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Home , DLX4, DLX gene family, Homeobox, Hox gene, Transcription factor

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10858-10863, October 1996

The evolution of the Dlx family (///Hox cluster/ factor) DAVID W. STOCK*t, DEBRA L. ELLIESt, ZHIYONG ZHAO*, MARC EKKERt, FRANK H. RUDDLE§, AND KENNETH M. WEISS*¶ *Department of Anthropology and 1Graduate Program in , Pennsylvania State University, University Park, PA 16802; iLoeb Institute for Medical Research, Ottawa Civic Hospital and University of Ottawa, Ottawa, ON Canada K1Y 4E9; and §Department of , Yale University, New Haven, CT 06520 Contributed by Frank H. Ruddle, July 18, 1996

ABSTRACT The vertebrate Dlx gene family consists of branchial arches, limb buds, forebrain, otic and olfactory homeobox-containing transcription factors distributed in vesicles, teeth, and hair follicles. Functional studies of Dlx pairs on the same as the Hox . To inves- genes are only beginning to appear (30), and their role in the tigate the evolutionary history of Dlx genes, we have cloned development of these structures is not well understood. An five new zebrafish family members and have provided addi- understanding of the evolutionary relationships among verte- tional sequence information for two mouse genes. Phyloge- brate Dlx genes, both for the paralogues within a species as well netic analyses of Dlx gene sequences considered in the context as the orthologues between species, may allow the integration of their chromosomal arrangements suggest that an initial of developmental data from divergent species as well as suggest tandem duplication produced a linked pair of Dlx genes after experiments for testing functional interactions among genes. the divergence of chordates and but prior to the To investigate these relationships, we have determined the divergence oftunicates and . This pair ofDlx genes sequence of five new Dlx genes from the zebrafish and have was then duplicated in the chromosomal events that led to the obtained additional sequence data from two mouse Dlx genes four clusters of Hox genes characteristic of bony fish and whose cloning but not has previously been re- tetrapods. It is possible that a pair of Dlx genes linked to the ported. Phylogenetic analyses of these and other Dlx genes are Hoxc cluster has been lost from mammals. We were unable to consistent with an initial tandem duplication to give two linked distinguish between independent duplication and retention of genes, followed by at least two duplications of this cluster. It the ancestral state of bony vertebrates to explain the presence is most parsimonious to assume that these duplications were of a greater number of Dlx genes in zebrafish than mammals. the same as those that gave rise to the multiple Hox clusters to Determination of the linkage relationship of these additional which the Dlx genes are linked. zebrafish Dlx genes to Hox clusters should help resolve this issue. MATERIALS AND METHODS Ohno that RNA Extraction. RNA was extracted from the head of a (1) proposed multiple rounds of chromosomal single E14.5 mouse (Swiss Webster) (EO.5 = finding duplication (polyploidization) early in vertebrate evolution of the vaginal plug). Zebrafish were obtained from a pet store were directly involved in the morphological diversification of in Monterey, CA, and and larvae were raised at 26°C. the group. This hypothesis has received dramatic support in Zebrafish RNA was extracted from a pool of ten 103-hr recent years with the cloning, mapping, and elucidation of (postfertilization) larvae, as well as from the of an adult developmental function of the -class Hox genes female. All RNA extractions used the RNAzol B reagent (2-8). Mammals have =40 Hox genes arranged in four clusters (Biotecx Laboratories, Houston) according to the manufac- on separate chromosomes. These genes are believed to be turer's instructions. involved in pattern specification along the anterior-posterior Reverse Transcription-PCR. A series of degenerate oligo- axis, with the domain of function of a gene related to its primers was designed from an alignment of position