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Molecular cloning and developmental expression of Tlx (Hox11) genes in zebrafish (Danio rerio)
Mechanisms of Development 117 (2002) 243–248 www.elsevier.com/locate/modo
Gene expression pattern
Molecular cloning and developmental expression of Tlx (Hox11) genes in zebrafish (Danio rerio) D.M. Langenau a,b,1, T. Palomero a,b,1, J.P. Kanki a,b,*, A.A. Ferrando a,b, Y. Zhou c, L.I. Zon c, A.T. Look a,b a
Department of Pediatric Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA b Department of Pathology, Harvard Medical School, Boston, MA 02115, USA c Howard Hughes Medical Institute, Division of Hematology/Oncology, Children’s Hospital, Boston, MA 02115, USA Received 25 February 2002; received in revised form 19 April 2002; accepted 19 April 2002
Abstract Tlx (Hox11) genes are orphan homeobox genes that play critical roles in the regulation of early developmental processes in vertebrates. Here, we report the identification and expression patterns of three members of the zebrafish Tlx family. These genes share similar, but not identical, expression patterns with other vertebrate Tlx-1 and Tlx-3 genes. Tlx-1 is expressed early in the developing hindbrain and pharyngeal arches, and later in the putative splenic primordium. However, unlike its orthologues, zebrafish Tlx-1 is not expressed in the cranial sensory ganglia or spinal cord. Two homologues of Tlx-3 were identified: Tlx-3a and Tlx-3b, which are both expressed in discrete regions of the developing nervous system, including the cranial sensory ganglia and Rohon–Beard neurons. However, only Tlx-3a is expressed in the statoacoustic cranial ganglia, enteric neurons and non-neural tissues such as the fin bud and pharyngeal arches and Tlx-3b is only expressed in the dorsal root ganglia. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tlx-1; Tlx-3a; Tlx-3b; Tlx-A; Pax2.1; Krox20; Narrowminded; Zebrafish; Cranial sensory ganglia; Dorsal root ganglia; Rohon–Beard neurons; Spleen
1. Results and discussion The Hox11-related genes are a family of orphan homeobox transcription factors located outside the Hox genes paralog clusters and involved in a variety of developmental pathways. While Tlx-1/Hox11 is essential for spleen development (Roberts et al., 1994, 1995; Dear et al., 1995), Tlx-2 regulates enteric neuron specification (Hatano et al., 1997; Shirasawa et al., 1997) and Tlx-3 affects development of the nucleus of the solitary tract and noradrenergic centers that control respiration rate (Shirasawa et al., 2000; Qian et al., 2001). 1.1. Molecular analysis of zebrafish Tlx genes A degenerate PCR strategy was used to isolate members of the zebrafish Tlx gene family. All three identified genes encode proteins with high amino acid similarity to their corresponding vertebrate orthologues (Fig. 1A, B). However, Tlx-3b shares a higher degree of amino acid iden-
tity with vertebrate Tlx-3 proteins than Tlx-3a. Phylogenetic analysis (Fig. 2A) places zebrafish Tlx-1 and Tlx-3b within different clades, clustered with other Tlx-1 and Tlx-3 family members, respectively, while Tlx-3a falls outside either of these groups. Recently, Andermann and Weinberg (2001) described a zebrafish Tlx gene (Tlx-A) corresponding to Tlx3a in amino acid sequence and phylogenetic assignment. All zebrafish Tlx family members map to conserved syntenic regions in the human genome (Fig. 2B, C). Specifically, zebrafish Tlx-1 maps to zebrafish chromosome 13, in a chromosomal region sharing partial synteny with the human chromosome 10q24 region where HOX11/TLX-1 is mapped (Dube et al., 1991; Hatano et al., 1991; Kennedy et al., 1991) (Fig. 2B). Similarly, zebrafish Tlx-3a and Tlx-3b map to zebrafish chromosomes 14 and 10, respectively, in regions with partial synteny to human chromosome 5q3435, where HOX11L2/TLX-3 is located (Fig. 2C) (Lee-Kirsch et al., 2001; Cinti et al., 2001). 1.2. Expression of zebrafish Tlx genes
* Corresponding author. Tel.: 11-617-632-4476; fax: 11-617-632-3546. E-mail address: [email protected] (J.P. Kanki). 1 These authors contributed equally to this work.
Tlx-1 expression is detected by 24 hour postfertilization (hpf) in the presumptive pharyngeal arches and neurons
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00187-9
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D.M. Langenau et al. / Mechanisms of Development 117 (2002) 243–248
Fig. 1. Amino acid alignment of vertebrate Tlx family members. (A) Alignment of Tlx-1 proteins from zebrafish (z), Xenopus (x), chicken (c) and human (h). Two zebrafish Tlx-1 transcripts of 2223 and 2187 bp were obtained, representing two alternative spliced RNAs that presumably encode 302 and 290 amino acid proteins, respectively. Both splice forms have been identified in human and mouse. Amino acids deleted in the alternative spliced form are indicated ( ^ ). (B) Alignment of zebrafish, Xenopus, chicken, human Tlx-3 proteins and human TLX-2. The 1780 bp Tlx-3a cDNA encodes a 301 amino acid protein, while the 2289 bp Tlx-3b cDNA encodes a protein 300 amino acids in length. In both (A) and (B), the FPWM-PBX binding domain is shown by (#) and gaps introduced to maximize alignment are indicated (·). Homeobox domain is underlined, and gray boxes indicate four highly conserved TH domains. Amino acids conserved across all species are indicated (*), while amino acid residues conserved among Tlx-3 family members that are not conserved in TLX-2 are designated (1). Sequence alignments were made using GCG. The GenBank accession numbers for the zebrafish Tlx genes are Tlx-1/Hox11 #AF398517, Tlx-3a #AF398518 and Tlx-3b #AF398519.
