THE JOURNAL OF EXPERIMENTAL ZOOLOGY 280:73–85 (1998)

© 1998 WILEY-LISS, INC.

Molecular Evolution of Hox Gene Regulation:Cloning and Transgenic Analysis of theLamprey HoxQ8 Gene

JANET L. CARR,1 COODUVALLI S. SHASHIKANT,1 WENDY J. BAILEY,1AND FRANK H. RUDDLE1,2*1Department of Molecular, Cellular, and Developmental Biology, YaleUniversity, New Haven, Connecticut 06520

2Department of Genetics, Yale University, New Haven, Connecticut 06520

ABSTRACT The mammalian Hox clusters arose by duplication of a primordial cluster. Theduplication of Hox clusters created redundancy within cognate groups, allowing for change infunction over time. The lamprey, Petromyzon marinus, occupies an intermediate position withinthe chordates, both in terms of morphologic complexity and possibly cluster number. To determinethe extent of divergence among Hox genes after duplication events within vertebrates, we ana-lyzed Hox genes belonging to cognate group 8. Here we report characterization of the HoxQ8 gene,which shows conservation with mammalian genes in its amino-terminal, homeobox and hexapeptidesequences, and in the position of its splice sites. A β-galactosidase reporter gene was introduced inthe HoxQ8 genomic region by targeted recombinational cloning using a yeast-bacteria shuttle vec-tor, pClasper. These reporter gene constructs were tested for their ability to direct region-specificexpression patterns in transgenic mouse embryos. Lamprey enhancers direct expression to poste-rior neural tube but not to mesoderm, suggesting conservation of neuronal enhancers. In the pres-ence of the mouse heat shock promoter, lamprey enhancers could also direct expression to theposterior mesoderm suggesting that there has been some divergence in promoter function. Ourresults suggest that comparative studies on Hox gene structure and analysis of regulatory ele-ments may provide insights into changes concomitant with Hox cluster duplications in the chor-dates. J. Exp. Zool. 280:73�85, 1998. © 1998 Wiley-Liss, Inc.

W.J. Bailey’s current address is Dept. of Bioinformatics, Merck andCo., Inc., West Point, Pennsylvania 19486

Contract Grant Sponsors: This work was supported by the Na-tional Science Foundation (NSF IBN 96-30567) and the National In-stitutes of Health (NIH GM09966).

*Correspondence to: Frank H. Ruddle, 1010 KBT/Dept. of MCDB,P.O. Box 208103, New Haven, CT 06520

Hox genes play a critical role in the patterning ofthe embryonic axis in a diverse array of animals.They encode transcription factors with a highlyconserved functional domain, the homeodomain. Ex-perimental exchanges of Hox genes and their cis-regulatory regions between different organisms havedemonstrated a high degree of functional conserva-tion among metazoans. For instance, mouse, human,and chicken Hox genes and their regulatory regionshave been shown to function in Drosophila in a man-ner similar to the homologous genes from Drosophila(Malicki et al., ’90; McGinnis et al., ’90; Malicki etal., ’92; Zhao et al., ’93; Frasch et al., ’95; Haerryand Gehring, ’96; Lutz et al., ’96; Haerry andGehring, ’97; Keegan et al., ’97). Similarly, regula-tory regions from Drosophila, pufferfish, chicken, andhuman have been shown to direct expression pat-terns in developing mouse embryos resembling theirrespective homologous mouse genes (Awgulewitschand Jacobs, ’92; Marshall et al., ’94; Studer et al.,’94; Aparicio et al., ’95; Knittel et al., ’95; Morrisonet al., ’95; Beckers et al., ’96; Gerard et al., ’97).

In all animals examined to date, the Hox genesare organized in clusters (Lewis, ’78; Krumlauf,’92; Garcia-Fernandez and Holland, ’94; Ruddleet al., ’94; Popodi et al., ’96; Aparicio et al., ’97). Amodel of Hox gene cluster evolution suggests thata primordial Hox cluster was formed by serial du-plication of an ancestral gene. Invertebrates andlower chordates such as amphioxus possess onesuch Hox cluster, whereas higher vertebrates in-cluding mouse, human, and pufferfish possess fourclusters. The four clusters result from duplicationof the original cluster, with occasional gain or lossof Hox genes occurring independently in differentlineages (Kappen et al., ’89; Ruddle et al., ’94;Bailey et al., ’97).

74 J.L. CARR ET AL.

Expansion of the number of Hox gene clustersamong deuterostomes has resulted in an increasein the number of genes within cognate groups withsome redundancy in function (Fig. 1). This redun-dancy in Hox genes may have contributed to ac-quisition of novel functions leading to an increasedcomplexity of body organization. Comparativestudies of Hox gene expression and regulation bothwithin and between species therefore should shedlight on how new functions may have evolvedwithin cognate groups.