along the . It has been suggested that Dll and vertebrate Dlx sequences obtained from tandem duplications produced an array of Hox genes along a GenBank. The primers 5'-GCCGGGATCCAARCCNMG- single chromosome in a common ancestor of insects and NACNATHTAYTC-3' and 5-TTYTGRAACCADATY- vertebrates, followed by at least two duplications of this array TTNAC-3' (the underlined sequence is a restriction site early in vertebrate evolution (5, 6, 8-12). A number of other added to the 5' end) bind to conserved sequences gene families have members located on multiple Hox- near the amino and carboxy ends, respectively, of the containing chromosomes in vertebrates, suggesting that the homeobox of all Dlx genes examined. Additional primers Hox cluster duplications may have involved entire chromo- located outside of the homeobox and specific for particular somes or (5, 6). One such gene family is a group of Dlx orthologues or groups of orthologues were as follows: homeobox-containing transcription factors homologous to the 5 '-GCCGGATCCGGNAARAARATHMGNAARCC-3' Drosophila Distal-less (Dll) gene (refs. 5, 6, 13-16 and 64; 5 '-GCCGGGATCCGARGARCCNGARNCNGARGT-3', D.L.E., G. Giroux and M.E., unpublished data). 5' -GCCGGAATTCGGNMCYTCNCCRTTYTTRTA-3' The Dll gene of Drosophila is required for the formation of 5 '-GCCGGGATCCATGCAYCAYCCNWSNCARGA-3' the distal portion of the legs, antennae, and mouthparts (17). and 5'-GCCGGAATTCTGNGGNSWRTTRCANGCCAT-3'. Multiple homologues ofDll (Dlx or distal-less-like genes) have Reverse transcription of total RNA was carried out at 42°C been isolated from zebrafish (18), newt (19), (20-23), using Moloney murine leukemia virus reverse transcriptase mouse (24-28, 64), and human (14, 29, 64). These genes are and either random hexamers or an adaptor-dT primer expressed in a variety of sites, many of which are regions of (5'GATGCTGCAGAGCTCAAGC(T)17-3'). PCR of the sin- epithelial-mesenchymal interaction, including the developing gle-stranded cDNA from this reaction typically used the

The publication costs of this article were defrayed in part by page charge Data deposition: The sequences reported in this paper have been payment. This article must therefore be hereby marked "advertisement" in deposited in the GenBank data base (accession nos. U67840-U67846). accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

10858 Downloaded by guest on September 26, 2021 Evolution: Stock et al. Proc. Natl. Acad. Sci. USA 93 (1996) 10859 following thermocycling protocol: denaturation at 940C for 2 assemble these subalignments sequentially into a global align- min, followed by 40 cycles of 1 min at 940C, 1 min at 45-55°C, ment. At each stage of the alignment process, manual adjust- and 1 min at 720C. A final extension was performed at 72°C for ments were made. The final alignment is available from the 5 min. authors on request. PCR Amplification of 5' and 3' Ends of Dlx cDNAs. The 5' Alignment of the sequence upstream of the homeodomain and 3' ends of Dlx cDNAs were obtained by one of two was more difficult than downstream of it because some se- different PCR-based methods. The 5' and 3' ends of mouse quences were incomplete in the former region and there is Dlx5 and the 3' ends of mouse Dbc6 and zebrafish dlxS and evidence for upstream of the homeodo- were obtained by the rapid amplification of cDNA ends main and not downstream of it (ref. 64 and this study). For this (RACE) method essentially as described by Frohman (31). reason we report a phylogenetic analysis using only the ho- The 5' RACE of mouse DlxS used a gene-specific primer for meodomain and the sequence downstream of it. However, reverse transcription, followed by a single PCR reaction on the analysis of the complete sequence gave similar results. Phylo- dA-tailed first strand cDNA with a nested gene-specific primer genetic analyses were as described above except that distances and the dT-adaptor primer described above. PCR cycling were y-corrected (a = 2.0; ref. 38). conditions were similar to those described above except that 35 cycles were used. The 3' RACE PCR was carried out in two steps with an initial 30- PCR being used as the template RESULTS for