D.M. Langenau et al. / Mechanisms of Development 117 (2002) 243–248
Fig. 2. Phylogenetic analysis of vertebrate (see Fig. 1) and Drosophila Tlx genes and chromosomal synteny between zebrafish and human TLX family members. (A) Phylogenetic analysis places the Tlx-1 proteins within one clade and Tlx-3 proteins in another. Tlx-3a fails to group with either Tlx-1 or Tlx-3 family members. (B) Synteny between the Tlx-1 genes in human and zebrafish. Zebrafish Tlx-1 maps to chromosome 13 in the vicinity of pax2.1 (Accession # x63961), TATA-binding protein associated factor 170 (TAFII, fb79b10), Lim-domain binding factor 4 (LDB4, af031378), fibroblast growth factor 8 (fgf8, af034264) and retinol-binding protein III (rbpIII, fb69e02), whose human homologues map close to Hox11, on chromosome 10q24. (C) Zebrafish Tlx-3a and Tlx-3b map to chromosome 14 and 10, respectively. Zebrafish Tlx-3a lies in close proximity to homologues of protocadherin gamma A11 (fc45f08), CD74 antigen (fb12a01), heterogeneous nuclear riboprotein A/B (hnrnpA/B, fa18g02), histidyl-tRNA synthetase (RH-zonmapper# CHUNP228, 255), and ubiquitin-conjugating enzyme E2D2 (RH-zonmapper# CHUNP 235, 248). Tlx-3b lies close to eukaryotic peptide chain release factor subunit 1 (eRF, fb52g05) and nucleophosmin (fb38d12). The human homologues of these genes map to chromosome 5, close to Hox11L2 at 5q34-35. Phylogenetic analysis was completed using Jotun Hein and Clustal multiple alignment algorithms (MegAlign). Tlx genes were mapped on the Goodfellow Radiation hybrid panel using 3 0 untranslated sequences (ZonRHmapper).
segmentally distributed in the hindbrain and cerebellum (Fig. 3A–C, Table 1). Between 48 and 72 hpf, neural
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Fig. 3. Tlx-1 expression during zebrafish development. In situ hybridization analysis of Tlx-1 RNA expression in 30 hpf (A–C), 48 hpf (D–F) and 5 dpf zebrafish embryos (G,H). (A,D,G): lateral views with anterior to the left. (B,E,H): dorsal views with anterior to the left. (C,F): frontal views with dorsal up. The abbreviations correspond to: hb, hindbrain (arrowhead); cb, cerebellum (arrowhead); PA, pharyngeal arches (arrow); Sp, spleen primordia (asterisk). Scale bars (lower left) ¼ 100 mm.
expression decreases overall (Fig. 3D–F), and is barely detectable by 5 days postfertilization (dpf), except for a subset of cerebellar neurons (Fig. 3G, H). By 5 dpf, Tlx-1 is detected asymmetrically on the left side of the developing anterior gut, in a position corresponding to the splenic primordia. The developmental expression pattern of Tlx-1 is highly conserved between species (Raju et al., 1993; Logan et al., 1998; Patterson and Krieg, 1999) and similar to its vertebrate homologues, zebrafish Tlx-1 is expressed in the developing hindbrain, branchial arches and splenic primordia. Notably, expression of Tlx-1 in the cranial
Table 1 Summary of zebrafish Tlx genes expression domains a Stage
Tlx-1/Hox11
Tlx-3a
Tlx-3b
3-somite 8-somite 18 hpf 24 hpf 35 hpf 48 hpf 72 hpf 5 dpf
N/D N/D PA, hb PA, hb, cb N/A PA, hb, cb PA, hb, cb PA, hb, cb, Sp
RB, Tg RB, Tg RB, Tg, ALL RB, Tg, ALL, SA, hb, PA RB, Tg, ALL, SA, hb, PA, npc, E Tg, ALL, SA, hb, PA, npc, E, pf, cb,en Tg, SA, hb, PA, npc, E, pf, cb, en Tg, PA, E, pf
RB, Tg RB, Tg RB, Tg, ALL RB, Tg, ALL, hb RB, Tg, ALL, hb, npc, E, DRG Tg, ALL, hb, npc, E, DRG, cb Tg, hb, npc, E, cb Tg, hb, cb
a Tlx genes are indicated at the top of each column and the stage of development is listed to the left. Abbreviations: RB, Rohon–Beard neurons; Tg, trigeminal ganglia (V); hb, hindbrain; PA, pharyngeal arches; ALL, anterior lateral line; cb, cerebellum; SA, statoacoustic ganglia (VIII); npc, nucleus of the posterior commissure; E, epibranchial ganglia (IX/X); DRG, dorsal root ganglia; pf, pectoral fin; en, enteric neurons; Sp, spleen; N/A, not assayed; N/D, not detected.