A variety of organisms have been surveyed toestimate or determine Hox cluster number in a con-tinuing effort to determine how and when expan-

sion of Hox genes and clusters have occurred dur-ing deuterostome evolution (Pendleton et al., ’93;Garcia-Fernandez and Holland, ’94; Pavell andStellwag, ’94; Ruddle et al., ’94; Di Gregorio et al.,’95; Misof et al., ’96; Misof and Wagner, ’96; Popodiet al., ’96; Aparicio et al., ’97). Among these, thelamprey, Petromyzon marinus, occupies an inter-mediate phylogenetic position that allows for com-parisons between it and both more primitive andmore derived chordates. Lampreys are true verte-brates possessing a neural crest, cranium, and seg-mented vertebral column. However, they are muchmore primitive than their sister taxon, the gnath-ostomes, lacking the innovations of jaws and paired

Fig. 1. Expansion and duplication of the Hox gene clus-ters through evolution. Systematic relationships of the chor-

dates based on paleontological data are illustrated along withthe known Hox cluster numbers in extant species.

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Lateral expansionof Hox cluster

Hox clusterduplicationsAdditional lateral

expansions

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Bony Fish

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Pufferfish4

LAMPREY HOXQ8 SEQUENCE AND REGULATION 75

fins (Forey and Janvier, ’93; Gee, ’96; Northcutt,’96). We have shown that the lamprey, Petromyzonmarinus, may have three Hox clusters (Pendletonet al., ’93; J.L.C., unpublished results), making itthe most primitive vertebrate known to have mul-tiple clusters. Thus, the lamprey is an ideal organ-ism for comparing the function of Hox genes beforeand after a duplication event. As a first step to-wards this goal, we describe the sequence of a lam-prey Hox cognate group 8 gene, HoxQ8. Further,we have identified its cis-regulatory regions by em-ploying reporter gene analysis in transgenic mouseembryos as an assay.

MATERIALS AND METHODSIsolation and sequencing of cognate group 8

Hox genes from lampreyA genomic library of lamprey DNA was con-

structed with 15 to 22 kb Sau3AI-digested lam-prey genomic DNA ligated to LambdaGEM®-11BamHI arms (Promega, Madison, WI). The unam-plified library was probed with a digoxigenin la-beled lamprey group 11 homeobox. The probe waslabeled via PCR, using primer set II from Misofand Wagner (’96) and digoxigenin-11-dUTP (10XDNA labeling mix: Boehringer Mannheim, Indian-apolis, IN). Filters were then hybridized at 55°Cwith approximately 10 ng/ml denatured probe in5× SSC, 0.1% SDS, 5% dextran sulfate, and 5%liquid blocking reagent (Amersham, Cleveland, OH).Filters were washed to 0.5× SSC, 0.1% SDS at roomtemperature. Digoxigenin signal was visualized bychemiluminescent detection of an alkaline phos-phatase conjugated anti-digoxigenin antibody.

Primary picks were amplified by PCR withHoxE and HoxF primers (Bartels et al., ’93). PCRproducts were cloned, and five clones from eachphage isolate were sequenced. This sequence wasused to give the phage clone a tentative identity.Identity of the clones was confirmed by directsequencing from phage clones using 32P-end-labeled primers designed to the homeobox and theThermosequenase kit (Amersham, Cleveland, OH).Isolation and sequencing of the HoxQ8 gene

The cosmid Pmcos2B was previously isolated byhybridization to a lamprey homeobox sequence(Pendleton et al., ’93; Fig. 2A). It was restrictionmapped to determine the locations of the Hox cog-nate group 7, 8, and 9 genes: HoxN7, HoxQ8, andHoxV9, respectively.

Three contiguous fragments from Pmcos2B weresubcloned into pBSSK+ (Stratagene, La Jolla, CA):a 1.9 kb XhoI fragment (XX1.9) covering the 3´

half of the homeobox and extending 1.9 kb down-stream; a 1.6 kb NotI/XhoI fragment (XN1.6) ex-tending 5´ from the beginning of the homeobox to1.6 kb upstream of the homeobox; and a 7.5 kbXhoI/NotI fragment (XN7.5) covering the next 7.5kb 5´ of XN1.6. XN7.5 was sequenced from theNotI site in the 5´ direction. The other two cloneswere sequenced from both ends by primer walking.

The sequence was translated and aligned to mouseHoxb8, Hoxc8 and Hoxd8 sequences manually.

