Prince V - The Hox Paradox - 2002

REVIEW
TheHox Paradox: MoreComplex(es) Than Imagined

VictoriaE. Prince
1
Department of Organismal Biology and Anatomy, Committees on Developmental Biology,
Neurobiology, Genetics and Evolutionary Biology, The University of Chicago,
1027 E. 57th Street, Chicago, I llinois 60637
An understandingof the origin of different body plans requires knowledge of how the genes and genetic pathways that
control embryonic development haveevolved. TheHox genesprovideanappealingstartingpoint for suchstudiesbecause
they play a well-understood causal role in the regionalization of the body plan of all bilaterally symmetric animals.
Vertebrateevolutionhasbeencharacterizedby gene, andpossibly genome, duplicationevents, whicharebelievedtohave
providedrawgenetic material for selectiontoact upon. It hasrecently beenestablishedthat theHox geneorganizationof
ray-nnedshes, such as thezebrash, differs dramatically fromthat of their lobe-nnedrelatives, agroupthat includes
humans andall theother widely usedvertebratemodel systems. This unusual Hox geneorganization of zebrash is the
result of a duplication event within the ray-nned sh lineage. Thus, teleosts, such as zebrash, have more Hox genes
arrayedover moreclusters(or complexes)thandotetrapodvertebrates. Here, I reviewour understandingof Hox cluster
architectureindifferent vertebratesandconsider theimplicationsof geneduplicationfor Hoxgeneregulationandfunction
andtheevolutionof different body plans. 2002 Elsevier Science (USA)
Key Words: Hox genes; Hox clusters; geneduplication; vertebrateevolution; teleosts; zebrash; hindbrain.
CLUSTERED ORGANIZATION
OF HOX GENES
The Hox genes were rst characteri zed i n the frui ty,
Drosophila melanogaster, where ei ght l i nked Antennapedia
cl ass homeobox genes make up the Homeoti c compl ex
(Lewi s, 1978). These ei ght genes encode homeodomai n tran-
scri pti on factors that are characteri zed by thei r rol e i n confer-
ral of segmental i denti ty al ong the pri mary body axi s, from
anteri or to posteri or (AP; revi ewed by McGi nni s and Krum-
l auf, 1992). Thus, mutati ons i n the y Hox genes l ead to
dramati c homeoti c phenotypes, where one body segment
takes on the i denti ty of another. Homol ogous Hox genes have
been found i n every bi l ateri an ani mal i nvesti gated (de Rosa et
al., 1999), and i n al l cases anal yzed, the genes show a cl ustered
organi zati on, al though gene and cl uster number vary. Most
i mportantl y, wherever tests have been appl i ed, the Hox genes
have proven to pl ay cri ti cal rol es i n determi ni ng AP i denti ty.
Comparati ve anal yses of Hox cl uster organi zati on have
reveal ed that vari ati ons i n Hox gene number between
speci es reect an evol uti onary hi story characteri zed by two
types of dupl i cati on events: tandem dupl i cati on and whol e
cl uster dupl i cati on. Current model s suggest that si ngl e
cl uster organi zati ons, l i ke that of D. melanogaster, arose
vi a the tandem dupl i cati on of ancestral Hox genes (Kappen
et al., 1989; Kmi ta-Cuni sse et al., 1998). A si ngl e cl uster
organi zati on appears to be common to al l protostomes, and
a si ngl e cl uster wi th seven genes was i n pl ace i n the
ancestor of al l bi l ateri ans (Fi g. 1) (de Rosa et al., 1999). A
si ngl e Hox cl uster i s al so assumed to be characteri sti c of
pri mi ti ve deuterostomes, wi th the cephal ochordate am-
phi oxus havi ng the cl uster wi th the l argest number of genes
(Fi gs. 1 and 2) (Ferri er et al., 2000).
It has l ong been supposed that gene dupl i cati on events
coul d have pl ayed a vi tal rol e i n al l owi ng vertebrates to
achi eve thei r compl exi ty of form through evol uti on of new
gene functi ons (Ohno, 1970). The ori gi n of vertebrates was
associ ated wi th major expansi ons i n gene number, possi bl y
as a resul t of two rounds of whol e genome dupl i cati on vi a
pol ypl oi di zati on (often referred to as the 2R hypothesi s,
for two rounds of dupl i cati on; Fri edman and Hughes,
2001; Si dow, 1996). Such dupl i cati ons woul d resul t i n
1
Fax: (773) 702-0037. E-mai l : vpri nce@mi dway.uchi cago.edu.
