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Identification and developmental expression pattern of van gogh-like 1, a second zebrafish strabismus homologue
Gene Expression Patterns 4 (2004) 339–344 www.elsevier.com/locate/modgep
Identification and developmental expression pattern of van gogh-like 1, a second zebrafish strabismus homologue Jason R. Jessen, Lilianna Solnica-Krezel* Department of Biological Sciences, Vanderbilt University, 1210 Medical Research Building III, VU Station B, Box 35-1634, Nashville, TN 37235-1634, USA Received 29 August 2003; received in revised form 30 September 2003; accepted 30 September 2003
Abstract Cell movement plays a central role in both normal embryogenesis and the development of diseases such as cancer. Therefore, identification and analysis of proteins controlling cell movement is of special importance. The zebrafish trilobite locus encodes a Van Gogh/Strabismus homologue, which regulates diverse cell migratory behaviors during embryogenesis. Trilobite is most similar to human Van Gogh-like 2 (VANGL2)/Strabismus 1 and mouse Loop-tail associated protein/Lpp1. Both human and mouse genomes encode a second Strabismus homologue referred to as VANGL1/Strabismus 2 and Lpp2, respectively. This prompted us to ask whether another van gogh/strabismus gene, one more closely related to human VANGL1, exists in the zebrafish genome. This paper describes the identification of zebrafish vangl1 and provides the first spatiotemporal expression and functional analysis of a vertebrate vangl1 homologue. Our data indicate that vangl1 and trilobite/vangl2 are expressed in largely non-overlapping domains during embryogenesis. Injection of synthetic vangl1 RNA partially suppressed the gastrulation defect in trilobite mutant embryos, suggesting that Vangl1 and Trilobite/Vangl2 have similar biochemical activities. q 2003 Elsevier B.V. All rights reserved. Keywords: Trilobite; Van gogh; Van gogh-like 2; STB1; STB2; Loop-tail associated protein; Gastrulation; Nervous system; Hindbrain; Cell movement; Orthologue; Embryogenesis; Convergence and extension; Neural retina
1. Results and discussion Mutations in the zebrafish trilobite locus cause defects in gastrulation movements and migration of hindbrain motor neurons (Bingham et al., 2002; Hammerschmidt et al., 1996; Jessen et al., 2002; Sepich et al., 2000; Solnica-Krezel et al., 1996). Positional cloning revealed that trilobite encodes a homologue of the Drosophila melanogaster polarity protein Van Gogh/Strabismus (Jessen et al., 2002). The gene disrupted in trilobite mutant embryos is identical in sequence to the previously reported van gogh-like 2 (vangl2)/strabismus (GenBank accession number AF428249; Park and Moon, 2002). In keeping with guidelines set forth by the Zebrafish Nomenclature Committee, this gene is properly referred to as trilobite (tri) and its encoded protein as Trilobite (Tri). To facilitate comparisons with other Van Gogh/Strabismus homologues, here we will refer to this gene (and protein) as tri/vangl2. * Corresponding author. Tel.: þ 1-615-343-9413; fax: þ1-615-343-6707. E-mail address: [email protected] (L. SolnicaKrezel). 1567-133X/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2003.09.012
The initially cloned Drosophila Van Gogh/Strabismus was described as a novel protein with two-four putative transmembrane domains and a C-terminal PDZ domainbinding motif (Taylor et al., 1998; Wolff and Rubin, 1998). The C-terminal region likely protrudes into the cytoplasm because it was shown to interact with the intracellular proteins Dishevelled and Prickle (Bastock et al., 2003; Jenny et al., 2003; Park and Moon, 2002). However, depending on the actual number of transmembrane domains, the N-terminus may prove to be intracellular or extracellular. Zebrafish Tri/Vangl2 is most similar to human VANGL2/Strabismus 1 (STB1; GenBank accession number AB033041; Katoh, 2002a) and mouse Loop-tail associated protein (Ltap)/Lpp1/Vangl2 (GenBank accession number AF365875; Kibar et al., 2001; Murdoch et al., 2001). In the mouse, loss of Ltap/Lpp1/Vangl2 function disrupts neural tube closure resulting in a condition known as craniorachischisis (Kibar et al., 2001; Murdoch et al., 2001). Consistent with its diverse roles in gastrulation cell movements and neuronal migration (Jessen et al., 2002), the expression of zebrafish tri/vangl2 is widespread
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throughout embryonic development (Park and Moon, 2002; and this report). Current data suggests Tri/Vangl2 controls cell movement behaviors by directly or indirectly regulating noncanonical Wnt signaling (Carreira-Barbosa et al., 2003; Jessen et al., 2002; Park and Moon, 2002). The presence of a second Strabismus homologue in both mice and humans suggests that this protein family may control numerous cell movement behaviors during vertebrate development. However, neither the expression patterns nor the in vivo functions of human VANGL1/STB2 and mouse Lpp2 have been described. Interestingly, human VANGL1/STB2 was shown to be upregulated in hepatocellular carcinoma (Yagyu et al., 2002) and either up or down regulated in different gastric and pancreatic cancer cell lines (Katoh, 2002b). Here, we report identification of vangl1, the zebrafish orthologue of human VANGL1/STB2, and a comparison of its expression and function with tri/vangl2.