Splice site confirmationRandom hexamer primed cDNA was prepared

from stage 14 and 15 lamprey embryos (Piavis,’61). Primers QI-1 (5´-AGCTACACGGCGGCGCAAATGTTC-3´, covering nucleotides 469 to 492)and QI-2 (5´-GGAACTCCTTCTCCAGCTCGAG-3´, reverse complement of nucleotides 580 to 559)were used to amplify cDNA to confirm the splicesite. The 120 bp PCR product was cloned intopGEM-T (Promega, Madison, WI), and sequencedusing SP6 and T7 primers.

Construction of reporter genes fortransgenic analysis

A 29 kb SpeI fragment of Pmcos2B was clonedinto the AvrII site in the polylinker of pClasper(Bradshaw et al., ’95), using conventional cloningtechniques. This construct was isolated from bacte-ria and transformed into yeast strain Y724 (MATa,ura3-52, lys2-801, ade2-101, leu2-∆98, trp1∆, his3∆200, cyhr, can1r; Bradshaw et al., ’95) by the one-step method (Chen et al., ’92), and plating on drop-out media lacking leucine. Leu+ colonies wereconfirmed by Southern blot analysis. The yeast straincarrying this construct is referred to as Y-SpSp29.

LacZ was inserted in-frame into the coding se-quence of HoxQ8 by homologous recombination inyeast (Bradshaw et al., ’96). Homologous regionsof DNA used as recombinogenic ends were ampli-fied by PCR. The 5´ recombinogenic end consistedof 270 bp from nt position 20 to 291 (Fig. 4) andwas fused to the SmaI site of pLZURA (Bradshawet al., ’96) to create a chimaeric HoxQ8/LacZ gene.The 3´ recombinogenic end covered 240 nt fromnucleotide 469 to 51 nucleotides 3´ of the splicesite. This fragment was cloned into the SacII siteof pLZURA, 3´ of the URA3 gene. The entire re-placement construct (QX1LZR) was linearized andseparated from the vector using SalI and trans-formed into Y-SpSp29. Transformants were se-lected on dropout media lacking leucine and uracil.Leu+, Ura+ colonies were screened by PCR andconfirmed by Southern blot analysis. This yielded

76 J.L. CARR ET AL.

Construct 1 (Fig. 2B), which then was isolatedfrom yeast and transformed into E. coli strainDH10B for preparation of DNA for microinjection.

An additional construct was made in which LacZwas inserted by homologous recombination into thesecond exon of HoxQ8, in frame after amino acid266. For this construct the 5´ recombinant end was270 nucleotides long and the 3´ recombinant endconsisted of 280 nucleotides of sequence from 110to 390 nucleotides 3´ of the stop codon.

Construct 2 was a HindIII/XbaI deletion of con-struct 1, which was cloned into pBSSK+ (Fig. 2B).This deleted 12.5 kb of 5´ sequence and 9 kb of 3´sequence, including the intron and all sequences

3´ of it. An additional construct (construct 1a) wasa direct deletion of 9 kb of 3´ sequence by XbaIdigestion of construct 1.

The 3 kb BamHI fragment of QX1LZR that con-tained the promoterless LacZ was replaced by a4 kb BamHI fragment containing hsp68 and LacZ(Kothary et al., ’89) to yield QhspLZR. This waslinearized and also used to transform Y-SpSp29as described above, yielding construct 3 (Fig. 2B).

Construct 4 (Fig. 2B) was constructed by clon-ing the XN7.5 fragment of Pmcos2B in front of acassette containing the hsp68 minimal promoterand LacZ containing the SV40 polyadenylationsite (Kothary et al., ’89).

Fig. 2. A lamprey cosmid and derived constructs used fortransgenic mice. (A) The lamprey cosmid Pmcos2B. The sitesof the Hox genes and selected restriction enzyme sites areindicated. (B) Constructs used for the production of transgenic

mice. For details see Materials and Methods. Abbreviationsfor restriction sites: E, EcoRI; H, HindIII; N, NotI; S, SpeI;Xb, XbaI; Xh, XhoI.

LAMPREY HOXQ8 SEQUENCE AND REGULATION 77

A 6 kb fragment containing the intergenic regionbetween Hoxb9 and Hoxb8 was amplified from amouse Hoxb YAC clone (Bentley et al., ’93) usingprimers based on the published sequences of Hoxb9(Bogarad et al., ’89) and Hoxb8 (Kongsuwan et al.,’89). A 1.7 kb Hind III fragment of this (Charite etal., ’95) was isolated and subcloned in front of thehsp68/LacZ cassette (construct 5). A 1 kb BspEI-Hind III fragment containing the Hoxc8 early en-hancer region was similarly cloned in front of hsp68/LacZ (construct 6; Shashikant et al., ’95).

