Prince V_The Hox Paradox_2002

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REVIEWThe Hox Paradox: More Complex(es) Than Imagined

Victoria E. Prince 1Depar tment of Organismal Bio logy and Anatomy, Commit tees on Developmenta l Bio logy,N eurobio logy, Genet i cs and Evolu t ionary Bio logy, Th e U n iv ersi t y of Ch icago ,

027 E. 57th Street , Chicago, I l l inois 60637

An understanding of the origin of different body plans requires knowledge of how the genes and genetic pathways that

ontrol embryonic development haveevolved. TheHox genes providean appealingstartingpoint for such studiesbecausehey play a well-understood causal role in the regionalization of the body plan of all bilaterally symmetric animals.Vertebrateevolution has been characterizedby gene, andpossibly genome, duplication events, which arebelieved to haverovided raw genetic material for selection to act upon. It has recently been established that theHox geneorganization of ay-nned shes, such as the zebrash, differs dramatically from that of their lobe-nned relatives, a group that includesumans and all theother widely used vertebrate model systems. This unusual Hox gene organization of zebrash is theesult of a duplication event within the ray-nned sh lineage. Thus, teleosts, such as zebrash, have more Hox genesrrayed over moreclusters (or complexes)than do tetrapodvertebrates. Here, I review our understandingof Hox clusterrchitecturein different vertebratesandconsider theimplicationsofgeneduplication for Hox generegulation andfunctionndtheevolution ofdifferent body plans. 2002 Elsevier Science (USA)

K ey W ords: Hox genes; Hox clusters; geneduplication; vertebrate evolution; teleosts; zebrash; hindbrain.

CLUSTERED ORGANIZATIONOF HOX GENES

The Hox genes w ere rst characterized in the fruit y,D rosophil a m elanogaster , where eight linked A ntennapedia

lass homeobox genes make up the Homeotic complexLewis, 1978). These eight genes encode homeodomain tran-cription factors that are characterized by their role in confer-al of segmental identity along the primary body axis, fromnterior t o posterior (AP; review ed by McG innis and Krum-

auf, 1992). Thus, mutat ions in the y Hox genes lead toramatic homeotic phenotypes, w here one body segmentakes on the identity of another. Homologous Hox genes haveeen found in every bilaterian a nim al inv estigated (de Rosa et l., 1999), and in all cases analyzed, the genes show a clusteredrganization, although gene and cluster number vary. Mostmportant ly, w herever tests have been applied, the Hox genesave proven to play critical roles in determining AP identity.Com parat ive analyses of Hox cluster organizat ion have

evea led tha t varia t ions in Hox gene number betw een

species re ect an evolutiona ry hist ory cha racterized by t w otypes of duplication events: t andem duplication and w holecluster duplicat ion. Current models suggest that s inglecluster organizations, like that of D . m elanogaste r , arosevia the tandem duplication of ancestral Hox genes (Kappenet al . , 1989; Kmita-Cunisse et al . , 1998). A single clusterorganizat ion appears to be comm on to all protostom es, anda s ingle c luste r w i th seven genes w as in place in theancestor of all bilaterians (Fig. 1) (de Rosa e t al . , 1999). Asingle Hox cluster is a lso assumed to be character ist ic of

primit ive deuterostomes, w i th the cephalochordate am-phioxus havin g the clust er w ith th e largest n um ber of genes(Figs. 1 and 2) (Ferrier e t al . , 2000).

It has long been supposed that gene duplication eventscould have played a vi tal role in al low ing vertebrates toachieve their complexity of form through evolution of new gene functions (Ohno, 1970). The origin of vertebrates w asassociat ed w ith m ajor expansions in gene number, possiblyas a result of t w o rounds of w hole genome duplication viapolyploidization (often referred to as th e 2R hypot hesis,for tw o rounds of duplicat ion; Friedman and Hughes2001; Sidow , 1996). Such duplications w ould result in1 Fax: (773) 702-0037. E-mail: vprince@midw ay.uchicago.edu.

evelopmental Biology 249, 115 (2002)oi :10.1006/dbi o.2002.0745

012-1606/02 $35.002002 Elsevier Science (USA)

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upl icat ion of ent i re Hox clusters , and consistent w i th theR hypothesis , te t rapod ver tebrates have four c lusters of

Hox genes (review ed by H olland et al., 1994; Sid ow , 1996).Mouse and hum an ha ve had their Hox cluster organizat ionsully described, and they share an identical 39-gene organi-at ion over 4 c lusters , A D (review ed by McG innis a nd

Krum lauf, 1992; see also Z eltser et al., 1996). The genes falln to 13 para logue g roups , w i th mos t pa ralogue g roupsaving less than a full com plem ent of 4 genes as a result ofecondary gene losses (Fig. 1). A la rge num ber of H ox genesav e also been isolat ed from frog ( Xenopus laevi s )and chick

(G a l l u s g a l l u s ) , and in each case , there is no evidence tosuggest differences from the 39-gene mam ma lian organiza-tion (e.g., G odsave e t al . , 1994; Ladjali-Mohammedi e t al . ,2001). Thus, available data strongly suggest that a 4-Hoxcluster organizat ion is the pr imit ive condi t ion of crown-group te t rapods. Similar ly, a PCR survey of Hox genes inthe more basal lung sh (Longhurst and Joss, 1999) is also

consis tent w i th a 4-cluster condi t ion. N evertheless , w eneed more com plete data from lung sh , as wel l as f rom thecoelacan th , before w e can conclude tha t the re has beencomplete conservat ion of the 4-Hox cluster organizat ionthroughout the sarcopterygians (lobe- nned sh es).

If 2R h a ppen ed , c a n w e es t im a t e w h en e ac h g en o m eduplication event w ould hav e occurred? The cephalochor-date amphioxus may provide the best approximation of thepreduplication vertebrate ancestor. The rst 10 Hox genesin t he s ingle am phioxus Hox cluster show clear hom ologyto the rst 10 ma m m alian paralogue groups (Fig. 1) (G arcia-Fernandez and Holland, 1994). The most basal group of thever tebra tes i s the cyc lostomes, compr is ing hag sh a n dlampreys (Fig. 2), w hich probably form a monophylect icgroup (Ma llatt and Sullivan, 1998). These jaw less verte-brates (Agnat ha) m ight be expected to fall into an interme-diate state between an ancestral, single Hox cluster organi-zat ion and a der ived, 4-cluster organizat ion. Recent ly, 2groups have published extensive analyses of the Hox clus-ters of the sea lamprey ( Pet r o m y z o n m a r i n u s ) (Force et al.,2002; Irvin e et a l . , 2002). Both groups have isolated andmapped genomic Hox clones, to extend previous analysesbased on PC R surveys (Pendelton et al., 1993; Sharman andHolland, 1998). In both of the n ew studies, the data point toa m inimu m of 3 Hox clusters, w ith a 4th cluster considered

likely. Irvine a nd colleagues (2002) built neighbor-joinin gtrees based on sequences of individual homeodomains andw ere unable to distinguish betw een m odels w here only one

IG. 1. Hox gene organization in D rosophi la m elanogaster , a m -hioxus, mouse, and z ebra sh. A hypothetical ancestral conditions also show n. Shades of gray indicate the most closely related genes.