zebrafish vangl1 is AY279079. Phylogenetic and syntenic analyses suggest this Van Gogh/Strabismus homologue is more closely related to human VANGL1/STB2/LPP2 (GenBank accession number AB075805; Katoh, 2002b; Yagyu et al., 2002) and mouse Lpp2 (referred to as mStrb in Wolff and Rubin (1998)), and therefore the genes encoding these proteins are orthologues (Fig. 2A,B). Utilizing both radiation hybrid mapping and an analysis of the zebrafish Ensembl Genome Server, we have assigned vangl1 to chromosome 9 (previously called linkage group 9). Similar to human VANGL1/STB2 (Katoh, 2002b), zebrafish vangl1 is located adjacent to calsequestrin 2 in a tail-to-tail genomic organization (Fig. 2B). It is clear that the current vertebrate Van Gogh/Strabismus homologues make up two subfamilies, Vangl1-related and Vangl2-related (Fig. 2A,C).
1.1. Cloning and mapping of zebrafish vangl1
Unlike tri/vangl2 transcripts, vangl1 is not maternally expressed in blastula stage embryos (data not shown). Transcripts for vangl1 are first detected at the 15-somite stage in a population of trigeminal ganglion cells (Fig. 3A,F). In embryos that are overstained, vangl1 expression can also be detected in neural cells of the trunk (Fig. 3A, inset). We do not believe this staining represents probe cross-reactivity with tri/vangl2 because even several days of overstaining with vangl1 does not label hatching gland or neural tube, two sites of prominent tri/vangl2 expression. After one day of development, vangl1 expression is detected in the hindbrain (Fig. 3B), and
To identify additional zebrafish Strabismus homologues, we searched the EST database and the Ensembl Genome Server using tri/vangl2 DNA sequence. We found several EST’s and genomic traces encoding a single Strabismus homologue highly similar to Tri/Vangl2. Because the DNA sequence was incomplete, 50 RACE was performed to obtain the full-length coding region. The predicted amino acid sequence indicates this second zebrafish Van Gogh/ Strabismus homologue is 60% identical (74% similar) to Tri/Vangl2 (Fig. 1). The GenBank accession number for
1.2. Developmental expression of zebrafish vangl1
Fig. 1. Alignment of the deduced amino acid sequences of zebrafish Van Gogh/Strabismus homologues Vangl1 and Tri/Vangl2. Identical residues are indicated by periods and the four putative transmembrane domains are underlined. Grey shaded boxes denote residues conserved among vertebrate Van Gogh/Strabismus family members and for which missense mutations have been described (D255E, mouse Loop-tailm1Jus; S464N, mouse Loop-tail, Kibar et al., 2001; Murdoch et al., 2001; M437T, zebrafish trib626; J.R.J. and L.S.-K., unpublished observations). The boxed region denotes the PDZ domain-binding motif conserved among all identified Van Gogh/Strabismus homologues.
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Fig. 2. Zebrafish vangl1 represents the orthologue of human VANGL1/STB2/LPP2 and perhaps mouse Lpp2. (A) Phylogenetic tree depicting evolutionary relationships between zebrafish Vangl1 and other Van Gogh/Strabismus family members, in which proteins with greater sequence similarity cluster together. The GenBank accession number for frog strabismus is AF427792 (Park and Moon, 2002). (B) Schematics of chromosomal locations of zebrafish and human van gogh-like 1 genes. (C) Protein sequence comparison of zebrafish Vangl1 and selected Van Gogh/Strabismus family members (percent amino acid identity/similarity). N-terminal denotes amino acids 50 of the first putative transmembrane domain; C-terminal denotes amino acids 30 of the fourth putative transmembrane domain.