Preparation of DNA for microinjection, produc-tion of transgenic mouse embryos, staging of em-bryos, Southern blot analysis, and detection ofβ-galactosidase activity have been described else-where (Shashikant et al., ’95).

RESULTSIsolation of three group 8 homeobox

sequences from lamprey genomic DNAPreviously, we conducted a PCR survey using de-

generate primers designed to the most conservedregions of the homeobox to detect partial homeoboxsequences in the lamprey (Pendleton et al., ’93).That study identified two homeobox sequences inthe lamprey belonging to Hox cognate group 8(Hox8). Sequences were classified as Hox8 basedon amino acid sequence; most vertebrate Hox8genes have a proline at position 24, a lysine at 29,a serine at 35, and a glycine at 39, among others.The original designations of ‘‘q’’ and ‘‘r’’ were re-placed by the more descriptive HoxQ8 and HoxR8.

When we sequenced the entire homeobox, wedetected a group of phage clones that had a thirdsequence, containing the same 82 bp partial se-quence as HoxQ8, but differing in nucleotide se-quence in other regions of the homeobox, and witha distinct restriction map (J.L.C., unpublished re-sults). We named this sequence HoxQ8a. Thehomeobox of HoxQ8a is identical to HoxQ8 inamino acid sequence and shares 95.6% nucleotidesimilarity; this similarity drops off outside thehomeobox region (data not shown). Amino acid

alignments of the lamprey Hox8 sequences areshown in Figure 3. The presence of three Hox8genes suggests at least three unlinked Hox clus-ters in lamprey.

Isolation and sequencing of HoxQ8Our previous studies (Pendleton et al., ’93) also

yielded a cosmid from lamprey genomic DNA, whichPCR analysis indicated had three homeobox genes.We have now mapped the 41 kb cosmid to deter-mine the locations of the HoxN7, HoxQ8, and HoxV9homeoboxes (Fig. 2A). The HoxN7 and HoxQ8homeoboxes are separated by 8 kb, and the HoxQ8and HoxV9 homeoboxes are separated by 20 kb.

A NotI restriction site was detected within thehomeobox of HoxN7, and an XhoI site was dis-covered within the homeobox of HoxQ8. Subclonesneighboring these sites were sequenced to deter-mine the orientation of the coding strand withinthe homeoboxes. The orientation is the same asfor all other vertebrate Hox genes, with 5´ to theposterior end of the complex and 3´ to the ante-rior. The orientation of HoxV9 was not determined.

Based on the observation that most vertebrateHox introns are approximately 1 kb long, wedecided to sequence HoxQ8 from genomic DNA.We sequenced about 2 kb in either directionfrom the XhoI site within the homeobox ofHoxQ8. The first exon sequence was recognizedby similarity to mouse Hox8 genes, specificallywithin the hexapeptide and amino terminal se-quences. The splice site also was estimated bysequence comparisons. To determine the exactlocation of the splice site, the putative splicesite was amplified from lamprey cDNA by PCR.This product was then cloned, sequenced, andaligned with sequences from genomic DNA. Thefull length sequence of HoxQ8 is shown in Fig-ure 4; nucleotides 469 to 580 represent the re-gion amplified from cDNA. The black triangleindicates the position of the 1.2 kb intron. Thesplice site was similar among all Hox8 genes(Fig. 5B). The position of the HoxQ8 splice site

Fig. 3. Homeodomain sequences of Hox cognate group 8genes from lamprey, mouse, and amphioxus. Identities areindicated by dashes. HoxQ8, HoxQ8a, and HoxR8 are lam-

prey Hox8 genes. Hoxb8, Hoxc8, and Hoxd8 are from mouse,and AmphiHox8 is from amphioxus.

78 J.L. CARR ET AL.

is identical to that of mouse Hoxb8 and Hoxc8(Breier et al., ’88; Kongsuwan et al., ’89) and is threenucleotides closer to the homeobox compared to theposition in Hoxd8 (Izpisua-Belmonte et al., ’90).

Once the entire sequence was known, it wastranslated and the deduced protein sequencewas aligned with that of mouse Hox8 genes (Fig.5A). There was sequence conservation at theamino terminus, the hexapeptide region,the homeodomain, and a few additional shortstretches of amino acids. Extreme conservationwithin the homeodomain, especially at aminoacid positions 24, 29, 35, and 39 of the homeo-domain, allowed us to determine that it is in-deed a cognate group 8 gene.