a second 30-cycle PCR with nested adaptor and gene- Dlx Genes Detected in the Mouse and Zebrafish. We specific primers. Amplification of the 5' ends of zebrafish dlxS sequenced -75 independent homeobox clones from the and dlx6 and the 5' and 3' ends of zebrafish dlxl, dlb7, and dlx8 mouse. We did not detect any new Dlx genes, but found clones was carried out using a Marathon cDNA amplification matching Dlxi, DL&2, D&3, DMS, and D&x6. The cloning, but not (Clontech) according to the manufacturer's instructions, and the sequence, of the latter two genes had been reported essentially as described (64). previously, and the homeodomain amino acid sequences we Cloning and Sequencing. PCR products of interest (identi- determined were identical to the human genes of the same fied by size or by Southern blotting with a labeled oligonucle- name (14). The only known mammal Dlx gene that was not otide) were cloned into pBluescript SK+ (Stratagene) by detected in our survey was Dlx7 (64). We determined the standard methods (32). Double-stranded plasmid DNA was complete cDNA sequence ofDlx5 and the sequence of most of sequenced on an Applied Biosystems model 373 automated the homeobox and 3' end of DLb6. sequencer. Errors inherent in the sequencing of cloned PCR Sequencing a total of =60 homeobox clones from zebrafish products were minimized by sequencing multiple clones of larvae and ovary revealed all three previously reported Dlx each PCR product so that each nucleotide of the coding genes (, , and ; ref. 18) as well as five new ones. We regions was determined from at least five different clones determined the complete cDNA sequence of the five new representing both strands of the DNA. Sequences were ma- genes and have named them based on the phylogenetic anal- nipulated and translated using the programs in the DNASTAR yses reported below. dlxl and dlx6 are obvious orthologues of package (DNAstar, Madison, WI). the mammal genes of the same name (14, 25). We have Phylogenetic Analyses. Amino acid sequences of the follow- designated one new gene dlxS that is a member of a clade ing Dlx genes were obtained from GenBank data base: nem- containing mammal Dlx2, Dlx3, and Dlx5. The previously atode C28A5.4 (accession no. Z32683); Drosophila Dll (17); reported zebrafish dlx4 is the likely orthologue of mammal tunicate (Ciona intestinalis) DII-A and DII-B (33); zebrafish DlxS, but we have not attempted to revise the nomenclature at dlx2, dlx3, and dlx4 (18); Xenopus Xdll (20), X-DLL1 (22), this point. We have designated one new gene dlx7 despite some Xdll-2 (23), X-d112, X-d113, and X-d114 (21); newt (Notophthal- difficulty in inferring its orthology from sequence analyses mus viridescens) NvHBox-4 and NvHBox-5 (19); chicken Dlx5 because it is linked to dbx3 (D.L.E. et al., unpublished), as is (34); rat rDlx (35); mouse Dbcl (25), Dlx2 (24, 26), Dlx3 (27, 28), mammalian Dlx7 (64). The remaining gene was named dlx8 by and Dlx7 (64); and human DLX1, DLX5, DLX6 (14), DLX2 accession. (29), and DLX7 (64). Members of the related Msx (36) and We obtained multiple transcripts differing in sequences Barxl (37) gene families were used as outgroups to upstream of the homeodomain for dLbS, dlx6, dlx7, and dlx8 phylogenetic trees. The necessary assumption that these genes (data not shown). In the case of dlx6, where 5' sequences were are outside of the Dlx family is based on homeodomain amino obtained from both larvae and adult ovary, multiple transcripts acid identities of at least 67% within the Dlx family (77% when were found in the larvae and all of these differed from the type only Drosophila and vertebrate members are considered) found in the ovary. In all cases, the differing transcripts predict compared with identities of 49-62% for Msx and Dlx genes amino acid sequence differences upstream of the homeodo- and 48-51% for Barxl and Dlx genes. main. We have reported the sequences that match most closely Because it was not possible to obtain reliable alignments of other vertebrate Dlx genes, but it is possible that alternative all Dlx and outgroup amino acid sequences outside of the splicing accounts for some of the difficulties