IG. 2. Vertebrate phylogeny show ing Hox cluster num ber and putat ive duplication events. (Based on C arroll, 1988).

Vic t or ia Pr ince

2002 Elsevier Science (USA). All right s reserved.


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ound of dupl icat ion occurred before divergence of theaw ed (gnathostome) and jaw less vertebrates , versus a l lupl icat ions predat ing the spl i t . By contrast , Force andolleagues (2002) concata nat ed t he lam prey hom eodomainequences that lay in cont igs , to provide addi t ional infor-

mative characters. Using this strategy, their extensive treenalysis of Hox genes across the vertebrates suggests thatnly one duplication event occurred prior to t he divergencef the agnathan s and gnathostom es, w i th a second dupl ica-ion event occurring w ithin t he lineage leading to lam preys.

Cons i st en t w i th th i s m odel , bo th g roups found pa i rs o famprey Hox genes tha t a re more c lose ly re la ted to onenother than to an y H ox gene from a gnathostom e. Takenogether, these data support the idea that 1 round of Hoxuplication occurred before the divergence of th e agnat hansnd gnathostomes, and a 2nd occurred in the gnathostomein ea ge. U l t i m a t el y, a c om p let e l in k a ge m a p o f a ll t h eamprey Hox genes w il l help to con rm this m odel .

An al ternat ive model to t he 2R hy pothesis has been putorw ard by Ruddle and col leagues (Bai ley et a l . , 1997).

Accord ing t o th i s m ode l, t he re w ere no t tw o rounds o fupl icat ion in the l ineage leading to te t rapods, but three.

The existence of only four Hox clusters in the tetrapods isxplained either by incomplete dupl icat ions (of a s ingleluster) or by losses of c lusters fol lowing whole genomeuplication events. Bailey and colleagues (1997) used se-uence f rom collagen genes l inked to Hox c lus ter s toeconstruct a l ikely dupl icat ion scenar io w hereby the an-est ra l H ox cluster w as D -l ike, w hich dupl icated to createn A-like cluster from which the B and C clusters arose inurn (D (A(B,C ))).

The Ruddle group has recent ly expanded i ts s tudies t o

nclude th e horn shark ( H eterodontu s f ranc isc i ). This spe-ies is carti laginous, a member of Chondrichthyes, anotherasal group of ver tebrates and a s is ter group to the bonyshes (Osteicthyes; Fig. 2). Investigations of the horn sharkave so far revealed the presence of only two Hox clusters,

M and N. However the sequences and organizat ion of theHox genes wit hin th ese clusters suggest that M is hom olo-

ous to the A cluster, w hi le N is homologous to the C or Dluster, as described in mammals (Kim e t al . , 2000). If thehree-dupl icat ion event model is correct , the horn shark

may u l t ima te ly prove to have on ly th ree H ox c luste rs inotal , represent ing a s tage before dupl icat ion of the com-

mon ancestor of the B and C clusters. How ever, there haseen li t t le a dditional dat a supporting th e th ree-duplicationvent model, and the recent extensive tree building of Forcend col leagues (2002) s t rongly supports tw o sequent ia luplicat ion events [(AB)(C D )] leading to a four-clust er H oxrganizat ion in Sarcopterygia. It th erefore seem s likely th athondrichthy ans (cartilaginous sh) and sarcopterygianslobe- nned osteichthya ns) w ill eventua lly prove to share aery s imilar four-cluster H ox organizat ion, in w hich casehe putat ive tw o rounds of duplication both occurred beforehe origin of chondrichthyans.

Despite the prevalence of the 2R hypothesis, the phylo-enetic analyses of Hughes and colleagues (e.g., Friedman

and Hughes, 2001; Hughes et a l . , 2001) do not providestrong support for tw o rounds of genome duplication in t hevertebrate s tem l ineage. The Hox genes t hemselves arenotoriously uninformative for detailed phylogenetic analy-s is because of their remarkable sequence conservat ion.Thus, Hu ghes e t a l . (2001) constructed phylogenetic treesfor other sets of duplicated genes lying on the Hox-bearingchromosomes of human. The trees for different gene fami-lies have different topologies, which the authors interpretas revealing that the duplicated genes did not arise simul-taneously and are ra ther the resul t of numerous indepen-dent sm all-scale duplication events. How ever, t hese differ-ent topologies could also re ect recom binat ion/conv ersionevents betw een closely related genes shortly aft er duplica-tion or may be an artefact of rapid evolution subsequent todupl icat ion. Furthermore, the newly avai lable human ge-nom e sequence reveals extensive synt eny betw een the fourHo x-bearing clu sters (alt hough less evidence for larger-scaleduplication events; International H um an G enom e Sequenc-ing Consortium, 2001). Whether or not the four Hox clus-ters of th e sarcopterygian v ertebrates arose as a result of tw orounds of genome dupl icat ion within the ver tebrate s teml ineage, the appearance o f more than one H ox c lus tercorrela tes w ell w i th the origin of ver tebrate speci c char-acters, such as neural crest, epibranchial placodes, and anelaborated brain. If th e role of Hox genes in regionalizationof the body plan is considered in the l ight of the idea th atgene dupl icat ions can provide n ew genet ic m ater ia l forselection t o a ct upon (Ohn o, 1970), i t can be hypoth esizedthat some of the special ized characters of the vertebrates ,a n d t h e v a ri at i on w i t h in t h e v ert eb ra t e b od y pl an , a reessen t i a lly a r esu lt o f the ava il abi li ty o f addi t iona l Hox

genes (Holland et al . , 1994). A more simplistic notion isthat there is a di rect rela t ionship betw een number of H oxgenes and complexity of morphology.

IMPLICATIONS OF HOX CLUSTERORGANIZATION FOR GENEREGULATION AND FUNCTION

The clustered organization of Hox genes show s an obvi-ous relationship t o their m ode of expression. Thus, expres-sion domain s along th e prima ry axis of developing embryos

re ect t he locations of individual genes w ithin t he clusters,such that m ore 3 -located genes have more anterior expres-sion domains. This orderly relationship is termed spatialco linea ri ty, and in ver tebra tes, the re i s a l so a t emporalco linea ri ty, such tha t the m ost 3 genes have the earliestonsets of express ion, wi th a sequent ia l act ivat ion of adja-c en t m o re 5 genes . The s tage of th is in i t ia l sequent ia lactivat ion of Hox genes is the most conserved developm en-ta l s tage among the ver tebrates, the phylot ypic sta ge.