Analysis of lamprey HoxQ8 regulatoryregions in transgenic mice

The resemblance of the coding sequence of thelamprey HoxQ8 gene to mammalian Hox8 genesprompted us to investigate whether its regulatorysequences were also conserved. For this purposea 29 kb SpeI fragment of Pmcos2B containing re-gions surrounding HoxQ8 gene and extending upto the neighboring Hox cognate group 9 and 7genes, was cloned into pClasper, a yeast-bacteriashuttle vector (Bradshaw et al., ’95). A LacZ re-porter gene was introduced by homologous recom-bination in yeast (see Materials and Methods) inframe either in the first exon or in the second exon

Fig. 4. Sequence of HoxQ8. NotI and XhoI restriction sitesare indicated. The open triangle indicates the site of fusionto LacZ in constructs 1 and 3 (see Fig. 2B). The black tri-

angle indicates the splice site. The outlined region is thehomeobox.

LAMPREY HOXQ8 SEQUENCE AND REGULATION 79

of the HoxQ8 gene. Since many regulatory ele-ments are typically located 5´ of the coding se-quences, a fragment containing 7.5 kb of sequence(extending upstream from the coding sequence tothe HindIII site) ligated to the reporter gene (con-struct 2, Fig. 2) was tested for activity in trans-genic mouse embryos. As shown in Figure 6A, thisconstruct directed LacZ expression in a 9.5 day

p.c. embryo in a region-specific manner in the pos-terior neural tube. The expression extendedrostrally from the caudal-most region with an an-terior boundary at the level of somite 9–10. Nosignificant expression was observed in the meso-derm, except in the caudal-most region. To deter-mine if mesoderm-specific elements lie outsideconstruct 2, we examined two larger constructs,

Fig. 5. Alignment of Hox cognate group 8 genes. Gapsare indicated by dots and identities by dashes. (A) Alignmentof complete amino acid sequences from lamprey HoxQ8 andall mouse Hox8 genes: Hoxb8, Hoxc8, and Hoxd8. The se-quence highlighted in dark gray is the homeodomain. The

light gray box indicates the conserved hexapeptide sequence.(B) Alignment of splice sites from Hox cognate group 8 genes.The black triangles indicates the splice sites. The dark graybox identifies the beginning of the homeobox.

80 J.L. CARR ET AL.

constructs 1 (Fig. 2B) and 1a (not shown), for re-porter gene activity in transgenic mice. Construct1a contained 20 kb of the upstream region where-as construct 1 contained 20 kb upstream, theentire coding region of HoxQ8, and 9 kb of down-stream sequence. Both constructs directed expres-sion to the posterior neural tube, but neitherconstruct directed significant expression to meso-derm (construct 1a not shown; construct 1, Fig.6B). The anterior boundary of the neural tubeexpression was at the level of the 14th somite,several somites posterior to the expression ob-served with the smaller construct 2. The posi-tion of LacZ either in the first or the secondexon (not shown) did not significantly changethe expression pattern.

To determine if the mesodermal expression ofthe transgene required a mouse promoter, we in-troduced the mouse hsp68 promoter into the re-porter construct (Fig. 2B, constructs 3 and 4). Inthe absence of enhancer elements, the mousehsp68 promoter does not direct any region-spe-cific expression in the developing embryo, but pro-vides basic transcriptional machinery for a robustexpression of the reporter in the presence of cis-regulatory regions (for example, Shashikant et al.,’95; Shashikant and Ruddle, ’96). In the presenceof the mouse promoter, both the smaller and largerconstruct showed considerable enhancer activityin the posterior mesoderm (Fig. 6C, D). The ex-pression was particularly pronounced in the cau-dal-most region in the unsegmented mesodermand lateral plate mesoderm. However, expressionin the somites was not very consistent among thetransgenic embryos that were examined. Theseresults may reflect a requirement of additionalelements for mesoderm expression, species-specificdifferences in the regulatory machinery, or a genu-ine lack of mesodermal expression of HoxQ8 inthe lamprey. The neural tube expression waslargely unaffected by the presence or absence ofthe mouse hsp68 promoter.

To compare enhancer activity of the lampreyHoxQ8 gene with enhancer activities of othermembers of cognate group 8 enhancers isolatedfrom Hoxb8 and Hoxc8 genomic regions were ana-lyzed for their ability to direct expression of thereporter genes in transgenic mice. As shown in Fig-ure 6E, a mouse Hoxb8 enhancer directs expres-sion of the reporter gene to posterior regions of theembryo in neural tube and mesoderm. The expres-sion in the neural tube was anteriorly restrictedto the level of somites 10–11. Similarly, a Hoxc8enhancer directs expression to the neural tube and

Fig. 6. Expression patterns of reporter genes in trans-genic mouse embryos. Expression of the β-galactosidase geneis detected in the posterior regions of the embryos stagedbetween 9.0 and 9.5 days p.c. Arrowheads indicate the an-terior boundary of expression in the neural tube. Reporterconstructs are shown in Figure 2B: (A) Construct 2; (B) Con-struct 1; (C) Construct 4; (D) Construct 3; (E) Construct 5;(F) Construct 6. fl, forelimb; m, mesoderm; n, neural tube;s, somites.