in aligning Dlx homeodomain, phylogenetic analyses of the entire gene family genes upstream of the homeodomain. Nakamura et al. (64) were conducted on the homeodomains alone. Alignment of found evidence for alternative splicing upstream of the home- these 61 amino acid regions did not require the addition of odomain of mouse Dlx7. gaps. Pairwise distances were calculated as percent similarities Number of Dlx Paralogues in Vertebrates. Phylogenetic (p-values) and neighbor-joining trees were constructed using analyses of the homeodomain alone and the homeodomain the program MEGA (38). along with downstream amino acid sequences are shown in We have attempted to use phylogenetic information from Figs. 1 and 2, respectively. There are six strongly supported regions outside the homeodomain that are conserved among groups of vertebrate Dlx genes, each containing a single type many of the vertebrate Dlx genes to provide greater resolution of mammalian Dlx gene. These groups designated by their of the relationships among vertebrate Dlx genes. Alignments mammalian member(s) and their bootstrap support (from Fig. of complete Dlx amino acid sequences were constructed using 1; from Fig. 2) are Dlxl (not determined because of identical CLUSTALW (39). Unsatisfactory results from simultaneous sequences; 99%), Dlx2 (86%; 99%), Dlx3 (91%; 100%), Dlx5 multiple alignment of the entire data set led us to construct (83%; 100%), Dlx6 (56%; 100%), and Dlx7 (36%; 83%). independent multiple alignments for the each of the strongly Xenopus Dlx genes fall into four of these six groups, and two supported clades from the phylogenetic analysis of homeodo- of the groups (Dlx2 and Dlx3) contain two Xenopus members. mains. The profile alignment feature of CLUSTALW was used to In both cases, these are highly similar sequences that are most Downloaded by guest on September 26, 2021 10860 Evolution: Stock et al. Proc. Natl. Acad. Sci. USA 93 (1996)

Relationship ofVertebrate Dlx Genes to Protochordate and 66 Xenopus X-DLL1 Human DLX2 Invertebrate Genes. Phylogenetic analysis of potential inver- 86 Xenopus X-dll4 2 tebrate and protochordate homologues ofvertebrate Dlx genes Zebrafish (Fig. 1) shows that the () gene 3 64 Zebrafish dlx2 C28A5.4 clusters outside the remaining eucoelomate Dlx Zebrafish dlx4 genes. This result is not strongly supported (49% of the bootstrap trees), but matches the phylogenetic relationships of 8 Xenopus X-dll3 the . Drosophila Dll and one of the tunicate genes 64 Human DLX5 Chicken (D11-A) form a weakly supported cluster (33% of bootstrap DlxS trees) that itself clusters weakly (25%) with the vertebrate 91 Mouse Dlx3 Dlxl-Dlx6-Dlx7 clade. The remaining tunicate gene (D11-B) Xenopus X-d112 clusters outside all other Dlx genes except that of C. elegans 5'7- Xeebnopfuss Xddbl12 3 with ZebrfishdIx3 moderately high support (74%).

Newt NvHBox4 _ Zebrafish dlx6 DISCUSSION 56 Human DLX6 6 Tandem Duplication of Dlx Genes Followed by Cluster 42 Xenopus Xdll Duplication. Fig. 3 presents a hypothesis of the evolutionary Zebrafish dxi1 history of the vertebrate Dlx gene family, the evidence for Human DLX1 J which is discussed below. An important clue to understanding 99 r Mouse Dbc7 Dlx gene evolution is provided by linkage data. The six L Human DLX7 mammalian Dlx genes are arranged as three pairs of closely 36 88 Newt NvHBox5 7 linked (10-30 kb), convergently transcribed genes (14, 64). Zebrafish dlx7 Each of these clusters is linked to one of the four Hox clusters. 90 70 Zebrafish dlx8 This linkage to Hox clusters is relatively distant where known. Tunicate DII-A The DLX3/DLX7 cluster is 1-2 Mb from HOXB in humans Drosophila DIl (64) and the DLX5/DLX6 cluster and HOXA are on opposite Tunicate DII-B chromosome arms (5, 14). The DLXl/DLX2 and DLX3/DLX7 Nematode C28A5.4 clusters are known to be on the side of the posteriorly 100 I..... -Drosophila msh expressed members of their respective linked Hox clusters (15, Mouse 64). The convergently transcribed configuration of Dlx genes L Mouse Barx-1 Xenopus X-dll2 0.05 Xenopus Xdll-2 Newt NvHBox4 3 FIG. 1. Phylogenetic analysis of Dlx and related homeodomains. Neighbor-joining tree