Devel opmental Bi ol ogy 249, 115 (2002)
doi :10.1006/dbi o.2002.0745
0012-1606/02 $35.00
2002 El sevi er Sci ence (USA)
Al l ri ghts reserved. 1
dupl i cati on of enti re Hox cl usters, and consi stent wi th the
2R hypothesi s, tetrapod vertebrates have four cl usters of
Hox genes (revi ewed by Hol l and et al., 1994; Si dow, 1996).
Mouse and human have had thei r Hox cl uster organi zati ons
ful l y descri bed, and they share an i denti cal 39-gene organi -
zati on over 4 cl usters, AD (revi ewed by McGi nni s and
Kruml auf, 1992; see al so Zel tser et al., 1996). The genes fal l
i nto 13 paral ogue groups, wi th most paral ogue groups
havi ng l ess than a ful l compl ement of 4 genes as a resul t of
secondary gene l osses (Fi g. 1). A l arge number of Hox genes
have al so been i sol ated from frog (Xenopuslaevis)and chi ck
(Gallus gallus), and i n each case, there i s no evi dence to
suggest di fferences from the 39-gene mammal i an organi za-
ti on (e.g., Godsave et al., 1994; Ladjal i -Mohammedi et al.,
2001). Thus, avai l abl e data strongl y suggest that a 4-Hox
cl uster organi zati on i s the pri mi ti ve condi ti on of crown-
group tetrapods. Si mi l arl y, a PCR survey of Hox genes i n
the more basal l ungsh (Longhurst and Joss, 1999) i s al so
consi stent wi th a 4-cl uster condi ti on. Neverthel ess, we
need more compl ete data from l ungsh, as wel l as from the
coel acanth, before we can concl ude that there has been
compl ete conservati on of the 4-Hox cl uster organi zati on
throughout the sarcopterygi ans (l obe-nned shes).
If 2R happened, can we esti mate when each genome
dupl i cati on event woul d have occurred? The cephal ochor-
date amphi oxus may provi de the best approxi mati on of the
predupl i cati on vertebrate ancestor. The rst 10 Hox genes
i n the si ngl e amphi oxus Hox cl uster show cl ear homol ogy
to the rst 10 mammal i an paral ogue groups (Fi g. 1) (Garci a-
Fernandez and Hol l and, 1994). The most basal group of the
vertebrates i s the cycl ostomes, compri si ng hagsh and
l ampreys (Fi g. 2), whi ch probabl y form a monophyl ecti c
group (Mal l att and Sul l i van, 1998). These jawl ess verte-
brates (Agnatha) mi ght be expected to fal l i nto an i nterme-
di ate state between an ancestral , si ngl e Hox cl uster organi -
zati on and a deri ved, 4-cl uster organi zati on. Recentl y, 2
groups have publ i shed extensi ve anal yses of the Hox cl us-
ters of the sea l amprey (Petromyzon marinus) (Force et al.,
2002; Irvi ne et al., 2002). Both groups have i sol ated and
mapped genomi c Hox cl ones, to extend previ ous anal yses
based on PCR surveys (Pendel ton et al., 1993; Sharman and
Hol l and, 1998). In both of the new studi es, the data poi nt to
a mi ni mum of 3 Hox cl usters, wi th a 4th cl uster consi dered
l i kel y. Irvi ne and col l eagues (2002) bui l t nei ghbor-joi ni ng
trees based on sequences of i ndi vi dual homeodomai ns and
were unabl e to di sti ngui sh between model s where onl y one
FIG. 1. Hox gene organi zati on i n Drosophila melanogaster, am-
phi oxus, mouse, and zebrash. A hypotheti cal ancestral condi ti on
i s al so shown. Shades of gray i ndi cate the most cl osel y rel ated genes.
FIG. 2. Vertebrate phyl ogeny showi ng Hox cl uster number and putati ve dupl i cati on events. (Based on Carrol l , 1988).
2 Victoria Prince
2002 El sevi er Sci ence (USA). Al l ri ghts reserved.
round of dupl i cati on occurred before di vergence of the
jawed (gnathostome) and jawl ess vertebrates, versus al l
dupl i cati ons predati ng the spl i t. By contrast, Force and
col l eagues (2002) concatanated the l amprey homeodomai n
sequences that l ay i n conti gs, to provi de addi ti onal i nfor-
mati ve characters. Usi ng thi s strategy, thei r extensi ve tree
anal ysi s of Hox genes across the vertebrates suggests that
onl y one dupl i cati on event occurred pri or to the di vergence
of the agnathans and gnathostomes, wi th a second dupl i ca-
ti on event occurri ng wi thi n the l i neage l eadi ng to l ampreys.