LAMPREY HOXQ8 SEQUENCE AND REGULATION 81

mesoderm (Shashikant et al., ’95; Fig. 6F) withthe anterior limits of neural tube expression at thelevel of somites 13–14. Thus the lamprey HoxQ8enhancer directs expression in the neural tube tocomparable levels seen in Hoxb8 and Hoxc8. How-ever, the mouse enhancers showed much strongerexpression in the mesoderm as compared with thelamprey enhancer.

In summary, lamprey regulatory regions are ca-pable of directing region-specific expression in theposterior neural tube and mesoderm in a patterncharacteristic of Hox genes from other species.

DISCUSSIONThe lamprey occupies a position at the base of

the vertebrates that allows for investigation intothe evolution of several morphological innovationswithin the vertebrates (Fig. 1). It has neural crestand neurogenic placodes that contribute to the de-velopment of the cartilaginous branchial basket,the cranial nerves, cranial ganglia, sensory recep-tors, and other parts of the vertebrate head (Foreyand Janvier, ’93; Gee, ’96; Northcutt, ’96). It alsoprovides an outgroup from which to study the in-novations of the gnathostomes, or jawed, verte-brates. Gnathostomes have paired appendagesand jaws, both of which were major innovationsleading to the successful predatory lifestyle of thegnathostomes. In addition to being intermediatein form, it is also likely to be intermediate in itsnumber of Hox clusters (Fig. 1; Pendleton et al.,’93; J.L.C., unpublished results).

The lamprey is likely to have three Hox clus-ters, which is intermediate between the one clus-ter of amphioxus and the four clusters of amniotes.Analysis of the duplication events leading to thefour clusters in mammals (Bailey et al., ’97) sug-gests that there was an intermediate with threeclusters. These hypothetical three clusters wouldbe the Hoxd cluster (the first cluster to diverge),the Hoxa cluster, and a cluster that was ances-tral to Hoxb and Hoxc.

Characterization of Hox genes has not been rig-orously pursued among the lower chordates withthe exception of the amphioxus. The expressionpatterns of two amphioxus Hox genes, AmphiHox-1 and AmphiHox-3, have been studied (Hollandet al., ’92; Holland and Holland, ’96). These genesare members of the anterior cognate groups 1 and3 and were found to be expressed in the anteriorneural tube. This expression was used to estab-lish a hypothesis of homology between that regionof the amphioxus neural tube and the hindbrainof higher chordates. These two Hox genes were

also found to be expressed in pre-somitic meso-derm in the most posterior part of the animal butnot in the somites, and mesodermal staining forAmphiHox-1 was undetectable in animals shortlyafter the neurula stage.

One reason lower chordates and vertebrates arenot studied more often is the difficulty in obtain-ing embryological material and developing specificmolecular methods for different taxa. An alterna-tive strategy is to do comparative studies withinthe context of a well-established model system.Conservation of basic developmental mechanismsamong metazoans permits us to examine the func-tion of genes and regulatory regions isolated fromvarious species in organisms that are more ame-nable to experimental manipulations. Regulatoryregions isolated from Drosophila, human, chick,zebrafish, and pufferfish have been shown to di-rect region-specific expression patterns in mice(Awgulewitsch and Jacobs, ’92; Marshall et al., ’94;Studer et al., ’94; Aparicio et al., ’95; Knittel etal., ’95; Morrison et al., ’95; Beckers et al., ’96;Gerard et al., ’97). The transgenic mouse systemprovides a powerful means of assaying both con-servation and divergence of regulatory systemsamong vertebrates.

Hox cognate group 8 (Hox8) genes are expressedin, among other places, the forelimbs, hindlimbs,and brachial region of the neural tube in mouseand chicken (Belting et al., ’98; Nelson et al., ’96).Within the neural tube, Hoxc8 is expressed in theregion from which motor neurons to the forelimbarise (Belting, ’97). Study of the Hox8 genes ofthe lamprey, therefore, may shed some light onthe origin of paired appendages.