again, overstained embryos reveal stronger expression in the forebrain and additional expression in the trunk (Fig. 3C,G). The expression of vangl1 in one day old embryos contrasts the more widespread expression of tri/vangl2 in the neuroectoderm, neural tube, and hatching gland (Fig. 3D). Moreover, vangl1 expression appears normal in trim209=m209 mutant embryos (compare Fig. 3E with C). By day 2 (Fig. 3H,I) and day 3 (Fig. 3J,K), vangl1 expression becomes restricted to the developing brain and neural retina with no detectable transcripts in the trunk. To characterize further vangl1 expression in anterior neural tissues, we generated sagittal and transverse sections of whole-mount stained embryos. We observed that in two day old embryos, vangl1 is expressed throughout the brain from the diencephalon to the caudal hindbrain, but is largely restricted to ventral domains (Fig. 3L– N). Expression is also detected in the cerebellum (Fig. 3L). The expression of vangl1 in post-otic neural tissue is restricted to the ventral hindbrain (Fig. 3L– N). In contrast to tri/vangl2, vangl1 expression is not detected in the hindbrain at 18 h postfertilization when branchiomotor neuron migration commences (Bingham et al., 2002; Chandrasekhar et al., 1997). However, according to our data, tri/vangl2 and vangl1 expression in the hindbrain may overlap at later stages of development, prior to the end of migration of this class of motor neurons. At day 3, vangl1 expression is detected in two of the three major laminae of the neural retina, the ganglion cell layer and the innermost portion of the inner nuclear layer (Fig. 3O,P). The restriction of vangl1 expression within the inner nuclear layer may reflect
staining of a specific cell type, such as a subset of amacrine cells, or it may label cells at different stages of neurogenesis, or mark cells that are post-mitotic (Malicki, 2000). Expression is not detected in the photoreceptor cell layer or the lens (Fig. 3P). For a comparative analysis, we used double-label whole-mount in situ hybridization against vangl1 and pax2a (also called pax2.1), a marker of neural structures including the midbrain-hindbrain boundary, optic stalks, otic capsules, and hindbrain (Krauss et al., 1991). Whereas vangl1 is excluded from the midbrain-hindbrain boundary and otic capsules of one day old embryos, vangl1 and pax2a expression overlap extensively throughout the hindbrain (Fig. 3Q,R), where pax2a is expressed in different classes of primary commissural interneurons (Mikkola et al., 1992). By day 2, vangl1 transcripts appear within the midbrain-hindbrain boundary, specifically in the cerebellum, and continue to co-localize with pax2a in the hindbrain (Fig. 3S,L). 1.3. Zebrafish Vangl1 can compensate for Tri/Vangl2 function in vivo The largely nonoverlapping expression patterns of vangl1 and tri/vangl2 during zebrafish development led us to pose the question of whether the encoded proteins possess similar or distinct activities. To begin to determine whether Vangl1 functions similarly to Tri/Vangl2, we injected synthetic vangl1 RNA into trim209=m209 mutant embryos and determined whether there was suppression of the mutant phenotype. Injection of 10– 60 pg/embryo of vangl1 RNA
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Fig. 3. Expression of vangl1 during embryogenesis as detected by whole-mount in situ hybridization. All images are of wild-type embryos, except (E). (A) vangl1 expression at the 15 somite stage, lateral view, arrow denotes expression in trigeminal ganglion. Boxed area denotes tail region depicted by inset. Inset, in overstained embryos, vangl1 expression is detected in neural cells of the tail (arrowhead). (B,C) Expression of vangl1 in one day old embryos stained with minimal background (B) and overstained (C), arrowhead indicates neural cells in tail. (D) tri/vangl2 expression in a one day old embryo. (E) vangl1 expression in a one day old trim209=m209 mutant embryo. (F,G) vangl1 expression in 15 somite stage (F) and one day old (G) overstained and flat mounted embryos with anterior to the left, arrows denote staining in trigeminal ganglion. (H,I) vangl1 expression in two day old embryos: (H) lateral view, (I) dorsal view of head. (J,K) vangl1 expression in three day old embryos: (J) lateral view of anterior region of embryo, (K) dorsal view of head. (L– N) vangl1 expression in two day old embryos. (L) Sagittal section of head, lateral view, an asterisk denotes vangl1 expression in cerebellum, 10 £ magnification. The dashed lines mark the anteroposterior position of the sections in (M) and (N). (M) Transverse section of anterior hindbrain at the level of the otic capsules, 20 £ magnification. (N) Transverse section of caudal hindbrain, 20 £ magnification. (O,P) vangl1 expression in a three day old embryo. (O) Transverse section of forebrain at the level of the eyes, 10 £ magnification. (P) Larger view of eye marked by black box in (O), arrow denotes ganglion cell layer, arrowhead denotes inner nuclear layer, the asterisks mark absence of staining in photoreceptor cell layer and lens, 20 £ magnification. (Q-S) Double-label in situ hybridization staining with probes for vangl1 (blue) and pax2a (red) in one day old embryos (Q,R) and a two day old embryo (S). (Q) Dorsal view of a flat mounted embryo with anterior to the left, arrows denote overlapping vangl1 and pax2a expression in the hindbrain. (R) Lateral view. (S) Lateral view, the asterisk denotes overlapping vangl1 and pax2a expression in the midbrain-hindbrain boundary. Abbreviations: ch, caudal hindbrain; d, diencephalon; fb, forebrain; hb, hindbrain; hg, hatching gland; mhb, midbrain-hindbrain boundary; ms, mesencephalon; mt, metancephalon; my, myelencephalon; nc, notochord; nt, neural tube; oc, otic capsule; op, optic primordium; ot, optic tectum.
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2. Methods
Fig. 4. Ectopic vangl1 partially suppresses the trim209 gastrulation defect. (A –C) Lateral views of embryos at approximately 30 h post-fertilization. (A) Uninjected wild-type control embryo. (B) Uninjected trim209=m209 mutant control embryo. (C) trim209=m209 embryo injected with 20 pg of synthetic vangl1 RNA. (D) Tabular presentation of the injection data.
partially suppressed the trim209 gastrulation convergence and extension defect (Jessen et al., 2002; Sepich et al., 2000; Solnica-Krezel et al., 1996), as assessed by anteroposterior length at day 1 (Fig. 4A – D). For comparison, higher doses of vangl1 RNA were needed to suppress the trim209 gastrulation phenotype with the same efficiency as tri/vangl2 RNA (Jessen et al., 2002). Nevertheless, these results indicate that, in this assay, Vangl1 can function similarly to Tri/Vangl2. 1.4. Summary We have shown that the zebrafish genome encodes two Van Gogh/Strabismus homologues, Vangl1 and Tri/Vangl2, which differ in their expression patterns but may have similar biochemical activities. While Tri/Vangl2 is broadly expressed throughout embryogenesis and functions in diverse processes such as gastrulation movements and neuronal migration, vangl1 expression begins later and appears to be restricted to the nervous system. It will now be important to dissect further the function of Vangl1 using loss-of-function experiments.