Consi stent wi th thi s model , both groups found pai rs of
l amprey Hox genes that are more cl osel y rel ated to one
another than to any Hox gene from a gnathostome. Taken
together, these data support the i dea that 1 round of Hox
dupl i cati on occurred before the di vergence of the agnathans
and gnathostomes, and a 2nd occurred i n the gnathostome
l i neage. Ul ti matel y, a compl ete l i nkage map of al l the
l amprey Hox genes wi l l hel p to conrm thi s model .
An al ternati ve model to the 2R hypothesi s has been put
forward by Ruddl e and col l eagues (Bai l ey et al., 1997).
Accordi ng to thi s model , there were not two rounds of
dupl i cati on i n the l i neage l eadi ng to tetrapods, but three.
The exi stence of onl y four Hox cl usters i n the tetrapods i s
expl ai ned ei ther by i ncompl ete dupl i cati ons (of a si ngl e
cl uster) or by l osses of cl usters fol l owi ng whol e genome
dupl i cati on events. Bai l ey and col l eagues (1997) used se-
quence from collagen genes l i nked to Hox cl usters to
reconstruct a l i kel y dupl i cati on scenari o whereby the an-
cestral Hox cl uster was D-l i ke, whi ch dupl i cated to create
an A-l i ke cl uster from whi ch the B and C cl usters arose i n
turn (D(A(B,C))).
The Ruddl e group has recentl y expanded i ts studi es to
i ncl ude the horn shark (Heterodontus francisci). Thi s spe-
ci es i s carti l agi nous, a member of Chondri chthyes, another
basal group of vertebrates and a si ster group to the bony
shes (Ostei cthyes; Fi g. 2). Investi gati ons of the horn shark
have so far reveal ed the presence of onl y two Hox cl usters,
M and N. However the sequences and organi zati on of the
Hox genes wi thi n these cl usters suggest that M i s homol o-
gous to the A cl uster, whi l e N i s homol ogous to the C or D
cl uster, as descri bed i n mammal s (Ki m et al., 2000). If the
three-dupl i cati on event model i s correct, the horn shark
may ul ti matel y prove to have onl y three Hox cl usters i n
total , representi ng a stage before dupl i cati on of the com-
mon ancestor of the B and C cl usters. However, there has
been l i ttl e addi ti onal data supporti ng the three-dupl i cati on
event model , and the recent extensi ve tree bui l di ng of Force
and col l eagues (2002) strongl y supports two sequenti al
dupl i cati on events [(AB)(CD)] l eadi ng to a four-cl uster Hox
organi zati on i n Sarcopterygi a. It therefore seems l i kel y that
chondri chthyans (carti l agi nous sh) and sarcopterygi ans
(l obe-nned ostei chthyans) wi l l eventual l y prove to share a
very si mi l ar four-cl uster Hox organi zati on, i n whi ch case
the putati ve two rounds of dupl i cati on both occurred before
the ori gi n of chondri chthyans.
Despi te the preval ence of the 2R hypothesi s, the phyl o-
geneti c anal yses of Hughes and col l eagues (e.g., Fri edman
and Hughes, 2001; Hughes et al., 2001) do not provi de
strong support for two rounds of genome dupl i cati on i n the
vertebrate stem l i neage. The Hox genes themsel ves are
notori ousl y uni nformati ve for detai l ed phyl ogeneti c anal y-
si s because of thei r remarkabl e sequence conservati on.
Thus, Hughes et al. (2001) constructed phyl ogeneti c trees
for other sets of dupl i cated genes l yi ng on the Hox-beari ng
chromosomes of human. The trees for di fferent gene fami -
l i es have di fferent topol ogi es, whi ch the authors i nterpret
as reveal i ng that the dupl i cated genes di d not ari se si mul -
taneousl y and are rather the resul t of numerous i ndepen-
dent smal l -scal e dupl i cati on events. However, these di ffer-
ent topol ogi es coul d al so reect recombi nati on/conversi on
events between cl osel y rel ated genes shortl y after dupl i ca-
ti on or may be an artefact of rapi d evol uti on subsequent to
dupl i cati on. Furthermore, the newl y avai l abl e human ge-
nome sequence reveal s extensi ve synteny between the four
Hox-beari ng cl usters (al though l ess evi dence for l arger-scal e
dupl i cati on events; Internati onal Human Genome Sequenc-
i ng Consorti um, 2001). Whether or not the four Hox cl us-
ters of the sarcopterygi an vertebrates arose as a resul t of two
rounds of genome dupl i cati on wi thi n the vertebrate stem
l i neage, the appearance of more than one Hox cl uster
correl ates wel l wi th the ori gi n of vertebrate speci c char-
acters, such as neural crest, epi branchi al pl acodes, and an
el aborated brai n. If the rol e of Hox genes i n regi onal i zati on
of the body pl an i s consi dered i n the l i ght of the i dea that
gene dupl i cati ons can provi de new geneti c materi al for
sel ecti on to act upon (Ohno, 1970), i t can be hypothesi zed
that some of the speci al i zed characters of the vertebrates,
and the vari ati on wi thi n the vertebrate body pl an, are
essenti al l y a resul t of the avai l abi l i ty of addi ti onal Hox
genes (Hol l and et al., 1994). A more si mpl i sti c noti on i s
that there i s a di rect rel ati onshi p between number of Hox
genes and compl exi ty of morphol ogy.