We sequenced three Hox8 homeoboxes from thelamprey: HoxQ8, HoxQ8a, and HoxR8 (Fig. 3).Two of these, HoxQ8 and HoxQ8a, are strikinglysimilar to each other. They differ by only 8 out of180 nucleotides and are identical at the aminoacid level. However, they are not alleles. The se-quence is less similar outside the homeobox, andthe restriction maps over a region of more than15 kb are distinct.

This similarity between Hox cognate groupmembers is unprecedented. The only mammalianHox genes that share this much identity in theamino acid sequence are Hoxa5 and Hoxb5 (Utsetet al., ’87; Tournier-Lasserve et al., ’89; Galangand Hauser, ’92). They share an identical aminoacid sequence through the homeodomain in bothmouse and man, but their nucleotide sequencesare less similar than HoxQ8 and HoxQ8a. Withinmouse, the Hoxa5 and Hoxb5 homeoboxes differ

82 J.L. CARR ET AL.

by 29 out of 180 nucleotides, and within humanthey differ by 32 nucleotides. To approach thesimilarity seen within the lamprey, one can com-pare the orthologous genes across species. Eventhen, they are not quite as similar as HoxQ8 andHoxQ8a are: the Hoxa5 homeoboxes differ by 11nucleotides between mouse and human, and theHoxb5 homeoboxes differ by 13 nucleotides. Wesuspect that a mechanism such as gene conver-sion may have contributed to this similarity of theHoxQ8 and HoxQ8a sequences.

We mapped a cosmid containing HoxQ8 as wellas a Hox cognate group 9 gene (HoxV9) and a cog-nate group 7 gene (HoxN7). The distance betweenthe HoxV9 and HoxQ8 homeoboxes was around20 kb, while the HoxQ8 and HoxN7 homeoboxeswere separated by 8 kb. The intergenic distancesseen were comparable to intergenic distanceswithin the amphioxus and mammalian Hox clus-ters (Acampora et al., ’89; Bentley et al., ’93;Garcia-Fernandez and Holland, ’94) but did notmatch the intergenic distances of any particularcluster. We also investigated the orientation oftranscription for HoxQ8 and HoxN7 relative totheir position in the cluster. The 5´ ends of thegenes are oriented toward the more posteriorlyexpressing genes, as has been seen in all chor-date Hox genes investigated to date.

We sequenced the coding regions of the lampreyHoxQ8 gene. Expression of the HoxQ8/LacZchimaeric protein in transgenic mice using thelamprey promoter and amplification of the regionsurrounding the splice site provide confirmationthat the sequence is from a functional gene. Thesequence and genomic organization of HoxQ8 aresimilar to mammalian Hox8 genes but are notsimilar enough to any single Hox8 gene to assignHoxQ8 to a particular cluster.

We wanted to examine further the relationshipbetween HoxQ8 and the mouse Hox8 genes. Weconducted a transgenic analysis of HoxQ8 to in-vestigate whether or not functional differenceshave emerged since lamprey and mouse lastshared a common ancestor. Our results suggestthat, like mouse Hox8 genes, the lamprey genecontains regulatory elements that can direct re-gion-specific expression patterns of a reporter genein mouse. Thus, basic principles of regulatory in-teractions involving cis-acting elements and trans-acting factors have been conserved betweenlampreys and mammals.

Many regulatory elements isolated from Hoxgenes direct expression of the reporter gene to theposterior regions of the embryo during early stages

of development (reviewed in Lufkin, ’96). Consis-tent with these findings, cis-regulatory regions oflamprey also direct expression to the posterior re-gions of the embryo. When using the native lam-prey HoxQ8 promoter, cis-regulatory elements arecapable of directing expression to the neural tube,whereas a mouse promoter is required for any ex-pression in the posterior mesoderm. Variations inthe mesodermal expression of heterospecific enhanc-ers have been reported previously (Morrison et al.,’95; Beckers et al., ’96). The requirement of a mousepromoter for expression of a zebrafish regulatoryelement in the mouse forelimb domain has also beenshown (Beckers et al., ’96). Thus, even thoughheterospecific enhancers can function in mice, a spe-cies-specific promoter may be necessary for an ac-curate representation of all the enhancer elements.It is also possible that the constructs which utilizethe native lamprey promoter are an accurate re-flection of the lamprey cis-regulatory elements. Inamphioxus, mesodermal expression of Hox genes isvery limited (Holland et al., ’92; Holland and Hol-land, ’96). Perhaps extensive expression of Hoxgenes in the posterior mesoderm, including thesomitic and lateral plate mesoderm, is a characterspecific to the gnathostomes.

