with T3 RNA polymerase (Boehringer Mannheim). Hybridization in situ was performed as described (Jowett, 1999). In situ hybridization using DIG-labeled 780bp 3 0 UTR fragment of fz7a immediately downstream of the translation termination site confirmed the observed expression pattern (data not shown). For Northern blotting, RNA from ten embryos frozen at each stage was purified and loaded on a denaturing agarose gel. Hybridization was performed as described (Hopwood et al., 1989). A Fz7a-T3TS/SpeI fragment was labeled with 32P and used as a probe. Ethidium bromide staining of ribosomal RNA was used as a loading control. References Bhanot, P., Brink, M., Samos, C.H., Hseih, J.-C., Wang, Y., Macke, J.P., Nathans, J., Nusse, R., 1996. A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 382, 225–230. Borello, U., Buffa, V., Sonnino, C., Melchionna, R., Vivarelli, E., Cossu, G., 1999. Differential expression of the Wnt putative receptors Frizzled during mouse somitogenesis. Mech. Dev. 89, 173–177. Djiane, A., Riou, J., Umbhauer, M., Boucaut, J., Shi, D., 2000. Role of frizzled 7 in the regulation of convergent extension movements during gastrulation in Xenopus laevis. Development 127, 3091–3100.
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