All work involving zebrafish was conducted in compliance with rules set forth by the Vanderbilt Institutional Animal Care and Use Committee. Zebrafish embryos were produced by natural matings and adults maintained as described (Solnica-Krezel and Driever, 1994). Embryo staging was performed according to morphology as described (Kimmel et al., 1995). To clone vangl1 we searched the zebrafish Ensembl Genome Server (www. ensembl.org/Danio_rerio/) and EST (www.ncbi.nih.gov/ BLAST/) databases using tri/vangl2 sequence. Two ESTs were identified that were similar to the 30 end of tri/vangl2 (fv22b07.y1 and fu40c01.y3). The vangl1 full-length open reading frame was obtained by performing 50 RACE. Primers flanking the ATG initiation and stop codons were used to PCR amplify the entire gene from cDNA for subsequent subcloning into an expression vector and DNA sequencing. Chromosomal mapping was performed by PCR screening of the Goodfellow T51 radiation hybrid panel (Invitrogen, Carlsbad, CA) using two sets of genomic primers corresponding to different vangl1 exons. The mapping results were confirmed using the zebrafish Ensembl Genome Server to identify mapped genomic markers located near vangl1. Whole-mount in situ hybridization was performed as described using full-length vangl1 cDNA as a probe (Westerfield, 1995). Phylogenetic analysis was carried out using the Phylodendron program (http://iubio.bio.indiana. edu/treeapp/treeprint-form.html). Frozen sections were made using a cryostat and embryos labeled by wholemount in situ hybridization. For rescue experiments, embryos from a clutch obtained from trim209=þ parents were injected with synthetic vangl1 RNA as described (Marlow et al., 1998). Sense-capped RNA was synthesized using mMessage mMachine (Ambion, Austin, TX) after template linearization by NotI digestion. Injected embryos scored for suppression of the trim209 mutant phenotype were PCR genotyped as described (Jessen et al., 2002). Acknowledgements We thank J. Clanton for skillful sectioning and excellent fish care. J.R.J. is supported by a National Institutes of Health Vascular Biology Training Grant (T32HL07751). Work in the L.S.-K. lab is supported by NIH grant GM55101. References Bastock, R., Strutt, H., Strutt, D., 2003. Strabismus is asymmetrically localised and binds Prickle and Dishevelled during Drosophila planar polarity patterning. Development 130, 3007– 3014. Bingham, S., Higashijima, S., Okamoto, H., Chandrasekhar, A., 2002. The zebrafish trilobite gene is essential for tangential migration of branchiomotor neurons. Dev. Biol. 242, 149 –160. Carreira-Barbosa, F., Concha, M.L., Takeuchi, M., Ueno, N., Wilson, S.W., Tada, M., 2003. Prickle1 regulates cell movements during
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gastrulation and neuronal migration in zebrafish. Development 130, 4037–4046. Chandrasekhar, A., Moens, C.B., Warren, J.T. Jr., Kimmel, C.B., Kuwada, J.Y., 1997. Development of branchiomotor neurons in zebrafish. Development 124, 2633–2644. Hammerschmidt, M., Pelegri, F., Mullins, M.C., Kane, D.A., Brand, M., van Eeden, F.J.M., et al., 1996. Mutations affecting morphogenesis during gastrulation and tail formation in the zebrafish, Danio rerio. Development 123, 143–151. Jenny, A., Darken, R.S., Wilson, P.A., Mlodzik, M., 2003. Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling. Eur. Mol. Biol. Org. J. 22, 4409–4420. Jessen, J.R., Topczewski, J., Bingham, S., Sepich, D.S., Marlow, F., Chandrasekhar, A., Solnica-Krezel, L., 2002. Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements. Nat. Cell Biol. 4, 610– 615. Katoh, M., 2002a. Structure and expression of Strabismus 1 gene on human chromosome 1q21-q23. Int. J. Oncol. 20, 1197– 1203. Katoh, M., 2002b. Molecular cloning and characterization of Strabismus 2 (STB2). Int. J. Oncol. 20, 993 –998. Kibar, Z., Vogan, K.J., Groulx, N., Justice, M.J., Underhill, D.A., Gros, P., 2001. Ltap, a mammalian homolog of Drosophila Strabismus/Van Gogh, is altered in the mouse neural tube mutant Loop-tail. Nat. Genet. 28, 251 –255. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253–310. Krauss, S., Johansen, T., Korzh, V., Fjose, A., 1991. Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis. Development 113, 1193–1206. Malicki, J., 2000. Harnessing the power of forward genetics-analysis of neuronal diversity and patterning in the zebrafish retina. Trends Neurosci. 23, 531 –541. Marlow, F., Zwartkruis, F., Malicki, J., Neuhauss, S.C.F., Abbas, L., Weaver, M., Driever, W., Solnica-Krezel, L., 1998. Functional