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sensory ganglia or spinal cord is not observed in the zebrafish. Although Tlx-1 transcripts were not detected in the developing spleen of Xenopus (Patterson and Krieg, 1999), our results strongly support the evolutionary conservation of Tlx-1 gene function in spleen development (Logan et al., 1998) and provide the earliest marker of splenogenesis to be identified in zebrafish. Tlx-3a and Tlx-3b transcripts are detected at the 3-somite stage in two bilateral stripes of cells corresponding to developing neurons within the neural plate. At this early stage, these cells are likely to be Rohon–Beard sensory neurons. The anterior group of cells represents the presumptive trigeminal sensory ganglia (Fig. 4A, B, G, H, Table 1). At 18 hpf, both genes are expressed in the Rohon–Beard neurons along the trunk, the trigeminal ganglia, and the
Fig. 5. Tlx-3a and Tlx-3b RNA expression during zebrafish development. Expression of Tlx-3a at 48, 72 hpf and 5 dpf, respectively (A–C). Expression of Tlx-3b at 48, 72 hpf and 5 dpf, respectively (D–F). Abbreviations: cb, cerebellum; pf, pectoral fin. Other abbreviations and scale bars are the same as in Fig. 4. Lateral views with dorsal upward and anterior to the left.
anterior lateral line. By 22–24 hpf, Tlx-3a and Tlx-3b expression partially overlaps, with both genes being expressed in the trigeminal and anterior lateral line ganglia, and segmentally distributed neurons of the hindbrain (Fig.
Fig. 4. Analysis of Tlx-3a and Tlx-3b RNA expression in early developing zebrafish. Expression of Tlx-3a in a 3-somite embryo (A,B). Expression of Tlx-3a (blue) and Pax2.1 and Krox20 (red) in a 22 hpf embryo (C,D). The box in (C) shows a magnified view of the dorsal spinal cord and the discrete expression of Tlx-3a in Rohon–Beard neurons and Pax2.1 in commissural neurons. Expression of Tlx-3a in the nrm mutant (F) and a wild-type sibling (E) at 20 hpf. Note the lack of expression in the trunk Rohon–Beard neurons in the mutant (asterisk) while anterior expression in the cranial sensory ganglia and hindbrain remains unaffected. Similar results were obtained for Tlx-3b (data not shown). Expression of Tlx-3b at the 3-somite stage (G,H). Expression of Tlx-3b (blue) at 24 hpf (J,L) and at 35 hpf (I,K). Expression of Pax2.1 and Krox20 in red in (J) and Pax2.1 (red) alone in (I,K). Panels A, C, E–G, I, K, L are lateral views with dorsal upwards and anterior to the left. Panel B and H are dorsal views with anterior upwards, and Panel D and J are dorsal views with anterior to the left. Abbreviations: Tg, trigeminal ganglia (V); RB, Rohon–Beard neurons; SA, statoacoustic ganglia (VIII); ALL, anterior lateral line; E, epibranchial ganglia (IX/X); PA, pharyngeal arches; Cm, commissural neurons; npc, nucleus of the posterior commissure; m/h, midbrain–hindbrain boundary; hb, hindbrain; r3, rhombomere 3; r5, rhombomere 5; ov, otic vesicle; os, optic stalk; pd, pronephric duct; DRG, dorsal root ganglia. Presumptive Rohon–Beard neurons and trigeminal ganglia are indicated in parentheses (A,B,G,H). Scale bars (lower left) ¼ 100 mm.
D.M. Langenau et al. / Mechanisms of Development 117 (2002) 243–248
4D, L). However, only Tlx-3a is detected in the statoacoustic ganglia and pharyngeal arches (Fig. 4C, D). Both genes are also specifically expressed in Rohon–Beard neurons (Fig. 4C, E, L), which can be distinguished from Pax2.1-expressing commissural interneurons in the spinal cord (Fig. 4C, insert) (Mikkola et al., 1992). Furthermore, Tlx-3a and Tlx3b expressions were not detected in the spinal cord of the narrowminded zebrafish mutant, which lack Rohon–Beard neurons in the trunk (compare Fig. 4E and F, asterisk) but exhibit normal commissural interneurons (Artinger et al., 1999). Both Tlx-3 genes are also expressed in a subset of diencephalic neurons, comprising the nucleus of the posterior commissure (npc) (Figs. 4C, I and 5A, B, D, E). Double in situ hybridization assays show that both Tlx-3 genes are coexpressed in the npc with islet-1, a gene known to be expressed in these neurons (data not shown) (Inoue et al., 1994). During pharyngula stages, expression of both genes is seen in prominent clusters of hindbrain neurons forming transversely oriented columns, and expression in the epibranchial ganglia increases dramatically (Fig. 5A, D). Between 30–48 hpf, Tlx-3b is distinguished by its segmental expression in the developing dorsal root ganglia (DRG) along the trunk, extending posteriorly to the end of the hindyolk (Fig. 4K). At least one Tlx-family member is expressed in the DRGs of zebrafish, mouse, and chicken, suggesting that the Tlx genes may have a conserved function in vertebrate DRG-development. By 48 hpf, the expression of Tlx-3 genes is lost in Rohon–Beard neurons. Between 48 and 72 hpf, Tlx-3a is detected in cells along the developing gut (data not shown), consistent with its reported expression in enteric neurons (Andermann and Weinberg, 2001). Unlike Tlx-3b, and other known vertebrate Tlx-3 genes (Logan et al., 1998, Uchiyama et al., 1999, Patterson and Krieg, 1999, Roberts et al., 1995), Tlx-3a is expressed in non-neuronal tissues including the pectoral fin buds and the pharyngeal arches, particularly those associated