The clustered organization of the Hox genes is assumedto play a vital role in the establishment of colinear expres-s ion , a l though the mechan isms o f th i s p rocess remainsom ew hat obscure (review ed by D uboule, 1998). Co nt rol of

3Hox Clus te r Archi tec ture and Ver tebra te Evolu t ion



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Hox gene expression has been well studied by using trans-en ic approaches in the mouse (rev iew ed by Capecch i,997) , and as with other eukaryot ic enhancers , the Hoxegulatory elements include series of independent modules.or example, retinoic acid response element s (RAREs) arerequent ly important for proper Hox gene regulat ion in

mouse, and in many cases , gene-speci c regulatory e le-ments have been shown to mediate positive autoregulation

r cross-regulation by ot her Hox genes. How ever, th e trans-enic approach has a lso revealed that adjacent Hox genesan share enhancer elements or compete for them (G ould et l. , 1997; Sharpe et a l . , 1998; Zak any et a l . , 1997). Asointed out by Duboule (1998), this kind of complex inter-w ining betw een regulatory element s of adjacent H ox genes

may be a consequence of the t ight c luster ing of the genesathe r than i t s cause: I t may expla in the main tenance o flustered organizat ion, yet not help us to understand how lustered organization arose in the rst place.

On this point, i t should also be noted that the vertebrateHox genes are c lustered far more t ight ly t han those of theruit y. The int ergenic distan ces in th e m ouse are an orderf m agni tude smal ler t han those o f Drosophi la , implying

hat the mechanisms of gene regulation must differ signi -antly between these species. For example, individual en-a n ce rs w i t hi n t h e D . m elanogaste r Bi thorax complex,

w hich co m prises a 300-kb region inclu ding th e U b x , abd-A ,nd A b d - B genes, a re funct iona lly separated f rom onenother by boundary elem ents, thus preventing the kindf enhancer sharing and com petition tha t a ppears prevalentn vertebrat e Hox clust ers (e.g., Ha gstrom et al., 1996; Zhout a l . , 1996). Furthermore, the Hox genes of D . m el a n o- aster a re separa ted in to two ind iv idua l c lus te r s , wi th a

pl i t betw een t he A n t p and U b x genes. In a related speciesD . v i r ul i s , t here are s imilar ly t w o individual c lusters, y ethe spl i t i s between U b x and abd-A (Von Allm en et a l . ,996). Such cluster breakdow n is not found in m ore primi-ive insects such as Tr i b o l i u m (Beeman et al . , 1993), em-hasizing that the drosophilids are highly derived insects.

Nevertheless, the differences in general cluster organiza-ion betw een insects an d ver tebrates suggest that the evo-utionary forces underlying retention of clustered organiza-ion may also vary between phyla .

The temporal aspect of colinearity is also likely to play anm po rt a n t ro le i n m a i n t a in i ng t h e o rga n iz a t io n o f t h e

ertebrate Hox clusters. Temporal colinearity may dependn chroma tin accessibi li ty. G ene t ransposit ion experi -ments have suggested that there is a progressive release of aepressive con gura t ion tha t a l lows Hox genes to be se-uentially activated in turn, from 3 t o 5 , as t heir chroma-in becomes accessible (Kondo et al., 1998; va n der H oevent a l . , 1996). In accord w ith this proposal , a long-rangeepressive element 5 to t he m ouse HoxD cluster has beendenti ed (Kondo and Duboule, 1999). These experimentsmphasize the importance of a gene s posi t ion w i th in theluster for establishment of colinear expression; thus, dele-ion o r t r ansposi t ion o f indiv idua l genes w ould have aegative impact on t he entire cluster and be selected against.

Extensive functional studies in both ies and m ice haveestabl ished that the basic funct ions of H ox genes are w ellconserved: Hox genes act as selectors of regional identityalong the pr imary body axis . Mutat ional analysis in D r o -sophila has establ ished that gain-of-funct ion mut at ionstend to cause poste rio ri z ing homeot ic t r ansformat ions ,where the iden t i ty o f a segment an te r io r to the normalexpress ion domain of the gene is a l tered to resemble themore posterior segment ; conv ersely, loss-of-function m uta-t ions cause anter ioriz ing t ransformat ions. In the te t rapodvertebrates , m isexpress ion and nul l m utant analyses haverevealed that similar rules apply, although the situation isrendered signi cantly m ore complex by the existence of notone but four Hox clusters . Also, unl ike Drosophi la , t h evertebrate Hox gene expression domains often overlap inthe posterior; however, the genes tend to act at or close totheir an terior expression lim its. The vertebrate Hox genesare expressed primarily in the CNS and the paraxial meso-derm. Cons i st en t w i th these express ion pat t e rns , a l t er-ations to Hox expression lead to changes in morphology ofth e m esoderm -derived vertebrae (review ed by Burke, 2000),the segmental ly organized neurons of the hindbrain (re-viewed by Lumsden and Krumlauf, 1996), and the deriva-tives of the cranial neural crest (reviewed by Trainor andKrumlauf, 2001).

More than one member o f a ve r t ebra te Hox para loguegroup i s o ft en expressed in a g iven loca t ion , and theseparalogous genes tend t o have part ia l ly redundant func-t ions . For example, nul l mut ants of the m ouse H o x a3 genehave defects in neural crest-derived structures (Chisaka andC apecchi, 1991; Man ley a nd C apecchi, 1995). By contrast,nu l l mutan t s o f the H o x d 3 gene show transformat ions in

t he r st tw o cervica l ver t ebrae (Condie and Capecch i,1993). Although these phenotypes are nonoverlapping,doub le mutan t s o f bo th H o x a 3 and H o x d 3 reveal redun-dancy betw een th e genes (C ondie and C apecchi, 1994). Inthis particular case, the differences in the phenoty pes of th eindividual mutations must be a consequence of differencesin the c i s -regulatory con trol of the t w o genes, rather than ofdifferences in their coding sequences, a s H o x a3 and H o x d 3 are funct ional ly interchangeable in gene-sw ap experi-ments , w here one cod ing sequence i s r eplaced w i th theother in the normal genomic context (G reer et al . , 2000).Although the overall expression patterns of the two genes

appear similar, the details of t heir ci s -regulation, includingvar ia t ions in level of expression, have profound conse-quences. Disparate functions of individual paralogues mayoften depend largely on their ci s - regulat ion; th is in turnsuggests that novel Hox regulat ion m echanisms must havear isen during evolut ion of the vertebrates a s Hox genescame t o pat tern new features of the ver tebrate body plan.

A recent s tudy (Manzanares et a l . , 2000) has begun toaddress th e degree of conservation betw een Hox gene regu-lation in vertebrates (mouse or chick) and in the cephalo-chordate amphioxus, w hich as described above approxi-mates a preduplication ancestral condition. As amphioxusdoes not have vertebrate-speci c structures, such a s neural

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These ndings a lso leave open the quest ion of w hen th eupl icat ion event that produced more tha n 4 Hox clustersn the teleosts occurred. The major vertebrate groups, theobe- ned and ray- nned shes , d iverged more than 400

My a (Fig. 2)(C arroll, 1988); thu s, the duplicat ion m ust h ave

ccurred subsequent to this t ime. The teleost species forwhich Hox cluster organization is known represent a radia-ion that s tar ted more t han 100 M ya (Nelson, 1994); t h isepresents the most recent date a t which the dupl icat ionould have occurred. However, Taylor e t a l . (2001a) h av eecent ly es t imated that the genome dupl icat ion may haveccurred more than 300 Mya, based on analysis of nucleo-ide subs t itu t ion ra tes in the 3rd codon posi t ion o f 15uplicated zebra sh genes . M ore accurate pinpoint ing ofhe t ime of the dupl icat ion wil l require analysis of basalctinopterygian shes, such as Polypteriform es (bichir),

Acipensiformes (sturgeon, paddle sh), or Am iiformes (bow -

n); several groups have already begun t o explore Hox genesn these basal species.