interactions of genes mediating convergent extension, knypek and trilobite, during partitioning of the eye primordium in zebrafish. Dev. Biol. 203, 383 –399. Mikkola, I., Fjose, A., Kuwada, J.Y., Wilson, S., Guddal, P.H., Krauss, S., 1992. The paired domain-containing nuclear factor pax[b] is expressed in specific commissural interneurons in zebrafish embryos. J. Neurobiol. 23, 933–946. Murdoch, J.N., Doudney, K., Paternotte, C., Copp, A.J., Stanier, P., 2001. Severe neural tube defects in the loop-tail mouse result from mutation of Lpp1, a novel gene involved in floor plate specification. Hum. Mol. Genet. 10, 2593–2601. Park, M., Moon, R.T., 2002. The planar cell-polarity gene stbm regulates cell behaviour and cell fate in vertebrate embryos. Nat. Cell Biol. 4, 20 –25. Sepich, D.S., Myers, D.C., Short, R., Topczewski, J., Marlow, F., SolnicaKrezel, L., 2000. Role of the zebrafish trilobite locus in gastrulation movements of convergence and extension. Genesis 27, 159–173. Solnica-Krezel, L., Driever, W., 1994. Microtubule arrays of the zebrafish yolk cell: organization and function during epiboly. Development 120, 2443–2455. Solnica-Krezel, L., Stemple, D.L., Mountcastle-Shah, E., Rangini, Z., Neuhauss, S.C.F., Malicki, J., et al., 1996. Mutations affecting cell fates and cellular rearrangements during gastrulation in zebrafish. Development 123, 117–128. Taylor, J., Abramova, N., Charlton, J., Adler, P.N., 1998. Van Gogh: a new Drosophila tissue polarity gene. Genetics 150, 199–210. Westerfield, M., 1995. The Zebrafish Book. University of Oregon Press, Eugene, OR. Wolff, T., Rubin, G.M., 1998. strabismus, a novel gene that regulates tissue polarity and cell fate decisions in Drosophila. Development 125, 1149–1159. Yagyu, R., Hamamoto, R., Furukawa, Y., Okabe, H., Yamamura, T., Nakamura, Y., 2002. Isolation and characterization of a novel human gene, VANGL1, as a therapeutic target for hepatocellular carcinoma. Int. J. Oncol. 20, 1173–1178.
Identification and developmental expression pattern of van gogh-like 1, a second zebrafish strabismus homologue Jason R. Jessen, Lilianna Solnica-Krezel* Department of Biological Sciences, Vanderbilt University, 1210 Medical Research Building III, VU Station B, Box 35-1634, Nashville, TN 37235-1634, USA Received 29 August 2003; received in revised form 30 September 2003; accepted 30 September 2003
Abstract Cell movement plays a central role in both normal embryogenesis and the development of diseases such as cancer. Therefore, identification and analysis of proteins controlling cell movement is of special importance. The zebrafish trilobite locus encodes a Van Gogh/Strabismus homologue, which regulates diverse cell migratory behaviors during embryogenesis. Trilobite is most similar to human Van Gogh-like 2 (VANGL2)/Strabismus 1 and mouse Loop-tail associated protein/Lpp1. Both human and mouse genomes encode a second Strabismus homologue referred to as VANGL1/Strabismus 2 and Lpp2, respectively. This prompted us to ask whether another van gogh/strabismus gene, one more closely related to human VANGL1, exists in the zebrafish genome. This paper describes the identification of zebrafish vangl1 and provides the first spatiotemporal expression and functional analysis of a vertebrate vangl1 homologue. Our data indicate that vangl1 and trilobite/vangl2 are expressed in largely non-overlapping domains during embryogenesis. Injection of synthetic vangl1 RNA partially suppressed the gastrulation defect in trilobite mutant embryos, suggesting that Vangl1 and Trilobite/Vangl2 have similar biochemical activities. q 2003 Elsevier B.V. All rights reserved. Keywords: Trilobite; Van gogh; Van gogh-like 2; STB1; STB2; Loop-tail associated protein; Gastrulation; Nervous system; Hindbrain; Cell movement; Orthologue; Embryogenesis; Convergence and extension; Neural retina
1. Results and discussion Mutations in the zebrafish trilobite locus cause defects in gastrulation movements and migration of hindbrain motor neurons (Bingham et al., 2002; Hammerschmidt et al., 1996; Jessen et al., 2002; Sepich et al., 2000; Solnica-Krezel et al., 1996). Positional cloning revealed that trilobite encodes a homologue of the Drosophila melanogaster polarity protein Van Gogh/Strabismus (Jessen et al., 2002). The gene disrupted in trilobite mutant embryos is identical in sequence to the previously reported van gogh-like 2 (vangl2)/strabismus (GenBank accession number AF428249; Park and Moon, 2002). In keeping with guidelines set forth by the Zebrafish Nomenclature Committee, this gene is properly referred to as trilobite (tri) and its encoded protein as Trilobite (Tri). To facilitate comparisons with other Van Gogh/Strabismus homologues, here we will refer to this gene (and protein) as tri/vangl2. * Corresponding author. Tel.: þ 1-615-343-9413; fax: þ1-615-343-6707. E-mail address: [email protected] (L. SolnicaKrezel). 1567-133X/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2003.09.012