with the epibranchial ganglia (Fig. 5A–C). The expression of Tlx-3a begins to decline at 72 hpf and by 5 dpf only remains in the trigeminal, epibranchial ganglia and gill arches while fading in the pectoral fin and statoacoustic ganglia (Fig. 5B, C). Similarly, Tlx-3b also diminishes in the cranial sensory ganglia between 72 hpf and 5 dpf, however, expression in the posterior hindbrain persists and expression in the dorsal cerebellum increases over this period. The temporal and spatial expression of Tlx3a is consistent with that of zebrafish Tlx-A (Andermann and Weinberg, 2001), however, we were unable to detect expression in the semicircular canals. Phylogenetic analysis suggests that Tlx-3a and Tlx-3b arose from a duplication of an ancestral Tlx-3-like gene. The mapping of both Tlx-3 genes to regions syntenic with human HOX11L2/TLX-3, and the similarity of expression patterns, supports this hypothesis and suggests that both are true members of the Tlx-3 gene family. However, only Tlx3b exhibits restricted, neural-specific expression and clus-
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ters phylogenetically with other vertebrate Tlx-3 homologues. Thus, Tlx-3b is more likely to be the functional Tlx3 homologue in zebrafish. Determining the developmental roles of these Tlx-3 genes may provide insight into the diversification of function resulting from the duplication of genes in the zebrafish genome.
2. Methods Degenerate PCR was used to amplify DNA fragments from cDNA prepared from 1–5 day zebrafish embryos. These fragments were then used to isolate full-length sequences, using RACE PCR (SMART RACE Marathon kit, Clontech) for Tlx-1 and Tlx-3a, or by screening a zebrafish brain cDNA library (RZPD, Heidelberg, Germany) for Tlx-3b. Whole-mount in situ hybridization assays were performed as described (Bennett et al., 2001).
Acknowledgements We would like to thank Dr Kristin B. Artinger for the zebrafish nmd mutants, Dr Steve Wilson for the Pax2.1 probe and Drs David Raible, Cecilia Moens and Qiufu Ma for helpful discussions. This work was supported by NIH grant CA68484. D.M.L. was supported by a NSF-predoctoral fellowship, T.P. was supported by a Postdoctoral Fellowship from Ministerio de Educacion (Spain) and A.A.F. is a Fellow of the Leukemia and Lymphoma Society.
References Andermann, P., Weinberg, E.S., 2001. Expression of zTlxA, a Hox11-like gene, in early differentiating embryonic neurons and cranial sensory ganglia of the zebrafish embryo. Dev. Dyn. 222, 595–610. Artinger, K.B., Chitnis, A.B., Mercola, M., Driever, W., 1999. Zebrafish narrowminded suggests a genetic link between formation of neural crest and primary sensory neurons. Development 126, 3969–3979. Bennett, C.M., Kanki, J.P., Rhodes, J., Liu, T.X., Paw, B.H., Kieran, M.W., Langenau, D.M., Delahaye-Brown, A., Zon, L.I., Fleming, M., Look, A.T., 2001. Myelopoiesis in the zebrafish, Danio rerio. Blood 98, 643– 651. Cinti, R., Fava, M., Sancandi, M., Matera, I., Ravazzolo, R., Ceccherini, I., 2001. Assignment(1) of the HOX11L2 gene to human chromosome band 5q35.1 and of its murine homolog to mouse chromosome bands 11A4-A5 by in situ hybridization. Cytogenet. Cell. Genet. 92, 354–355. Dear, T.N., Colledge, W.H., Carlton, M.B., Lavenir, I., Larson, T., Smith, A.J., Warren, A.J., Evans, M.J., Sofroniew, M.V., Rabbits, T.H., 1995. The Hox11 gene is essential for cell survival during spleen development. Development 121, 2909–2915. Dube, I.D., Kamel-Reid, S., Yuan, C.C., Lu, M., Wu, X., Corpus, G., Raimondi, S.C., Crist, W.M., Carrol, A.J., Minowada, J., Baker, J.B., 1991. A novel human homeobox gene lies at the chromosome 10 breakpoint in lymphoid neoplasias with chromosomal translocation t(10;14). Blood 78, 2996–3003. Hatano, M., Roberts, C.W., Minden, M., Crist, W.M., Korsmeyer, S.J., 1991. Deregulation of a homeobox gene, HOX11, by the t(10;14) in leukemia. Science 253, 79–82. Hatano, M., Aoki, T., Dezawa, M., Yusa, S., Iitsuka, Y., Koseki, H., Tani-
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guchi, M., Tokuhisa, T., 1997. A novel pathogenesis of megacolon in Ncx/Hox11L1 deficient mice. J. Clin. Invest. 100, 795–801. Inoue, A., Takahashi, M., Hatta, K., Hotta, Y., Okamoto, H., 1994. Developmental regulation of Islet-1 mRNA expression during neuronal differentiation in embryonic zebrafish. Dev. Dyn. 199, 1–11. Kennedy, M.A., Gonzalez-Sarmiento, R., Kees, U.R., Lampert, F., Dear, N., Boehm, T., Rabbits, T.H., 1991. HOX11, a homeobox-containing Tcell oncogene on human chromosome 10q24. Proc. Natl Acad. Sci. USA 88, 8900–8904. Lee-Kirsch, M.A., Engel, K., Paditz, E., Rosen-Wolff, A., Lee, Y.A., Gahr, M., 2001. Assignment(1) of the human homeobox 11-like 2 gene (HOX11L2) to chromosome 5q34-- . q35 by radiation hybrid mapping. Cytogenet. Cell. Genet. 92, 358. Logan, C., Wingate, R.J.T., McKay, I.J., Lumsden, A., 1998. Tlx-1 and Tlx3 homeobox gene expression in cranial sensory ganglia and hindbrain of the chick embryo: markers of patterned conectivity. J. Neurosci. 18, 5389–5402. 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. Patterson, K.D., Krieg, P.A., 1999. Hox11-family genes Xhox11 and Xhox11L2 in Xenopus: Xhox11L2 expression is restricted to a subset of primary sensory neurons. Dev. Dyn. 214, 34–43.