rooted with msh-like and Barxl homeodomains. Zebrfish dlx3 25 Mouse Dlx3 The scale bar indicates a p-distance of 0.05. Numbers at nodes indicate support (1000 Left set of - bootstrap replications). brackets indicate six 00 Zebrafish dIx4 orthologous groups of Dlx genes and the numbers indicate the single Xenopus X-dll3 mammal members of each group. Right set of brackets highlight the clustering of vertebrate Dlx genes into two major groups. Dotted lines 94 Chicken Dlx5 5 indicate adjustments to the tree that are necessary to make it consis- 57 100 Rat rDlx tent with the scenario for Dlx evolution depicted in Fig. 3. Mouse Dlx5 easily explained as gene duplications resulting from the tet- Zebrafish dIx5 raploidization event in the lineage leading to Xenopus (40). All Zeb-efish dlx2 six of the groups defined by a single mammal member contain Mouse Dlx2 2 zebrafish genes and two (Dlx2 and Dlx7) contain two zebrafish 79 99 Xenopus X-DLL1 genes. The phylogeny of the genes within the six groups in Fig. Xenopus X-dll4 2 matches that of the organisms except in the case of Dlx3 and Dlx7. Only in the latter case is the organismal phylogeny strongly contradicted (88% bootstrap support in Fig. 1; 97% in 6 Fig. 2). A Fundamental Split Among Vertebrate Dlx Genes. Fig. 1 1 shows that vertebrate Dlx genes fall into two major clades, one ofwhich is characterized by mammal Dlx2, Dlx3, and Dlx5, and Mouse Dlx7 the other of which contains mammal Dlxl, Dlx6, and Dlx7. - Newt NvHBox5 7 Bootstrap support for these groupings is moderate to low (68% Zebrafish for the former and 44% for the latter). The phylogenetic dIx7 analysis in Fig. 2 provides strong support (98%) for the Zebrafish dIx8 existence of an internal branch separating these two groups, but the absence of an outgroup leaves open the possibility that one (but only one) of the two groups is not monophyletic. As 0.1 discussed below, linkage arrangements of Dlx genes provide a FIG. 2. of homeodomain and strong indication that both of these groups are monophyletic. Phylogenetic analysis carboxy termi- nus of vertebrate Dlx genes. Neighbor-joining tree is rooted according The relationships of the three paralogue groups within each of to Fig. 1. The scale bar indicates a -y-corrected distance of 0.1. Numbers the two major divisions is unresolved because of low bootstrap indicate bootstrap support (1000 replications). Brackets indicate or- support and contradictory results between the analyses in Fig. thologous groups as described for Fig. 1. Arrows indicate evidence for 1 and Fig. 2. independent duplications of Dlx genes in zebrafish depicted in Fig. 3. Downloaded by guest on September 26, 2021 Evolution: Stock et al. Proc. Natl. Acad. Sci. USA 93 (1996) 10861

A P oD-D-o 1 FIG. 3. Scenario for the evolutionary origin of multiple pairs of Dlx genes linked to Hox clusters. Hypothesized genomic arrangement of Hox and Dlx genes are diagrammed at different stages of metazoan evolution. Open boxes, filled boxes, and x's represent Hox genes, Dlx genes, and Dlx gene losses, respectively. Broken lines represent the distant linkage of Hox and Dlx genes. A and P refer to anteriorly expressed and posteriorly expressed Hox genes, respectively. Numbers indicate evolutionary events as follows: 1, ancestral condition of a single Dlx gene distantly linked to the posteriorly-expressed side of a small Hox cluster (drawn as that of C. elegans); 2, tandem duplication of Dlx genes along a single Hox cluster-containing chromosome; 3, disruption of the HOM/Hox cluster and translocation of Dll to a different chromosome; 4, duplication of large genomic regions to produce four Hox clusters linked to four pairs of Dlx genes (drawn as the Hox clusters of mammals); 5, loss of Dlx pair (alternatively these genes remain to be isolated); and 6, two independent Dlx duplications lead to the eight locus condition in zebrafish. The linkage relationships cannot be predicted. Alternatively the "extra" zebrafish genes may represent a retention of the condition resulting from step 4. also applies to at least some of the eight zebrafish genes; HOM/Hox cluster and, like ceh-23,is located on the side of the zebrafish