The cis-regulatory elements of mouse Hoxc8 di-rect expression to the neural tube with an ante-rior limit at the level of somites 13–14, whereasthe cis-regulatory elements of Hoxb8 direct expres-sion to the level of somites 10–11. Different cis-regulatory regions surrounding the lampreyHoxQ8 gene directed expression to the neural tubeat two different anterior limits. A smaller en-hancer region that contained immediate 5´ up-stream region of HoxQ8 directed expression tomore anterior regions (somite levels 9–10; Fig. 6A,C) compared to the larger enhancer region thatcontained entire genomic regions between neigh-boring Hox9 and Hox7 genes (somite levels 13–14; Fig. 6B, D). These differences can be attributedto the presence of negative elements in the largerconstruct, which restrict the boundary of expres-sion to a more posterior region. Alternatively, twoindependent enhancers are present in the Hox8genomic region, one proximal and the other dis-tal. Deletion analysis indicates that distal ele-ments are located 5´ to the 7.5 kb XhoI site. It ispuzzling that even though the larger construct in-cludes the proximal enhancer, the distal elementsdominate in determining a more caudal anteriorboundary of expression. These results are, how-ever, consistent with the suggestions that ante-rior boundaries of expression are determined by

LAMPREY HOXQ8 SEQUENCE AND REGULATION 83

interactions among multiple elements (Bradshawet al., ’96; Shashikant and Ruddle, ’96). Synergis-tic interactions among separable enhancer ele-ments often determine expression domain of Hoxgene expression (Charite et al., ’95). In these stud-ies, additional elements have resulted in moreanterior boundaries. Our results suggest thatadditional elements may also result in moreposteriorized expression.

Although lamprey HoxQ8 regulatory regions di-rect expression reminiscent of mouse Hox8 genes(Fig. 6; Charite et al., ’95; Shashikant and Ruddle,’96), it is difficult to ascertain to what extent thelamprey gene resembles Hoxc8, Hoxb8 or Hoxd8,based on these studies. First, it is difficult to as-certain whether the complete repertoire of cis-regulatory regions of the lamprey HoxQ8 gene hasbeen captured in the reporter gene constructs. Itis possible that certain elements are farther awayin the cluster, which may further modify the ex-pression of the gene. Second, analysis of individualenhancers may be blurred by their interactions,which can result in a pattern which does not equalthe sum of the individual patterns. Third, someof the enhancers may direct regulation of theneighboring gene, which can be difficult to distin-guish. Nevertheless, these studies reveal a basicconservation of the regulatory information.

We have extensively characterized cis-acting el-ements of Hoxc8 gene by mutational analysis(Shashikant et al., ’95; Shashikant and Ruddle,’96). At least five distinct elements present in a200 bp region were shown to act in combinationto determine Hoxc8 early expression. Theseelements can be isolated by PCR from differentmammalian species reflecting a high degree of con-servation of the early enhancer (C.S.S., unpub-lished results). However, attempts to isolatelamprey elements by PCR were not successful,perhaps reflecting divergence of these elementsin agnathans. Alternatively, the lamprey HoxQ8gene may not be directly related to Hoxc8, but toHoxb8, Hoxd8, or even Hoxa8, which is notpresent in mammals. It may also turn out to bethe case that the multiple lamprey clusters arosethrough a set of duplications independent of themammalian clusters. Even though Hoxb8 andHoxc8 cis-regulatory regions can direct expressionto similar regions of the posterior neural tube, thesequence of the Hoxb8 enhancer that is being char-acterized does not resemble Hoxc8 enhancer ele-ments (C.S.S., unpublished results).

In conclusion, we have shown that the lampreyhas three unlinked Hox cognate group 8 genes on

at least three clusters. This positions the lampreyas the most primitive vertebrate shown to havemultiple Hox clusters. The entire coding sequenceof one of these genes, HoxQ8, is similar to allmouse Hox8 sequences but is not readily assign-able as an ortholog to any particular Hox8 gene.We used targeted recombinational cloning to in-sert LacZ into the coding region of HoxQ8, andused these constructs in the generation of trans-genic mice. This showed that although the lam-prey and the mouse promoters have divergedslightly over time, HoxQ8 does have neural tube–specific enhancers that can function in the mouseembryo. This study lays the groundwork for de-tailed analyses of regulatory sequences through-out a wide range of organisms, which is necessaryfor investigtion into the long-standing question ofhow gene and genome duplications may have con-tributed to the innovation of morphologic noveltywithin the vertebrates.

ACKNOWLEDGMENTSWe thank G.P. Wagner and H.-G. Belting for

critical review of the manuscript and StephanieAtiyeh for excellent technical assistance. Thiswork was supported by grants from the NationalScience Foundation (NSF IBN 96-30567) and theNational Institutes of Health (NIH GM09966).

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