Qian, Y., Fritzsch, B., Shirasawa, S., Chen, C.L., Choi, Y., Ma, Q., 2001. Formation of brainstem (nor)adrenergic centers and first-order relay visceral sensory neurons is dependent on homeodomain protein Rnx/ Tlx3. Genes Dev. 15, 2533–2545. Raju, K., Tang, S., Dube, I.D., Kamel-Reid, S., Bryce, D.M., Breitman, M.L., 1993. Characterization and developmental expression of Tlx-1, the murine homolog of HOX11. Mech. Dev. 44, 51–64. Roberts, C.W., Shutter, J.R., Korsmeyer, S.J., 1994. Hox11 controls the genesis of the spleen. Nature 368, 747–749. Roberts, C.W.M., Sonder, A.M., Lumsden, A., Korsmeyer, S.J., 1995. Developmental expression of Hox11 and specification of splenic cell fate. Am. J. Pathol. 146, 1089–1101. Shirasawa, S., Arata, A., Onimaru, H., Roth, K.A., Brown, G.A., Horning, S., Arata, S., Okumura, K., Sasazuki, T., Korsmayer, S.J., 2000. Rnx deficiency results in congenital central hypoventilation. Nat. Genet. 24, 287–290. Shirasawa, S., Yunker, A.M., Roth, K.A., Brown, G.A., Horning, S., Korsmeyer, S.J., 1997. Enx (Hox11L1)-deficient mice develop myenteric neuronal hyperplasia and megacolon. Nat. Med. 3, 646–650. Uchiyama, K., Otsuka, R., Hanaoka, K., 1999. Chox11L2, a Hox11 related gene, is expressed in the peripheral nervous system and subpopulation of the spinal cord during chick development. Neurosci. Lett. 273, 97– 100.
Gene expression pattern
Molecular cloning and developmental expression of Tlx (Hox11) genes in zebrafish (Danio rerio) D.M. Langenau a,b,1, T. Palomero a,b,1, J.P. Kanki a,b,*, A.A. Ferrando a,b, Y. Zhou c, L.I. Zon c, A.T. Look a,b a
Department of Pediatric Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA b Department of Pathology, Harvard Medical School, Boston, MA 02115, USA c Howard Hughes Medical Institute, Division of Hematology/Oncology, Children’s Hospital, Boston, MA 02115, USA Received 25 February 2002; received in revised form 19 April 2002; accepted 19 April 2002
Abstract Tlx (Hox11) genes are orphan homeobox genes that play critical roles in the regulation of early developmental processes in vertebrates. Here, we report the identification and expression patterns of three members of the zebrafish Tlx family. These genes share similar, but not identical, expression patterns with other vertebrate Tlx-1 and Tlx-3 genes. Tlx-1 is expressed early in the developing hindbrain and pharyngeal arches, and later in the putative splenic primordium. However, unlike its orthologues, zebrafish Tlx-1 is not expressed in the cranial sensory ganglia or spinal cord. Two homologues of Tlx-3 were identified: Tlx-3a and Tlx-3b, which are both expressed in discrete regions of the developing nervous system, including the cranial sensory ganglia and Rohon–Beard neurons. However, only Tlx-3a is expressed in the statoacoustic cranial ganglia, enteric neurons and non-neural tissues such as the fin bud and pharyngeal arches and Tlx-3b is only expressed in the dorsal root ganglia. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tlx-1; Tlx-3a; Tlx-3b; Tlx-A; Pax2.1; Krox20; Narrowminded; Zebrafish; Cranial sensory ganglia; Dorsal root ganglia; Rohon–Beard neurons; Spleen
1. Results and discussion The Hox11-related genes are a family of orphan homeobox transcription factors located outside the Hox genes paralog clusters and involved in a variety of developmental pathways. While Tlx-1/Hox11 is essential for spleen development (Roberts et al., 1994, 1995; Dear et al., 1995), Tlx-2 regulates enteric neuron specification (Hatano et al., 1997; Shirasawa et al., 1997) and Tlx-3 affects development of the nucleus of the solitary tract and noradrenergic centers that control respiration rate (Shirasawa et al., 2000; Qian et al., 2001). 1.1. Molecular analysis of zebrafish Tlx genes A degenerate PCR strategy was used to isolate members of the zebrafish Tlx gene family. All three identified genes encode proteins with high amino acid similarity to their corresponding vertebrate orthologues (Fig. 1A, B). However, Tlx-3b shares a higher degree of amino acid iden-