dlxl and dlx2 form a closely linked pair with this cluster containing the most posteriorly expressed gene (A arrangement, as do dlx4 and dlx6 (D.L.E. et al., unpublished Celegans Data Base on the worldwide web). The extensive observations). Analysis of zebrafish/mouse somatic hybrids sequence available flanking both the HOM/Hox cluster and (41) has indicated that the zebrafish dlx3 and dlx7 genes are C28A5.4 (ref. 43; ACEDB) does not contain any other poten- present on the same chromosome as the Hoxa cluster (G. tial Dlx homologues (c.f, the close linkage of vertebrate Dlx Giroux and M.E., unpublished) although the distance sepa- gene pairs). The presence of a single Dlx locus, a single rating dlx3 and dlx7 has yet to be determined precisely. HOM/Hox cluster and the phylogenetic result presented here Each of the three mammalian Dlx linkage groups contains suggest that all Dlx duplications occurred after the divergence a member of both of the major divisions of Dlx genes. This of and eucoelomates. pattern is consistent with a tandem duplication along a chro- A single Dlx homologue has been described to date in mosome to form a cluster of two Dlx genes, followed by Drosophila. Dll is located on a separate chromosome from the chromosomal duplication to give multiple pairs of linked Dlx homeotic genes, but these latter genes themselves have been genes with the same relative orientation. This pattern of separated during evolution by approximately 1 Mb (5, 44). The duplication resembles that proposed for Hox genes (5, 6). The vertebrate Hox cluster duplications appear to have occurred linkage of Dlx genes to chromosomes containing Hox clusters, after the divergence of insects and vertebrates (5, 8). The along with evidence for Hox cluster duplication involving large presence of the single Drosophila Dll gene is consistent with all chromosomal regions (6), makes it reasonable to assume that Dlx duplications occurring after this divergence. In the phy- Dlx cluster duplication occurred during these same genomic logenetic tree presented in Fig. 1, however, Dll groups with the events. Phylogenetic analyses of Dlx sequences do not provide Dlxl-Dlx6-Dlx7 clade. This result is not strongly supported and strong support for a particular order of cluster duplication; the because it would require evolutionary loss of or experimental trees in Figs. 1 and 2 yield conflicting results with respect to failure to detect an additional Drosophila Dlx gene, we tenta- relationships of paralogue groups within the two major Dlx tively conclude that the tandem duplication of Dlx genes clades. To date, it has also been difficult to reconstruct the occurred after insects and vertebrates diverged. order of chromosomal duplication using data from the Hox The duplication of Hox clusters has been proposed to have genes (refs. 5 and 6; W. Bailey, and F.H.R., unpublished). occurred after -the divergence of amphioxus and vertebrates The timing of the initial tandem duplication of Dlx genes can (8), and consistent with this, the tunicate Ciona intestinalis be estimated by considering the Dlx genes of invertebrates and appears to have a single Hox cluster (8, 33). Di Gregorio et al. protochordates. It is likely that the nematode C. elegans (33) isolated two Dlx genes from Ciona intestinalis from a contains a single Dlx orthologue. It has been suggested that single A phage clone. This linkage arrangement and the this orthologue is ceh23, which is closely linked to the HOM/ presence of a single Hox cluster is consistent with the tandem Hox cluster (6, 15, 42, 43). However, this gene, whose homeo- duplication of Dlx genes occurring before the divergence of domain is 50% identical to Dll, is actually slightly closer (52%) vertebrates and tunicates. This scenario would predict the to Drosophila Ems (42). More recent genomic sequencing in C. tunicate genes to be partitioned among the two main groupings elegans has revealed a gene (C28A5.4) with a homeodomain ofvertebrate Dlx genes. One of these genes, D1l-A, does cluster 74% similar to that of Dll. This gene is distantly linked to the specifically with the Dlxl-Dlx6-Dlx7 grouping (Fig. 1), but