FATES OF DUPLICATED GENES

It has long been th ought t hat gene duplication could playvi ta l role in providing new genet ic m ater ia l for nat ural

elect ion t o a ct upon, an d m ult iple m odels have been putorwa rd to predict the fates of duplicated genes. The clas-ical model of gene duplication holds that because dupli-a ted genes are ini t ia l ly ident ical they can be consideredunct ional ly redundant , and s t resses t he role of the acqui-

sit ion of novel function in the retention of gene duplicates(Oh no, 1970). This m odel suggests th at , follow ing duplica-t ion, only one of th e tw o copies needs to be m aintained forancestral function t o be retained. Thus, once one of the tw ogenes acquires a s t rongly deleter ious mut at ion, further

muta t ions can accumula te in tha t gene unchecked . Asdeleter ious mutat ions are far more l ikely than bene cialones (Lynch and Conery, 2000), the reten t ion o f bothduplicated genes as a result of acquisition of some key novelfunct ion (neo-funct ional izat ion) is thought to be an ex-tremely rare event. According to this model, loss of dupli-cated genes is a common and relatively rapid evolutionaryev en t . In a c co rd an c e w i t h t h e m o d el , t h e m a jo ri t y o fdupl icated H ox genes have been lost f rom the zebra shclusters. Furthermore, there remains evidence of pseudo-genes in some of the locat ions w here a dupl icate w ould beexpected to lie. For example, Amores et al . (1998) desc ribed

a pseudogene in the location of h o x A 1 0a . No t a l l the Hoxcluster sequence has yet been an alyzed, and so i t i s l ikelythat other pseudogenes remain to be discovered. Indeed,recent ly avai lable zebra sh genomic sequences (SangerCentre zebra sh genom e sequencing project; Kheirbek andV.E.P., unpublished data), have a llow ed me t o com pare theduplicate HoxA clusters of zebra sh w i th the HoxA c lus -ters of both human and horn shark, to recognize a previ-ously undescr ibed pseudogene at the locat ion of h o x A 2 a (Fig. 3).

D espite the predictions of the classical model, that m anymore dupl icates wil l be los t than re ta ined, ver tebrate ge-n o m es a ppea r t o b e ri fe w i t h a n ci en t ge ne d upl ic a t es

IG. 3. Pipmaker plot (Schw artz et al . , 2000) comparing hum an and horn shark H oxA clusters w ith the hoxAa and hoxAb clusters ofebra sh. This strategy allow s a hoxa2a pseudogene to be recognized. Blast analyses of this sequence show homology to previously isolatedo xa2 genes. However, there are STOPS in all three frames. The zebra sh hoxAa sequence w as primari ly derived from the zebra sh

equencing project at the Sanger Inst i tute (hoxAa genomic sequence at Accession No. AL645756; hoxAb genomic sequence att tp://w w w .sanger.ac.uk/cgi-bin/nph-getblast ?hum pub/zebra sh a ll dZ31B14.00422), w ith gaps l led by our own sequenc ing of the

HoxA1a A3a intergenic sequence (Kheirbek and V.E.P., unpublished data).

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Nadeau and Sankoff, 1997). To explain this conundrum,ew theories have been put forw ard. G ibson and Spring1998)have suggested that changes in multidomain proteinsre l ikely to have dominan t nega t ive e ffect s , and thusupl icate genes m ay be re ta ined inde nitely despite theirunctiona l redundancy , because altered forms ha ve negativempact. Force and colleagues (Force e t al . , 1999; Lynch andorce, 2000) have proposed a model of subfunctionaliza-ion that ma y be m ore general ly appl icable. The Force

model suggests that the modular nature of eukaryotic genenhancers m ay l ead to a par t it ion ing o f gene funct ionsollowin g duplication, such th at complementa ry expressionomain s (spatial or tem poral) are lost th rough degenerationf individual regulatory e lements for each dupl icate . En-ancers could also change w ith respect t o th e levels of genexpression, so that duplicates produce some low er amoun tf protein than did the ancestral, preduplicate gene. Suchhanges could lead to the duplicates retaining complemen-ary funct ions both dupl icates wil l then be required toecapitulate the original gene function (referred to as theuplication degeneration complementa t ion , o r D D C

m odel; Force et al., 1999). These complementary mutationsnsure that both gene copies are retained in the genome. Anmportant extension of th is model is that once gene func-ions are divided betw een the duplicates, each gene may bereed to evolve along a novel trajectory on ce the const raintf funct ioning in mult iple contexts is removed.The limited data available suggest that different teleosts

o not all share a comm on H ox cluster architecture. Rather,here appear to have been different pat terns of Hox geneosses subsequent to the genome dupl icat ion event . Forxample, the zebra s h h a s a hoxC1a a n d a hoxC3a gene

Amores et a l ., 1998), but in the puffer s h, t h es e a reseudogen es (Aparicio et al., 1997). Sim ilarly , Ma laga -Trillond Meyer (2001) have described several differences in therchitecture of the H oxA clusters of zebra sh, striped bass,uffer sh, and an African cichlid. This variability in clusterrganizat ion contrasts markedly w ith our understanding ofs table te t rapod Hox cluster organizat ion. Nevertheless ,

a ther than being an except ion w ithin t he ver tebrates , th isariable architecture should perhaps be considered the rule,s t e leos t s make up the majori ty o f ver t ebra te species

abou t 25,000 sh species have been described; Nelson,994). Indeed, i t has been suggested that the variable Hox

rga n iz a t io n s m a y h a v e a d ire ct re la t io n sh ip w i t h t h eiversi ty of morphologies am ong t he te leosts (Meyer andchartl, 1999; Wittbrodt et al., 1998).

RESOLUTION OF ZEBRAFISH HOXGENE DUPLICATES

The zebra sh provides a tracta ble model system to exam -ne the funct ional s igni cance of Hox gene dupl icat ions .

U sing com parat ive sequence, expression, and funct ionalt ud ies , w e c an b egi n t o i n ve st i ga t e w h a t ev en t s h a v el lowed reten t ion o f selec t pa i rs o f zebra s h H o x g en e

duplicates (although many duplicate genes have been lost,a t l eas t 10 dupl ica tes have been main ta ined). I f novelfunctions could be un covered for z ebra sh Hox genes, thisw ould be consistent w i th t he hypothesis that the avai labi l-i ty of duplicate Hox genes w as important in facili tat ing theteleost radiation. Ideally, w e w ould compare zebra sh H oxgenes to t hose of a species that approximat es the ancestral,preduplicat ion condit ion. U nfortunately, informa t ion onthe H ox genes of the basal actinopterygians, wh ich are mostl ikely to prov ide such a compar ison g roup, has no t ye treached t he li terature.