tity with vertebrate Tlx-3 proteins than Tlx-3a. Phylogenetic analysis (Fig. 2A) places zebrafish Tlx-1 and Tlx-3b within different clades, clustered with other Tlx-1 and Tlx-3 family members, respectively, while Tlx-3a falls outside either of these groups. Recently, Andermann and Weinberg (2001) described a zebrafish Tlx gene (Tlx-A) corresponding to Tlx3a in amino acid sequence and phylogenetic assignment. All zebrafish Tlx family members map to conserved syntenic regions in the human genome (Fig. 2B, C). Specifically, zebrafish Tlx-1 maps to zebrafish chromosome 13, in a chromosomal region sharing partial synteny with the human chromosome 10q24 region where HOX11/TLX-1 is mapped (Dube et al., 1991; Hatano et al., 1991; Kennedy et al., 1991) (Fig. 2B). Similarly, zebrafish Tlx-3a and Tlx-3b map to zebrafish chromosomes 14 and 10, respectively, in regions with partial synteny to human chromosome 5q3435, where HOX11L2/TLX-3 is located (Fig. 2C) (Lee-Kirsch et al., 2001; Cinti et al., 2001). 1.2. Expression of zebrafish Tlx genes
* Corresponding author. Tel.: 11-617-632-4476; fax: 11-617-632-3546. E-mail address: [email protected] (J.P. Kanki). 1 These authors contributed equally to this work.
Tlx-1 expression is detected by 24 hour postfertilization (hpf) in the presumptive pharyngeal arches and neurons
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00187-9
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D.M. Langenau et al. / Mechanisms of Development 117 (2002) 243–248
Fig. 1. Amino acid alignment of vertebrate Tlx family members. (A) Alignment of Tlx-1 proteins from zebrafish (z), Xenopus (x), chicken (c) and human (h). Two zebrafish Tlx-1 transcripts of 2223 and 2187 bp were obtained, representing two alternative spliced RNAs that presumably encode 302 and 290 amino acid proteins, respectively. Both splice forms have been identified in human and mouse. Amino acids deleted in the alternative spliced form are indicated ( ^ ). (B) Alignment of zebrafish, Xenopus, chicken, human Tlx-3 proteins and human TLX-2. The 1780 bp Tlx-3a cDNA encodes a 301 amino acid protein, while the 2289 bp Tlx-3b cDNA encodes a protein 300 amino acids in length. In both (A) and (B), the FPWM-PBX binding domain is shown by (#) and gaps introduced to maximize alignment are indicated (·). Homeobox domain is underlined, and gray boxes indicate four highly conserved TH domains. Amino acids conserved across all species are indicated (*), while amino acid residues conserved among Tlx-3 family members that are not conserved in TLX-2 are designated (1). Sequence alignments were made using GCG. The GenBank accession numbers for the zebrafish Tlx genes are Tlx-1/Hox11 #AF398517, Tlx-3a #AF398518 and Tlx-3b #AF398519.
D.M. Langenau et al. / Mechanisms of Development 117 (2002) 243–248
Fig. 2. Phylogenetic analysis of vertebrate (see Fig. 1) and Drosophila Tlx genes and chromosomal synteny between zebrafish and human TLX family members. (A) Phylogenetic analysis places the Tlx-1 proteins within one clade and Tlx-3 proteins in another. Tlx-3a fails to group with either Tlx-1 or Tlx-3 family members. (B) Synteny between the Tlx-1 genes in human and zebrafish. Zebrafish Tlx-1 maps to chromosome 13 in the vicinity of pax2.1 (Accession # x63961), TATA-binding protein associated factor 170 (TAFII, fb79b10), Lim-domain binding factor 4 (LDB4, af031378), fibroblast growth factor 8 (fgf8, af034264) and retinol-binding protein III (rbpIII, fb69e02), whose human homologues map close to Hox11, on chromosome 10q24. (C) Zebrafish Tlx-3a and Tlx-3b map to chromosome 14 and 10, respectively. Zebrafish Tlx-3a lies in close proximity to homologues of protocadherin gamma A11 (fc45f08), CD74 antigen (fb12a01), heterogeneous nuclear riboprotein A/B (hnrnpA/B, fa18g02), histidyl-tRNA synthetase (RH-zonmapper# CHUNP228, 255), and ubiquitin-conjugating enzyme E2D2 (RH-zonmapper# CHUNP 235, 248). Tlx-3b lies close to eukaryotic peptide chain release factor subunit 1 (eRF, fb52g05) and nucleophosmin (fb38d12). The human homologues of these genes map to chromosome 5, close to Hox11L2 at 5q34-35. Phylogenetic analysis was completed using Jotun Hein and Clustal multiple alignment algorithms (MegAlign). Tlx genes were mapped on the Goodfellow Radiation hybrid panel using 3 0 untranslated sequences (ZonRHmapper).