Downloaded by guest on September 26, 2021 10862 Evolution: Stock et aL Proc. Natl. Acad. Sci. USA 93 (1996) DII-B clusters outside of all chordate Dlx genes and that of It is difficult to apply this argument as an explanation for the Drosophila. These results would require that Ciona intestinalis conservation of Dlx-Hox linkage. Mouse Evx-1 and Evx-2 are has lost a Dlx2-Dlx3-DlxS homologue and that Drosophila and more closely linked to Hoxa (50 kb) and Hoxd (8 kb), vertebrates have independently lost a homologue of Dll-B respectively, than are the Dlx gene pairs and they are located (probably from multiple chromosomes in the case of verte- on the same side (54). Dolle et al. (54) showed that while brates). Because it is difficult to obtain reliable phylogenetic aspects ofEvx-2 expression are coordinately regulated with the trees with the 61 amino acid homeodomain alone, especially Hox cluster, coordinate regulation of Evx-1 and Hoxa was not with respect to the root which plays a large role in these detected. In the case of the HOM/Hox complex of C. elegans conflicting results, and because of the additional duplications and mammalian Hoxb and Hoxd, the Dlx genes are located on and losses required, we feel that it is more reasonable to the side of the most posteriorly expressed Hox genes, while Dlx assume that the phylogenetic tree is in error and that the Dlx genes are expressed more anteriorly in the CNS than are any tandem duplication occurred once in a common ancestor of of the Hox genes (14, 21, 24-26, 48, 55, 56). Ruddle et al. (6) vertebrates and tunicates. have noted the clustering of a number of additional develop- Dlx Gene Loss in the Mouse Versus Additional Duplications mentally important gene families around the Hox clusters, but in the Zebrafish. The proposed Dlx cluster duplications tied to the significance of these conserved linkage relationships re- that of the Hox clusters might suggest the presence of an mains elusive. additional pair of mammalian Dlx genes linked to Hoxc (only Functional Significance of Duplicated Dlx Genes. Dlx genes six Dlx genes have been cloned to date, exclusive ofDlx4, which have been proposed to participate in developmental "codes" is likely to be artifactual; ref. 27). Because the origin of four for specifying distintct regions of the developing (55-57), Hox clusters has been proposed to predate the divergence of branchial arches (18), fins (18), and dentition (28, 58). It has zebrafish and mammals (45), the five new zebrafish Dlx loci been argued that in an analogous "Hox code" for the branchial that we have reported in combination with the three previously region of the head, there is greater functional redundancy described ones would be more consistent with the hypothetical between trans-paralogues (related genes on different chromo- eight locus ancestral state in fish and tetrapods. The clustering somes) than between cis-paralogues (linked genes; ref. 3). of zebrafish dlx2 with dlxS and dlx7 with dlx8 in our phyloge- More recent analyses of mice deficient for multiple Hox genes netic analyses, however, suggests that these two pairs of genes suggest functional redundancy among cis- and nonparalogous may be the result of two independent duplication events along Hox genes as well (59-61). Because the linkage and sequence the lineage leading to zebrafish. The grouping of zebrafish dlx7 relationships of Dlx genes resemble a smaller version of the and dlx8 more closely with the newt NvHBox-5 than with Hox complexes, it is interesting to compare cis- and trans- mammalian Dlx7, contradicting the phylogenetic relationship paralogues for their functional similarity. Some functional of the organisms, would indicate that seven of the predicted redundancy probably does exist because the inactivation of ancestral eight groups of Dlx paralogues have been isolated mouse Dlx2 did not affect all regions in which the gene is from modern vertebrates. It is also possible, however, that the expressed (30). The few comparisons of expression that can be rapid rate of mammalian Dlx7 evolution postulated by Naka- made among Dlx paralogues actually seem to show greater mura et al. (64) has