The initially cloned Drosophila Van Gogh/Strabismus was described as a novel protein with two-four putative transmembrane domains and a C-terminal PDZ domainbinding motif (Taylor et al., 1998; Wolff and Rubin, 1998). The C-terminal region likely protrudes into the cytoplasm because it was shown to interact with the intracellular proteins Dishevelled and Prickle (Bastock et al., 2003; Jenny et al., 2003; Park and Moon, 2002). However, depending on the actual number of transmembrane domains, the N-terminus may prove to be intracellular or extracellular. Zebrafish Tri/Vangl2 is most similar to human VANGL2/Strabismus 1 (STB1; GenBank accession number AB033041; Katoh, 2002a) and mouse Loop-tail associated protein (Ltap)/Lpp1/Vangl2 (GenBank accession number AF365875; Kibar et al., 2001; Murdoch et al., 2001). In the mouse, loss of Ltap/Lpp1/Vangl2 function disrupts neural tube closure resulting in a condition known as craniorachischisis (Kibar et al., 2001; Murdoch et al., 2001). Consistent with its diverse roles in gastrulation cell movements and neuronal migration (Jessen et al., 2002), the expression of zebrafish tri/vangl2 is widespread
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throughout embryonic development (Park and Moon, 2002; and this report). Current data suggests Tri/Vangl2 controls cell movement behaviors by directly or indirectly regulating noncanonical Wnt signaling (Carreira-Barbosa et al., 2003; Jessen et al., 2002; Park and Moon, 2002). The presence of a second Strabismus homologue in both mice and humans suggests that this protein family may control numerous cell movement behaviors during vertebrate development. However, neither the expression patterns nor the in vivo functions of human VANGL1/STB2 and mouse Lpp2 have been described. Interestingly, human VANGL1/STB2 was shown to be upregulated in hepatocellular carcinoma (Yagyu et al., 2002) and either up or down regulated in different gastric and pancreatic cancer cell lines (Katoh, 2002b). Here, we report identification of vangl1, the zebrafish orthologue of human VANGL1/STB2, and a comparison of its expression and function with tri/vangl2.
zebrafish vangl1 is AY279079. Phylogenetic and syntenic analyses suggest this Van Gogh/Strabismus homologue is more closely related to human VANGL1/STB2/LPP2 (GenBank accession number AB075805; Katoh, 2002b; Yagyu et al., 2002) and mouse Lpp2 (referred to as mStrb in Wolff and Rubin (1998)), and therefore the genes encoding these proteins are orthologues (Fig. 2A,B). Utilizing both radiation hybrid mapping and an analysis of the zebrafish Ensembl Genome Server, we have assigned vangl1 to chromosome 9 (previously called linkage group 9). Similar to human VANGL1/STB2 (Katoh, 2002b), zebrafish vangl1 is located adjacent to calsequestrin 2 in a tail-to-tail genomic organization (Fig. 2B). It is clear that the current vertebrate Van Gogh/Strabismus homologues make up two subfamilies, Vangl1-related and Vangl2-related (Fig. 2A,C).
1.1. Cloning and mapping of zebrafish vangl1
Unlike tri/vangl2 transcripts, vangl1 is not maternally expressed in blastula stage embryos (data not shown). Transcripts for vangl1 are first detected at the 15-somite stage in a population of trigeminal ganglion cells (Fig. 3A,F). In embryos that are overstained, vangl1 expression can also be detected in neural cells of the trunk (Fig. 3A, inset). We do not believe this staining represents probe cross-reactivity with tri/vangl2 because even several days of overstaining with vangl1 does not label hatching gland or neural tube, two sites of prominent tri/vangl2 expression. After one day of development, vangl1 expression is detected in the hindbrain (Fig. 3B), and
To identify additional zebrafish Strabismus homologues, we searched the EST database and the Ensembl Genome Server using tri/vangl2 DNA sequence. We found several EST’s and genomic traces encoding a single Strabismus homologue highly similar to Tri/Vangl2. Because the DNA sequence was incomplete, 50 RACE was performed to obtain the full-length coding region. The predicted amino acid sequence indicates this second zebrafish Van Gogh/ Strabismus homologue is 60% identical (74% similar) to Tri/Vangl2 (Fig. 1). The GenBank accession number for
1.2. Developmental expression of zebrafish vangl1
Fig. 1. Alignment of the deduced amino acid sequences of zebrafish Van Gogh/Strabismus homologues Vangl1 and Tri/Vangl2. Identical residues are indicated by periods and the four putative transmembrane domains are underlined. Grey shaded boxes denote residues conserved among vertebrate Van Gogh/Strabismus family members and for which missense mutations have been described (D255E, mouse Loop-tailm1Jus; S464N, mouse Loop-tail, Kibar et al., 2001; Murdoch et al., 2001; M437T, zebrafish trib626; J.R.J. and L.S.-K., unpublished observations). The boxed region denotes the PDZ domain-binding motif conserved among all identified Van Gogh/Strabismus homologues.