How ever, Hox genes are unusual ly conserved in theirsequence, c lustered organizat ion, and regulat ion, w hichpermits (even requires) comparisons to be m ade over w ideevolutionary distances. Indeed, H ox genes are so conservedat t he level of protein function tha t, in som e cases, they canbe functionally substitu ted for one another betw een differ-ent phyla (e.g., Lutz e t al . , 1996). Thus, informative com-parisons can be made between zebra sh a nd such phyloge-net ically dis tant os te ichthya ns as m ice, a l low ing us to takeadvant age of the w ealth of data concerning mouse Hox geneexpression and funct ion. This approach rel ies on the as-sumption that the four-cluster organizat ion, seeminglyw idespread in sarcopterygians, re ect s the ances t ra l os-teichthy an condition. While this assumption already seem sreasonable, i t w ould be even m ore strongly supported w eretw o addi t ional Hox clusters to be found in t he chondrich-thy an horn shark, bringing the tota l num ber of Hox clustersto four in th e s is ter group to Osteicthyes .

There a re a to ta l o f 48 Hox genes desc ribed fo r thezebra sh (compared w ith 39 for m ouse and human ), yetdespi t e th i s d iffe rence in gene number, t he majori ty o f

zebra sh Hox genes show express ion pat t e rns tha t a reessent ia l ly s imilar to those of their m urine or thologues(Prince et al . , 1998a,b). One interesting exception to thisrule is the z ebra sh h o x A 1 a gene (McC lintock et al., 2001),w hich i s d i scussed in more de ta il below . Although thezebra s h i s w el l k n o w n f or i t s t r a ct a bi li t y a s a gen et i cmodel system, no homeot ic m utants have been uncoveredin large-scale forw ard genetic screens. Z ebra s h H o x m u -tan ts w ould be expected, based on our know ledge of mouse,to cau se alterations in vertebral m orphology and h indbrainsegmental identity. The large-scale zebra sh mut agenesisscreens w ere not designed to identify such phenoty pes, and

thus i t i s unsurpr ising that homeot ic mut ants have not yetbeen found.Nevertheless, other ty pes of m utan t h ave provided useful

information about zebra sh H ox gene function. In particu-lar, the phenotype of the lazarus ( lzr) mutant has suggestedthat zebra sh Hox genes must play very similar functionalroles to m amm alian H ox genes (Po pperl e t al . , 2000). Thel zr mutant affects a Pbx gene, zebra sh pbx4 ; Pbx proteinsare Hox cofactors , b inding together with Hox proteins ontheir target sequences to provide proper speci city to regu-la t ion of the downstream targets ( reviewed by Mann andAffolt er, 1998). The zebra sh pbx4 gene provides the majorPbx cofactor act ing dur ing ear ly development , and in i ts




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bsence there are mult iple defects wi thin the developingindbrain region. Al l of the phenotypes have been inter-reted as corresponding to losses of Hox gene function, byn a lo gy t o k n ow n m o u se H o x m u t a n t s (P o pperl et a l . ,000). This in terpretat ion h as been furt her supported by ou rna lysi s o f Hox gene knock-dow n phenotypes, w here

Hox protein t ransla t ion is blocked by using ant isense re-gents (Hunter and Prince, 2002; McClintock e t al . , 2002,ee below ).

Comparison of the l zr m utan t pheno type w i th the phe-otypes of mouse Hox mutants does not reveal any majorebra sh-speci c Hox gene functions during early develop-

ment: The l zr phenotype largely phenocopies null mut ant sf m ouse Hox genes. I t should be remembered, h ow ever,hat any novel la te funct ions of these Hox genes w ould note recognized due to the le thal nature of the l zr m u t a n t .urthermore, zebra sh H ox genes m ay have evolved Pbx-ndependent funct ions that w ould be unaffected by the l zr

m utan t. Whether or not the zebra sh Hox genes have tak enn novel funct ions (a q uest ion t hat remains w ide open atresen t ), w e do know tha t some dupl ica te genes w ereeta ined . A comparat ive approach can be used to t ry tostablish the mechanisms underlying these retentions.

Bruce et al . (2001) perform ed th e rst study t o investigatew hy a pair of zebra sh H ox genes have been retained rath erha n one gene being lost from th e genom e. In th is study, thexpression pat terns of zebra sh hoxB5a and hoxB5b wereompared to tha t o f the s ing le m ouse H o x b 5 gene, andound to recapitulate i ts overall expression. The zebra sho x b 5 duplicates have different, but overlapping, expres-ion pat terns , y et appear to share ident ical biochemicalunctions as assessed by a gain-of-function approach. Thus,

t seems tha t in th i s case , zebra sh hoxb5a and hoxb5b epresent a partit ioning of expression doma ins w ith respecto t h e m u ri ne H o x b 5 gene . Assuming tha t the mur ine

H o x b 5 gene re ec ts the ancestra l os te ichthyan s ta te , theHox duplication in the teleost l ineage appears to have leado a subfunct ional izat ion for these zebra sh hoxB5 dupli-a tes in accordance w ith t he D D C model . Fur ther tes ts ofhe m odel w ould inc lude demonst ra t ing tha t these tw oebra sh genes are able to funct ional ly subst i tu te for onenot her, alth ough it should be remembered that , even w henhe DDC model is invoked to explain the xat ion of geneuplications, this does not rule out subsequent alterations

hat might obscure ini t ia l funct ional equivalence. It w ouldlso be of interest to explore the regulatory sequences of theebra sh hoxB5a and hoxB5b genes, to attempt to identifyegenerative changes in the zebra sh sequences tha t un der-ie t he presumed part i t ioning of the ancestra l express ionomain.C h i u e t al . (2002) have recently investigated the molecu-

ar evolut ion of HoxA clusters across the major gnathos-ome l ineages: They compared complete HoxA c lus terequences of zebra sh , huma n, and horn shark. Dupl icateenes have been re ta ined for three of the more 5 -locatedebra sh HoxA genes, yet t he duplicated zebra sh clustersid no t show evidence fo r the k ind o f complementa ry

degenerative changes in ci s -regulatory e lements that theD D C m o de l pre di ct s . In st ea d , t h e t w o z eb ra s h H o x Aclus te r s , a s we l l a s the one repor ted s t r iped bass HoxAcluster, showed a conspicuous loss of putat ive ci s -regula-tory elements that are conserved between human and hornshark . The au thors conclude tha t the changes they havefound in the zebra sh sequences are consistent w ith a dap-t ive modi cation rather than t he more passive m echanismsassoc ia ted w i th subfunct iona li za t ion . By cont rast , com-parative sequence analysis of t he int ergenic region betw eenH o x b 2 and H o x b 3 of human, mouse, zebra sh , fugu, andstr iped bass has revealed extensive conservat ion of t ran-scription factor binding sites (Scemama et al., in press). Theconserved sites have been show n t o be important for properexpression of mouse H o x b 2 , and consistent w ith conservedfunct ion of these e lements , the expression pat terns of t hevertebrate H o x b 2 orthologues a re largely conserved. Inter-estingly , in several cases, th e binding sites occur in differentorders in different species, and such reorganization of smallci s -regulatory element s may ma ke it dif cult for large-scaleal ignm ent t echniques t o pick up funct ional hom ology.