caused an artifactual clustering (46) of newt similarities among cis-paralogues. In the brain, mouse Dlxl and and zebrafish genes to the exclusion of mammal Dlx7. Linkage DLb2 (cis-paralogues) have very similar expression patterns of both mammalian and zebrafish Dlx7 genes to Dlx3 (ref. 64; (55), while Dlx3 (a trans-paralogue ofDlx2) is not expressed at G. Giroux and M.E., unpublished) does support the orthology all (27). DlxS and Dlx6 (cis-paralogues) have similar expression of the former genes. The possibility that zebrafish dlxS and dlx8 patterns throughout the embryo and share the unique feature represent the fourth pair of Dlx genes formed early in verte- of being expressed in most developing skeletal elements (14). brate history (contradicted in our tree) may be tested by There is clearly a need for more determining the linkage arrangements of these genes relative comparisons of expression and to Hox clusters. especially function among Dlx paralogues. Leaving aside the question of the orthology of the Dlx7 Dlx Nomenclature. The orthologous relationships among genes in mammals and zebrafish, it is intriguing that Dlx3 is vertebrate Dlx genes proposed here and by other researchers linked to Hoxb in mammals (64), and Hoxa in zebrafish (G. (18, 21, 62) would be best represented by changes to the Giroux and M.E., unpublished; D.L.E. et al., unpublished). nomenclature. This could be accomplished by renaming the This may be an example of a translocation between "home- Xenopus and zebrafish genes according to their mouse ortho- ologous" chromosomes formed during the polyploidization logues. In the case ofXenopus, this would require a designation events postulated to have occurred early in vertebrate evolu- for genes related by recent tetraploidization as well. Adjusting tion. Morizot (47) suggested that such translocations have the zebrafish nomenclature would require fewer changes than occurred for a number of families of metabolic enzymes and that of Xenopus-i.e., changing dlx4 to dbxS and giving a proposed a mechanism based on pairing of homeologous different designation to the dlxS gene reported here. Given the chromosomes during meiosis. similarity of genomic organization to that of the Hox clusters, Interestingly, zebrafish appear to have a larger number of a new system reflecting cis- and trans-paralogue relationships members of several additional developmentally important analogous to that in use for the Hox genes (63) might be gene families than the mouse-e.g. Msx (48), Hh (49), En (50), useful-e.g., Dlxal and Dlxa2 for mouse Dlx5 and Dbc6. and perhaps Evx (51). One possible explanation is tet- Because of the possibility of translocations of Dlx clusters raploidization, which has occurred in a number of members of relative to Hox clusters, however, such a system should not be the family Cyprinidae (52), to which the zebrafish belongs. undertaken until a better understanding of Dlx gene linkage in However, the chromosome number of zebrafish is very similar the zebrafish is obtained. to that of most diploid (53). The greater number of Dlx genes in zebrafish than in mammals may therefore be related We thank R. T. Merritt, G. Giroux, and Dr. A. Buchanan for to a technical assistance; Prof. D. Powers for facilities for raising zebrafish; the genomes of teleosts as group; Morizot (47) lists a and Dr. S. Kumar for helpful discussions. This work was supported by number of metabolic enzymes encoded by more gene family National Institutes of Health Grant DE10871 to K.M.W. and F.H.R., members in teleosts than in mammals. National Science Foundation Grant SBR 9408402 to K.M.W., and a Significance ofDix- Linkage. The members of the grant from the Medical Research Council of Canada to M.E. Hox clusters are relatively closely linked to each other (-10 kb) and the linkage conservation among species is believed to 1. Ohno, S. (1970) Evolution by Gene Duplication (Springer, Hei- be at least partly the result of shared regulatory regions (4-6). delberg). Downloaded by guest on September 26, 2021 Evolution: Stock et al. Proc. Natl. Acad. Sci. USA 93 (1996) 10863

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