segmentally distributed in the hindbrain and cerebellum (Fig. 3A–C, Table 1). Between 48 and 72 hpf, neural
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Fig. 3. Tlx-1 expression during zebrafish development. In situ hybridization analysis of Tlx-1 RNA expression in 30 hpf (A–C), 48 hpf (D–F) and 5 dpf zebrafish embryos (G,H). (A,D,G): lateral views with anterior to the left. (B,E,H): dorsal views with anterior to the left. (C,F): frontal views with dorsal up. The abbreviations correspond to: hb, hindbrain (arrowhead); cb, cerebellum (arrowhead); PA, pharyngeal arches (arrow); Sp, spleen primordia (asterisk). Scale bars (lower left) ¼ 100 mm.
expression decreases overall (Fig. 3D–F), and is barely detectable by 5 days postfertilization (dpf), except for a subset of cerebellar neurons (Fig. 3G, H). By 5 dpf, Tlx-1 is detected asymmetrically on the left side of the developing anterior gut, in a position corresponding to the splenic primordia. The developmental expression pattern of Tlx-1 is highly conserved between species (Raju et al., 1993; Logan et al., 1998; Patterson and Krieg, 1999) and similar to its vertebrate homologues, zebrafish Tlx-1 is expressed in the developing hindbrain, branchial arches and splenic primordia. Notably, expression of Tlx-1 in the cranial
Table 1 Summary of zebrafish Tlx genes expression domains a Stage
Tlx-1/Hox11
Tlx-3a
Tlx-3b
3-somite 8-somite 18 hpf 24 hpf 35 hpf 48 hpf 72 hpf 5 dpf
N/D N/D PA, hb PA, hb, cb N/A PA, hb, cb PA, hb, cb PA, hb, cb, Sp
RB, Tg RB, Tg RB, Tg, ALL RB, Tg, ALL, SA, hb, PA RB, Tg, ALL, SA, hb, PA, npc, E Tg, ALL, SA, hb, PA, npc, E, pf, cb,en Tg, SA, hb, PA, npc, E, pf, cb, en Tg, PA, E, pf
RB, Tg RB, Tg RB, Tg, ALL RB, Tg, ALL, hb RB, Tg, ALL, hb, npc, E, DRG Tg, ALL, hb, npc, E, DRG, cb Tg, hb, npc, E, cb Tg, hb, cb
a Tlx genes are indicated at the top of each column and the stage of development is listed to the left. Abbreviations: RB, Rohon–Beard neurons; Tg, trigeminal ganglia (V); hb, hindbrain; PA, pharyngeal arches; ALL, anterior lateral line; cb, cerebellum; SA, statoacoustic ganglia (VIII); npc, nucleus of the posterior commissure; E, epibranchial ganglia (IX/X); DRG, dorsal root ganglia; pf, pectoral fin; en, enteric neurons; Sp, spleen; N/A, not assayed; N/D, not detected.
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sensory ganglia or spinal cord is not observed in the zebrafish. Although Tlx-1 transcripts were not detected in the developing spleen of Xenopus (Patterson and Krieg, 1999), our results strongly support the evolutionary conservation of Tlx-1 gene function in spleen development (Logan et al., 1998) and provide the earliest marker of splenogenesis to be identified in zebrafish. Tlx-3a and Tlx-3b transcripts are detected at the 3-somite stage in two bilateral stripes of cells corresponding to developing neurons within the neural plate. At this early stage, these cells are likely to be Rohon–Beard sensory neurons. The anterior group of cells represents the presumptive trigeminal sensory ganglia (Fig. 4A, B, G, H, Table 1). At 18 hpf, both genes are expressed in the Rohon–Beard neurons along the trunk, the trigeminal ganglia, and the
Fig. 5. Tlx-3a and Tlx-3b RNA expression during zebrafish development. Expression of Tlx-3a at 48, 72 hpf and 5 dpf, respectively (A–C). Expression of Tlx-3b at 48, 72 hpf and 5 dpf, respectively (D–F). Abbreviations: cb, cerebellum; pf, pectoral fin. Other abbreviations and scale bars are the same as in Fig. 4. Lateral views with dorsal upward and anterior to the left.
anterior lateral line. By 22–24 hpf, Tlx-3a and Tlx-3b expression partially overlaps, with both genes being expressed in the trigeminal and anterior lateral line ganglia, and segmentally distributed neurons of the hindbrain (Fig.
Fig. 4. Analysis of Tlx-3a and Tlx-3b RNA expression in early developing zebrafish. Expression of Tlx-3a in a 3-somite embryo (A,B). Expression of Tlx-3a (blue) and Pax2.1 and Krox20 (red) in a 22 hpf embryo (C,D). The box in (C) shows a magnified view of the dorsal spinal cord and the discrete expression of Tlx-3a in Rohon–Beard neurons and Pax2.1 in commissural neurons. Expression of Tlx-3a in the nrm mutant (F) and a wild-type sibling (E) at 20 hpf. Note the lack of expression in the trunk Rohon–Beard neurons in the mutant (asterisk) while anterior expression in the cranial sensory ganglia and hindbrain remains unaffected. Similar results were obtained for Tlx-3b (data not shown). Expression of Tlx-3b at the 3-somite stage (G,H). Expression of Tlx-3b (blue) at 24 hpf (J,L) and at 35 hpf (I,K). Expression of Pax2.1 and Krox20 in red in (J) and Pax2.1 (red) alone in (I,K). Panels A, C, E–G, I, K, L are lateral views with dorsal upwards and anterior to the left. Panel B and H are dorsal views with anterior upwards, and Panel D and J are dorsal views with anterior to the left. Abbreviations: Tg, trigeminal ganglia (V); RB, Rohon–Beard neurons; SA, statoacoustic ganglia (VIII); ALL, anterior lateral line; E, epibranchial ganglia (IX/X); PA, pharyngeal arches; Cm, commissural neurons; npc, nucleus of the posterior commissure; m/h, midbrain–hindbrain boundary; hb, hindbrain; r3, rhombomere 3; r5, rhombomere 5; ov, otic vesicle; os, optic stalk; pd, pronephric duct; DRG, dorsal root ganglia. Presumptive Rohon–Beard neurons and trigeminal ganglia are indicated in parentheses (A,B,G,H). Scale bars (lower left) ¼ 100 mm.