Recent s tudies in my own lab have a lso focused on thequestion of w hy som e Hox duplicates have been retained inthe zebra sh genome. We have concentrated on the fourzebra sh Hox genes comprising paralogue group (PG ) 1,w h i ch i n cl ud e a pa i r o f d upl ic a t es w i t h r es pec t t o t h efour-clust er sta te, hoxB1a a nd hoxB1b . In th is case, w e havefound good evidence for an ancient subfunct ional izat ionbetw een the dupl ica tes . How ever, w e addi t iona l ly n devidence for a subsequen t more complex s itua t ion o f funct ion shuf in g among t he m embers of the paraloguegroup.

FUNCTION SHUFFLING AMONGPG1 GENES

The PG 1 genes are a particularly good system in w hich t oinvest igate potent ia l subfunct ional izat ion because tw o ofthe th ree mouse genes have had bo th gene func t ion andregulation studied in great detail . These experiments haves ho w n t h a t m o us e H o x a 1 and H o x b 1 are necessary forproper development of the hindbrain . In zebra s h, a s i n

mouse and chick, hindbrain morphology is conceptual lysimple , w i th overt segmentat ion dividing the hindbraininto seven lineage-restricted compartments termed rhom-bom eres (r1 r7 f rom A t o P). This basic organizat ion isconserved across the vertebrates, and th ere are a w ealth ofmolecu la r and neuroana tomica l markers tha t a l low theident i ty of individual rhombomeres to be unam biguouslyrecognized (review ed by Moen s an d P rince, 2002).

H o x a 1 and H o x b 1 are coexpressed in the mouse hind-brain from t he early stages of gastrulation, w ith an identicalan te rior expression l imi t a t the p resumpt ive boundarybetw een r3 and r4 (Barrow et al., 2000; Frohm an et al., 1990;Murphy and Hil l , 1991; Wilkinson et a l ., 1989). This

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xpression is dependent on retinoic acid response elementsRAREs) that lie 3 of each gene. H o x a1 expression is veryransient in r4, retracting posteriorly out of the hindbrainuring early som ite stages. In contrast, H o x b 1 expression istably maintained in r4, while expression is gradually lostrom r5 and r6 to leave an r4 stripe of H o x b 1 expression.

This r4 H o x b 1 domain is mainta ined by an autoregulatoryositive feedback m echanism, w hich is dependent on t hreee ned Hox/Pbx binding sites upstream of H o x b 1 (P opperlt a l . , 1995).

Mutan t analysis of mouse H o x a1 a nd H o x b 1 has revealedha t these tw o paralogues p lay d ivergen t , bu t par t ia l lyedundant , roles in pat terning the hindbrain . The primeunct ion of the H o x b 1 gene is t o confer proper r4 identity ,s loss of H o x b 1 function results in ma jor alterations to th e4-derived facial m otor neurons, w hich no longer undergoheir normal migration behavior (G aufo e t al . , 2000; G od-ard et al . , 1996; Studer et al . , 1996). By contrast, loss of

H o x a 1 function ca uses a radical reduction in the AP extentf r4 and r5, with an accompanying reduction in the size ofhe adjacent otic vesicle (C arpenter et al., 1993; Chisaka et l. , 1992; Lufk in et al., 1991; Ma rk et al., 1993); thus, H o x a1 s ra ther unusual as i t i s important for se t t ing up properegmental organizat ion of the hindbrain , not jus t for con-er ra l o f segmenta l iden t ity. D ouble knockouts o f both

H o x b 1 and H o x a1 show synergistic phenotypes (Barrow et l. , 2000; G avalas e t al . , 1998; Ro ssel an d C apecchi, 1999;tuder et al . , 1998) revealing redundancy of function be-

ween the paralogues.In t h e z eb ra s h, t h e hoxB1 duplicates, hoxB1a and

oxB1b , ha ve expression pro les tha t a re intriguingly sim i-ar t o t h os e o f m o us e H o x b 1 and Hoxa1 , respectively

McClintock et al., 2001), although zebra sh hoxB1a lackshe early gastrula-stage expression show n by m ouse H o x b 1 .

By con t ras t , t he zebra sh o rtho logue o f mouse H o x a 1 ,ebra sh h o x A 1 a , is not expressed in presumptive r4, andhu s cannot play a role in early patterning of this hindbrainerritory (McC lintock et al., 2001; Sh ih et al., 2001). Hence,xpress ion da ta sugges t the hypothesi s tha t zebra shoxB1a and hoxB1b a re the funct iona l equ iva len t s o f

mouse H o x b 1 and H o x a 1 , respectively.A new knock-dow n technology, using stabilized anti-

ense m orpho l inos, has a l lowed us to t e st d irec t ly theunctions of t he zebra sh hoxB1 duplicates (McC lintock et

l. , 2002). We have dem onst rated t hat zebra sh hoxB1a a ndoxB1b do indeed play s imilar roles to m ouse H o x b 1 andH o x a 1 . Th u s, t h e z eb ra sh hoxB1a gene , l ike mouseH o x b 1 , i s r equ i red for proper migra t ion o f f acia l ne rve

eurons f rom r4 and for i t s own posi t ive regulat ion. TheoxB1b gene, l ike mouse H o x a 1 , i s required for properegmental organizat ion of the hindbrain , and for develop-

ment of a normal ly s ized ot ic vesicle . How can our ndingha t a zebra sh H o x B dupl icate gene and a mouse H o x Aene a re funct iona lly equ iva len t be reconc i led w i th the

D D C subfunct ional izat ion m odel? Data emerging from t heebra sh sequencing project have helped us to develop a

model to explain our ndings.

Accord ing to the D DC model , the dupl ica tes w ould beexpected to divide out the ancestral expression domain. Inaccord w ith th e model, the hoxB1b gene has an expressionpattern resem bling the gastrulation phase of m urine H o x b 1 expression, while the hoxB1a gene has an expression pat-tern resembling th e later r4 stripe phase of mouse H o x b 1 expression. The D D C m odel also predicts t he degenerationof discrete complementary ci s -regulatory elements in thetw o duplicates. We nd that , a l though hoxB1b possesses a3 RARE wi th a two-nuc leo t ide spacer be tween the ha l fs i t e s , s imi la r to the one which in mouse H o x b 1 confersgastrulation st age expression, w e are unable to detect suchan elem ent 3 of hoxB1a , consistent w ith its lack of an earlyexpression phase. Similarly, zebra sh hoxB1a retains per-fect copies of a l l t hree H ox/Pbx binding s i tes, w hich inmouse H o x b 1 confer autoregulation in r4, yet hoxB1b haspoint changes in each of the individual s i tes, consis tentw i th the absence o f a l a t e r4 expression domain fo r th i sgene. This degeneration of different regulatory modules ineach of the t w o duplicates is l ikely t o have been suf cientto allow preservation of the two genes as postulated by theD D C m odel (Fig. 4), but leaves open the quest ion of how th efunction of a H o x A gene could have shifted to a H o x B gene.