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Fig. 2. Zebrafish vangl1 represents the orthologue of human VANGL1/STB2/LPP2 and perhaps mouse Lpp2. (A) Phylogenetic tree depicting evolutionary relationships between zebrafish Vangl1 and other Van Gogh/Strabismus family members, in which proteins with greater sequence similarity cluster together. The GenBank accession number for frog strabismus is AF427792 (Park and Moon, 2002). (B) Schematics of chromosomal locations of zebrafish and human van gogh-like 1 genes. (C) Protein sequence comparison of zebrafish Vangl1 and selected Van Gogh/Strabismus family members (percent amino acid identity/similarity). N-terminal denotes amino acids 50 of the first putative transmembrane domain; C-terminal denotes amino acids 30 of the fourth putative transmembrane domain.
again, overstained embryos reveal stronger expression in the forebrain and additional expression in the trunk (Fig. 3C,G). The expression of vangl1 in one day old embryos contrasts the more widespread expression of tri/vangl2 in the neuroectoderm, neural tube, and hatching gland (Fig. 3D). Moreover, vangl1 expression appears normal in trim209=m209 mutant embryos (compare Fig. 3E with C). By day 2 (Fig. 3H,I) and day 3 (Fig. 3J,K), vangl1 expression becomes restricted to the developing brain and neural retina with no detectable transcripts in the trunk. To characterize further vangl1 expression in anterior neural tissues, we generated sagittal and transverse sections of whole-mount stained embryos. We observed that in two day old embryos, vangl1 is expressed throughout the brain from the diencephalon to the caudal hindbrain, but is largely restricted to ventral domains (Fig. 3L– N). Expression is also detected in the cerebellum (Fig. 3L). The expression of vangl1 in post-otic neural tissue is restricted to the ventral hindbrain (Fig. 3L– N). In contrast to tri/vangl2, vangl1 expression is not detected in the hindbrain at 18 h postfertilization when branchiomotor neuron migration commences (Bingham et al., 2002; Chandrasekhar et al., 1997). However, according to our data, tri/vangl2 and vangl1 expression in the hindbrain may overlap at later stages of development, prior to the end of migration of this class of motor neurons. At day 3, vangl1 expression is detected in two of the three major laminae of the neural retina, the ganglion cell layer and the innermost portion of the inner nuclear layer (Fig. 3O,P). The restriction of vangl1 expression within the inner nuclear layer may reflect
staining of a specific cell type, such as a subset of amacrine cells, or it may label cells at different stages of neurogenesis, or mark cells that are post-mitotic (Malicki, 2000). Expression is not detected in the photoreceptor cell layer or the lens (Fig. 3P). For a comparative analysis, we used double-label whole-mount in situ hybridization against vangl1 and pax2a (also called pax2.1), a marker of neural structures including the midbrain-hindbrain boundary, optic stalks, otic capsules, and hindbrain (Krauss et al., 1991). Whereas vangl1 is excluded from the midbrain-hindbrain boundary and otic capsules of one day old embryos, vangl1 and pax2a expression overlap extensively throughout the hindbrain (Fig. 3Q,R), where pax2a is expressed in different classes of primary commissural interneurons (Mikkola et al., 1992). By day 2, vangl1 transcripts appear within the midbrain-hindbrain boundary, specifically in the cerebellum, and continue to co-localize with pax2a in the hindbrain (Fig. 3S,L). 1.3. Zebrafish Vangl1 can compensate for Tri/Vangl2 function in vivo The largely nonoverlapping expression patterns of vangl1 and tri/vangl2 during zebrafish development led us to pose the question of whether the encoded proteins possess similar or distinct activities. To begin to determine whether Vangl1 functions similarly to Tri/Vangl2, we injected synthetic vangl1 RNA into trim209=m209 mutant embryos and determined whether there was suppression of the mutant phenotype. Injection of 10– 60 pg/embryo of vangl1 RNA
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Fig. 3. Expression of vangl1 during embryogenesis as detected by whole-mount in situ hybridization. All images are of wild-type embryos, except (E). (A) vangl1 expression at the 15 somite stage, lateral view, arrow denotes expression in trigeminal ganglion. Boxed area denotes tail region depicted by inset. Inset, in overstained embryos, vangl1 expression is detected in neural cells of the tail (arrowhead). (B,C) Expression of vangl1 in one day old embryos stained with minimal background (B) and overstained (C), arrowhead indicates neural cells in tail. (D) tri/vangl2 expression in a one day old embryo. (E) vangl1 expression in a one day old trim209=m209 mutant embryo. (F,G) vangl1 expression in 15 somite stage (F) and one day old (G) overstained and flat mounted embryos with anterior to the left, arrows denote staining in trigeminal ganglion. (H,I) vangl1 expression in two day old embryos: (H) lateral view, (I) dorsal view of head. (J,K) vangl1 expression in three day old embryos: (J) lateral view of anterior region of embryo, (K) dorsal view of head. (L– N) vangl1 expression in two day old embryos. (L) Sagittal section of head, lateral view, an asterisk denotes vangl1 expression in cerebellum, 10 £ magnification. The dashed lines mark the anteroposterior position of the sections in (M) and (N). (M) Transverse section of anterior hindbrain at the level of the otic capsules, 20 £ magnification. (N) Transverse section of caudal hindbrain, 20 £ magnification. (O,P) vangl1 expression in a three day old embryo. (O) Transverse section of forebrain at the level of the eyes, 10 £ magnification. (P) Larger view of eye marked by black box in (O), arrow denotes ganglion cell layer, arrowhead denotes inner nuclear layer, the asterisks mark absence of staining in photoreceptor cell layer and lens, 20 £ magnification. (Q-S) Double-label in situ hybridization staining with probes for vangl1 (blue) and pax2a (red) in one day old embryos (Q,R) and a two day old embryo (S). (Q) Dorsal view of a flat mounted embryo with anterior to the left, arrows denote overlapping vangl1 and pax2a expression in the hindbrain. (R) Lateral view. (S) Lateral view, the asterisk denotes overlapping vangl1 and pax2a expression in the midbrain-hindbrain boundary. Abbreviations: ch, caudal hindbrain; d, diencephalon; fb, forebrain; hb, hindbrain; hg, hatching gland; mhb, midbrain-hindbrain boundary; ms, mesencephalon; mt, metancephalon; my, myelencephalon; nc, notochord; nt, neural tube; oc, otic capsule; op, optic primordium; ot, optic tectum.