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4D, L). However, only Tlx-3a is detected in the statoacoustic ganglia and pharyngeal arches (Fig. 4C, D). Both genes are also specifically expressed in Rohon–Beard neurons (Fig. 4C, E, L), which can be distinguished from Pax2.1-expressing commissural interneurons in the spinal cord (Fig. 4C, insert) (Mikkola et al., 1992). Furthermore, Tlx-3a and Tlx3b expressions were not detected in the spinal cord of the narrowminded zebrafish mutant, which lack Rohon–Beard neurons in the trunk (compare Fig. 4E and F, asterisk) but exhibit normal commissural interneurons (Artinger et al., 1999). Both Tlx-3 genes are also expressed in a subset of diencephalic neurons, comprising the nucleus of the posterior commissure (npc) (Figs. 4C, I and 5A, B, D, E). Double in situ hybridization assays show that both Tlx-3 genes are coexpressed in the npc with islet-1, a gene known to be expressed in these neurons (data not shown) (Inoue et al., 1994). During pharyngula stages, expression of both genes is seen in prominent clusters of hindbrain neurons forming transversely oriented columns, and expression in the epibranchial ganglia increases dramatically (Fig. 5A, D). Between 30–48 hpf, Tlx-3b is distinguished by its segmental expression in the developing dorsal root ganglia (DRG) along the trunk, extending posteriorly to the end of the hindyolk (Fig. 4K). At least one Tlx-family member is expressed in the DRGs of zebrafish, mouse, and chicken, suggesting that the Tlx genes may have a conserved function in vertebrate DRG-development. By 48 hpf, the expression of Tlx-3 genes is lost in Rohon–Beard neurons. Between 48 and 72 hpf, Tlx-3a is detected in cells along the developing gut (data not shown), consistent with its reported expression in enteric neurons (Andermann and Weinberg, 2001). Unlike Tlx-3b, and other known vertebrate Tlx-3 genes (Logan et al., 1998, Uchiyama et al., 1999, Patterson and Krieg, 1999, Roberts et al., 1995), Tlx-3a is expressed in non-neuronal tissues including the pectoral fin buds and the pharyngeal arches, particularly those associated with the epibranchial ganglia (Fig. 5A–C). The expression of Tlx-3a begins to decline at 72 hpf and by 5 dpf only remains in the trigeminal, epibranchial ganglia and gill arches while fading in the pectoral fin and statoacoustic ganglia (Fig. 5B, C). Similarly, Tlx-3b also diminishes in the cranial sensory ganglia between 72 hpf and 5 dpf, however, expression in the posterior hindbrain persists and expression in the dorsal cerebellum increases over this period. The temporal and spatial expression of Tlx3a is consistent with that of zebrafish Tlx-A (Andermann and Weinberg, 2001), however, we were unable to detect expression in the semicircular canals. Phylogenetic analysis suggests that Tlx-3a and Tlx-3b arose from a duplication of an ancestral Tlx-3-like gene. The mapping of both Tlx-3 genes to regions syntenic with human HOX11L2/TLX-3, and the similarity of expression patterns, supports this hypothesis and suggests that both are true members of the Tlx-3 gene family. However, only Tlx3b exhibits restricted, neural-specific expression and clus-
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ters phylogenetically with other vertebrate Tlx-3 homologues. Thus, Tlx-3b is more likely to be the functional Tlx3 homologue in zebrafish. Determining the developmental roles of these Tlx-3 genes may provide insight into the diversification of function resulting from the duplication of genes in the zebrafish genome.
2. Methods Degenerate PCR was used to amplify DNA fragments from cDNA prepared from 1–5 day zebrafish embryos. These fragments were then used to isolate full-length sequences, using RACE PCR (SMART RACE Marathon kit, Clontech) for Tlx-1 and Tlx-3a, or by screening a zebrafish brain cDNA library (RZPD, Heidelberg, Germany) for Tlx-3b. Whole-mount in situ hybridization assays were performed as described (Bennett et al., 2001).
Acknowledgements We would like to thank Dr Kristin B. Artinger for the zebrafish nmd mutants, Dr Steve Wilson for the Pax2.1 probe and Drs David Raible, Cecilia Moens and Qiufu Ma for helpful discussions. This work was supported by NIH grant CA68484. D.M.L. was supported by a NSF-predoctoral fellowship, T.P. was supported by a Postdoctoral Fellowship from Ministerio de Educacion (Spain) and A.A.F. is a Fellow of the Leukemia and Lymphoma Society.
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