Our m odel for how hoxB1b cam e to take on the role thatin mouse is played by H o x a 1 has been in uenced by ourexpression analyses of vertebrate H o x a 1 orthologues. Wehave show n that the zebra sh h o x A 1 a gene is expressed atlate neurulation stages in a small, bilaterally located groupof neurons in th e ventral midbrain (McC lintock et al., 2001;Shih et al., 2001). As m idbrain expression ha s not generallybeen descr ibed for Hox genes , th is domain seems at rs tobserva t ion to re ect a poten t ia l neofunct iona li za t ion

even t . However, our compara t ive ana lyses have demon-strated that midbrain expression is more likely a primitivecharacter is t ic of the vertebrate PG 1 genes . Thus, w e n dexpression of H o x a1 orthologues in a similar group of cellsnot on ly in an other teleost, m edaka, but also in the sarcop-terygian ch ick (C . Jozefow icz. an d V.E.P., unpublished ob-servat ions). Fur thermore, w e have con rmed a previousdescript ion of midbrain express ion for X e n op u s H o x a1 (Kolm and Sive, 1995). As Xenopus and chick combine bothhindbrain and midbrain expression domains of H o x a1 , w ehypothesize th at these tw o separate expression domainsrepresent the a ncestral condition. In t he zebra sh , hoxB1b

has t aken on the h indbra in pa t t e rn ing ro le o f t e t rapodH o x a 1 , w hich may have f reed h o x A 1 a to lose its hin dbrainexpress ion domain w hi le re ta ining the ancestra l midbrainpatterning role (Fig. 4). We hav e t ermed t his phenomenonfunct ion shuf ing (McC lintock et al . , 2001, 2002), and itrel ies upon a phase of par t ia l funct ional redundancy be-tween nonorthologous genes , in this case the paralogoush o x A 1 a and hoxB1b . T he se d at a re ve al t h a t i t m a y b eessential to study an entire group of related genes to fullyunderstand the consequences of a part icular duplicat ionevent.

We have a l so been ab le to combine the morpho l inoknock-dow ns w ith m RN A misexpression to test th e degree




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f interchangeability of Hox PG 1 coding sequences (Mc-Clintock et al., 2002). In these experiments, w e att empted

rescue of knock-down phenotypes with different mRNAs.We found that mouse H o xb 1 can functionally substitute for

ither zebra sh hoxB1a or hoxB1b , consistent w ith th e modelhat the tw o zebra sh duplicates have subfunctionalized t hencestral roles that in mouse continue to be played by theingle H o x b1 gene. However, we also found that, althoughoxB1a can functionally substitute for hoxB1b , th e reciprocals not true. Thus, hoxB1b has lost t he capacity to allow proper

migration of facial nerve neurons. Once again, this is consis-ent w ith th e model of Force and colleagues (Force et al., 1999;ynch and Force, 2000): Their D D C model states that, al-

hough complementary degeneration of ci s -regulat ory ele-ments i s w ha t ini t ia lly a llow s maintenance o f a pa ir o f

uplicates, it does not prevent the individual genes from then

becoming ne-tuned to t heir separate functions or eventu-ally taking on novel functions.

Func t ion shuf i n g m a y pro v e t o b e c o m m o n a m o n gzebra sh pa ra logues . For example , i t has recen t ly beens h o w n u s in g m o r ph o l in o -b a se d k n o c k-d o w n t h a t t h ezebra sh en g 2 and eng3 genes have ear ly developmentalr ol es e q u i v a l en t t o t h a t o f t h e n o n o r t h o l og ou s m o u seEN 1 gene (Scholpp and Brand, 2001). Furthermore, func-t ion shuf i ng may no t be l imi t ed to t r ansc r ip t ion fac to rgenes: The secreted s ignal ing molecule bmp2a from ze-bra sh appears to p lay an equ iva len t func t iona l ro le tothe nonor tho logous X e n o p u s Bm p4 dur ing dorsoven t ra lp a t t er n in g o f g a st r u la -s t a ge e m b ry o s (N g u y e n et a l .,1998). O n a pract ical n ote , t hese ndings suggest t hat , incases w here o r tho logy rela t ionsh ips a re unc lea r, i t m aynot he lp to assume th a t com mon func t ion can he lp w i th

IG. 4. Model outlining the evolutionary mechan ism of Hox P G 1 gene funct ion shuf ing. The ci s -regulatory element s characterizedor the mouse and human H oxa1 a nd H o x b1 genes [3 RAREs (blue), H ox/Pbx bindin g sites (red)] are assum ed to be present in t he an cestral,re- third -duplication, condition. We also postulate the presence of a regulatory domain directing midbrain expression of H oxa1 (m au ve),lthough no such domain has yet been characterized. The duplication event in the lineage leading to teleosts produced redundant copiesf both Ho xa1 and H o x b 1 in an ancestor of the zebra sh. The hoxA1b duplicate was eventual ly lost by accumulat ion of deleter ious

mutat ions ( nonfunctionalization )a s predicted by classical models. In cont rast, the hoxB1a and hoxB1b genes accumulated complemen-ary degenerat ive changes in their ci s -regulatory elements such that hoxB1a lost early, RARE-mediated expression, a nd hoxB1b lostutoregulation. This led to retention of t he duplicate genes, as both w ere required to m ainta in th e expression pattern and function of t heingle H o x b 1 ancestral gene (subfunctional izat ion), as predicted by the DD C model . As hoxA1a and hoxB1b shared similar coding

equences and expression patterns, these two genes were now functionally redundant with respect to a role during gastrulation in settingp segmental organizat ion of the hindbrain. These nonorthologous genes w ere t hus able to go through anoth er subfunctionalization vent , such that ho xA1 a lost its early RARE-mediated expression, which was retained by hoxB1b . Thus, hoxB1b became essential forroper hindbrain segmentation, the role played in the ancestral state by H oxa1 . Retent ion of the hoxA1a gene in the lineage leading toebra sh w as presumably dependent on a funct ion that w as not redundant w ith hoxB1b , possibly a role in m idbrain patterning. We termhis rearrangement of P G 1 gene roles funct ion shuf ing.

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ss ignments synt eny re la t ionships are m ore l ikely to berel iable tool .

CONCLUSION AND FUTURE DIRECTIONS

There is no doubt that Hox gene functions are intima telyssociated w ith axial pat terning, an d therefore changes in