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2. Methods
Fig. 4. Ectopic vangl1 partially suppresses the trim209 gastrulation defect. (A –C) Lateral views of embryos at approximately 30 h post-fertilization. (A) Uninjected wild-type control embryo. (B) Uninjected trim209=m209 mutant control embryo. (C) trim209=m209 embryo injected with 20 pg of synthetic vangl1 RNA. (D) Tabular presentation of the injection data.
partially suppressed the trim209 gastrulation convergence and extension defect (Jessen et al., 2002; Sepich et al., 2000; Solnica-Krezel et al., 1996), as assessed by anteroposterior length at day 1 (Fig. 4A – D). For comparison, higher doses of vangl1 RNA were needed to suppress the trim209 gastrulation phenotype with the same efficiency as tri/vangl2 RNA (Jessen et al., 2002). Nevertheless, these results indicate that, in this assay, Vangl1 can function similarly to Tri/Vangl2. 1.4. Summary We have shown that the zebrafish genome encodes two Van Gogh/Strabismus homologues, Vangl1 and Tri/Vangl2, which differ in their expression patterns but may have similar biochemical activities. While Tri/Vangl2 is broadly expressed throughout embryogenesis and functions in diverse processes such as gastrulation movements and neuronal migration, vangl1 expression begins later and appears to be restricted to the nervous system. It will now be important to dissect further the function of Vangl1 using loss-of-function experiments.
All work involving zebrafish was conducted in compliance with rules set forth by the Vanderbilt Institutional Animal Care and Use Committee. Zebrafish embryos were produced by natural matings and adults maintained as described (Solnica-Krezel and Driever, 1994). Embryo staging was performed according to morphology as described (Kimmel et al., 1995). To clone vangl1 we searched the zebrafish Ensembl Genome Server (www. ensembl.org/Danio_rerio/) and EST (www.ncbi.nih.gov/ BLAST/) databases using tri/vangl2 sequence. Two ESTs were identified that were similar to the 30 end of tri/vangl2 (fv22b07.y1 and fu40c01.y3). The vangl1 full-length open reading frame was obtained by performing 50 RACE. Primers flanking the ATG initiation and stop codons were used to PCR amplify the entire gene from cDNA for subsequent subcloning into an expression vector and DNA sequencing. Chromosomal mapping was performed by PCR screening of the Goodfellow T51 radiation hybrid panel (Invitrogen, Carlsbad, CA) using two sets of genomic primers corresponding to different vangl1 exons. The mapping results were confirmed using the zebrafish Ensembl Genome Server to identify mapped genomic markers located near vangl1. Whole-mount in situ hybridization was performed as described using full-length vangl1 cDNA as a probe (Westerfield, 1995). Phylogenetic analysis was carried out using the Phylodendron program (http://iubio.bio.indiana. edu/treeapp/treeprint-form.html). Frozen sections were made using a cryostat and embryos labeled by wholemount in situ hybridization. For rescue experiments, embryos from a clutch obtained from trim209=þ parents were injected with synthetic vangl1 RNA as described (Marlow et al., 1998). Sense-capped RNA was synthesized using mMessage mMachine (Ambion, Austin, TX) after template linearization by NotI digestion. Injected embryos scored for suppression of the trim209 mutant phenotype were PCR genotyped as described (Jessen et al., 2002). Acknowledgements We thank J. Clanton for skillful sectioning and excellent fish care. J.R.J. is supported by a National Institutes of Health Vascular Biology Training Grant (T32HL07751). Work in the L.S.-K. lab is supported by NIH grant GM55101. References Bastock, R., Strutt, H., Strutt, D., 2003. Strabismus is asymmetrically localised and binds Prickle and Dishevelled during Drosophila planar polarity patterning. Development 130, 3007– 3014. Bingham, S., Higashijima, S., Okamoto, H., Chandrasekhar, A., 2002. The zebrafish trilobite gene is essential for tangential migration of branchiomotor neurons. Dev. Biol. 242, 149 –160. Carreira-Barbosa, F., Concha, M.L., Takeuchi, M., Ueno, N., Wilson, S.W., Tada, M., 2003. Prickle1 regulates cell movements during
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