Hox genes are l ikely to play a key role in the evolut ion ofew body plans . Many researchers have emphasized t hemportance of alterations in ci s -regulation of Hox and ot herevelopmental control genes for a l lowing differ ing mor-hologies to arise during evolution (reviewed by Carroll ,000). Stu dies in invertebrates have tended t o support theo t ion tha t ci s -regulation can be t inkered w i t h m o r easily than protein coding sequences, presumably becauseetrimental effects are less l ikely to result from sequencehanges. How ever, r ecen t w ork has revea led tha t a l t er-tions to Hox proteins, as opposed to alterations in regula-ion of Hox expression, can underlie major morphologicalransi t ions . Tw o s tudies (G alant and C arrol l, 2002; Ron-haugen et al . , 2002) have demonstrated that insects lostheir abdominal l im bs, such th at t hey have only six legs, as

resu lt o f func t iona l changes in the Hox p ro tein Ubx .These reports underscore the importance of consider ing

oth gene regu la t ion and p rote in funct ion as w e t ry t onravel how changes in Hox genes have in uenced verte-rate evolution.Consis tent wi th the idea that changes to Hox genes can

nderlie n ew m orphologies, t he large-scale gene duplica-ions in the ver tebrate s tem l ineage provided many addi-ional Hox genes, wh ich correlate w ith th e innovat ions that

haracter ize the vertebrates (review ed by Holland et a l . ,994). It has been suggested that the additional duplicationvent in t he lineage leading to teleosts such as th e zebra shrovided yet more raw genetic material for selection to actpon, and that this may have facili tated the broad radiationf teleosts (Meyer and Schartl , 1999). Although the radia-ion of t he te leosts has been underway for about 200 My,his t im e frame is re la t ively short in comparison w ith t heistant or igin of ver tebrates, more t han 520 M ya. Thus,tudies of teleost shes hold signi cant promise for a l low-ng us to tes t the importance of changes in Hox genes forhe genera t ion o f new fo rms . In o rder to pursue these

tudies , i t w i l l be important for the duplicat ion event thatas led to addi t ional Hox clusters in te leosts to be moreccurate ly dated. This w il l a l low us to recognize the las tommon ances to r o f an imals wi th and wi thou t the ex t rauplication, and provide an appropriate comparison pointor a l l future s tudies. To t his end, upcoming new data onasal teleosts and ray- nned shes will be invaluable.To fur ther invest igate the roles of Hox genes w ithin the

adiat ing te leosts , i t w i l l be vi ta l to s tudy species w ithin ahylogenetic framework. In particular, i t will be importanto correla te known morpho logical va ri a t ion w i th d iffe r-nces in Hox organizat ion an d function. Species suitable foruch s tudies m ight include the m embers of t he te t raodon-

t i fomes , w hich have a r emarkably va rian t morpho logy(Sant ini and Stellw ag, 2002; Tyler an d Sorbini, 1996). Thiswil l entai l much hard work in determining detai ls of Hoxcluster archit ecture for a range of species, and thu s it w ill beimportant to choose species wisely. It w ill also be useful tohave a r el iab le means o f d is rupt ing gene funct ion , andconven ien t ly, the new morpho l ino t echno logy fo r geneknock-down should be equal ly appl icable to any systemw here early embryos can be microinjected. As th e ma jorityof sh species have embryos that develop external ly, t h isopens up the p rospect o f b road comparat ive funct iona lanalyses.

Another approach to understanding t he genet ic basis ofmorphological evolution is to use variation among closelyrela ted species to ident i fy loci that contr ibute to the ob-served variation. Peichel et al . (2001)have used quantitativetrait locus (QTL) mapping to investigate variation in skel-e ta l armor and feeding morphologies of the threespinedst icklebacks, w ell -s tudied te leosts that have undergonerapid divergence and speciation over th e last 15,000 years.This w ork has identi ed a large num ber of QTL associatedwith the differ ing morphologies . I t wi l l be interes t ing toknow whether these QTL correla te with known develop-mental control genes , including Hox genes , a l though tar-geted s tudies of the expression pat terns of speci c H oxgenes in morphological ly dist inct populat ions of s t ickle-b ac ks h a v e n o t y et r ev ea le d a n y c orre la t io n s w i t h t h edifferent m orphologies (Ahn and G ibson, 1999).

I t i s important to not e that gene dupl icat ion events m aybe important for a l lowing speciat ion to occur via mecha-n is m s t h a t a re s epa ra bl e f ro m t h e gen era t io n o f n ew morphologies. Lynch and col leagues have postula ted t hat

divergent resolution of duplicate genes could cause spe-ciation within populations that are temporarily geographi-cally isolated (Lynch and Conery, 2000; Lynch and Force,2000; review ed by Tay lor e t al . , 2001a,b). This w ould relyupon speci c pairs of duplicated genes un dergoing differentfates in different populations, for example, loss of differentdupl icate genes , or subfunct ional izat ion versus nonfunc-t ional izat ion. Such events would reduce the fecundi ty offuture hybrids once the separated populations become re-u n it e d. C o n s is t en t w i t h t h i s h y po t h es is , t h e s al m o ni d shes, wh ich ha ve gone through a recent genom e duplica-t ion event , are s igni can t ly more speciose than a s is t er

taxon t hat has not (review ed by Taylor e t al . , 2001b). Themore divergently resolved loci present, the more effectivesuch an isola t ion m echanism w ould be, thus in t he case ofthe radiating teleosts this m odel is more relevant to a w holegenome dup l ica t ion even t than to a more l imi ted , Hox-speci c, duplication event.

Wh a t t h en c a n w e l e arn f ro m t h e H o x g en es o f t h ezebra sh , the te leost that is current ly best understood atboth t he m olecular genet ic level , and in t erms of i ts earlydevelopment? I t has been establ ished t hat zebra s h h a sretained at least 10 duplicated Hox genes, opening up thepossibil i ty that , in some cases , dupl icates w ere xed be-cause one o f them a t t a ined a nove l func t ion . In the two




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ases that have been investigated in detail , this appears noto be the case . The hoxB5 duplicates have subfunctional-zed in accordance wit h the D D C m odel (Force et al., 1999),

w hereas the PG 1 genes h ave gone t hrough an interest ingunct ion shuf ing, w hi le s t i l l not undergoing an y obviouseofunct iona li za t ion . How ever, i t shou ld be no ted tha teofunct ional izat ion may prove dif cult to recognize, es-ec ia l ly in the absence o f a comple te knowledge o f therimit ive condit ion. Important changes could be subt le or example, minor but cr i t ical changes in t iming of genexpression, concentrat ion of gene product , or or igin of aew la te expression pa t t e rn tha t w ould no t be detected

within the usual t ime frame of developmental express iontudies. Furthermore, the com parative sequence analysis of

C h i u e t al . (2002) has provided evidence for adapt ive m odi-cation in teleost Hox regulatory elements, suggesting th atew express ion domains may w el l have ar isen followingupl icat ion. Alternat ively, the 10 reta ined dupl icate Hoxenes m ay al l prove to have undergone some var ia t ion onhe subfunct ional izat ion theme. Nevertheless , th is wouldo t undermine Ohno s h ypothesis th at gene dupl icat ionacili tates evolution by providing new genetic material andllow ing genes to tak e on new functions. Rath er, there m aye o ther developmenta l con t ro l genes tha t have ga inedmportant novel funct ions subsequent to dupl icat ion, an dery good candidates for such genes would be the down-tream effectors of Hox function.

ACKNOWLEDGMENTS

I thank James McClintock, Chris Jozefowicz, Ed Stel lwag, andAnnie Burke for many helpful discussions and comments on themanuscript . I am grateful to Ed Stel lw ag for sharing unpublished

a ta and to Mazen Khei rbek for sequenc ing a l a rge par t o f theoxA 3ahoxA 1a intergenic element . The w ork discussed hererom my lab was funded by NSF G rant IBN 0091101 and by MOD

G rant # FY00-336.

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Received for publication March 8, 2002Revised May 28, 2002

Accepted May 28, 2002Published online August 7, 2002


Prince V_The Hox Paradox_2002

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