IMPLICATIONS OF HOX CLUSTER
ORGANIZATION FOR GENE
REGULATION AND FUNCTION
The cl ustered organi zati on of Hox genes shows an obvi -
ous rel ati onshi p to thei r mode of expressi on. Thus, expres-
si on domai ns al ong the pri mary axi s of devel opi ng embryos
reect the l ocati ons of i ndi vi dual genes wi thi n the cl usters,
such that more 3-l ocated genes have more anteri or expres-
si on domai ns. Thi s orderl y rel ati onshi p i s termed spati al
col i neari ty, and i n vertebrates, there i s al so a temporal
col i neari ty, such that the most 3 genes have the earl i est
onsets of expressi on, wi th a sequenti al acti vati on of adja-
cent more 5 genes. The stage of thi s i ni ti al sequenti al
acti vati on of Hox genes i s the most conserved devel opmen-
tal stage among the vertebrates, the phyl otypi c stage.
The cl ustered organi zati on of the Hox genes i s assumed
to pl ay a vi tal rol e i n the establ i shment of col i near expres-
si on, al though the mechani sms of thi s process remai n
somewhat obscure (revi ewed by Duboul e, 1998). Control of
3 Hox Cluster Architecture and Vertebrate Evolution
Hox gene expressi on has been wel l studi ed by usi ng trans-
geni c approaches i n the mouse (revi ewed by Capecchi ,
1997), and as wi th other eukaryoti c enhancers, the Hox
regul atory el ements i ncl ude seri es of i ndependent modul es.
For exampl e, reti noi c aci d response el ements (RAREs) are
frequentl y i mportant for proper Hox gene regul ati on i n
mouse, and i n many cases, gene-speci c regul atory el e-
ments have been shown to medi ate posi ti ve autoregul ati on
or cross-regul ati on by other Hox genes. However, the trans-
geni c approach has al so reveal ed that adjacent Hox genes
can share enhancer el ements or compete for them (Goul d et
al., 1997; Sharpe et al., 1998; Zakany et al., 1997). As
poi nted out by Duboul e (1998), thi s ki nd of compl ex i nter-
twi ni ng between regul atory el ements of adjacent Hox genes
may be a consequence of the ti ght cl usteri ng of the genes
rather than i ts cause: It may expl ai n the mai ntenance of
cl ustered organi zati on, yet not hel p us to understand how
cl ustered organi zati on arose i n the rst pl ace.
On thi s poi nt, i t shoul d al so be noted that the vertebrate
Hox genes are cl ustered far more ti ghtl y than those of the
frui ty. The i ntergeni c di stances i n the mouse are an order
of magni tude smal l er than those of Drosophila, i mpl yi ng
that the mechani sms of gene regul ati on must di ffer si gni -
cantl y between these speci es. For exampl e, i ndi vi dual en-
hancers wi thi n the D. melanogaster Bi thorax compl ex,
whi ch compri ses a 300-kb regi on i ncl udi ng the Ubx, abd-A,
and Abd-B genes, are functi onal l y separated from one
another by boundary el ements, thus preventi ng the ki nd
of enhancer shari ng and competi ti on that appears preval ent
i n vertebrate Hox cl usters (e.g., Hagstrom et al., 1996; Zhou
et al., 1996). Furthermore, the Hox genes of D. melano-
gaster are separated i nto two i ndi vi dual cl usters, wi th a
spl i t between the Antp and Ubx genes. In a rel ated speci es
D. virulis, there are si mi l arl y two i ndi vi dual cl usters, yet
the spl i t i s between Ubx and abd-A (Von Al l men et al.,
1996). Such cl uster breakdown i s not found i n more pri mi -
ti ve i nsects such as Tribolium (Beeman et al., 1993), em-
phasi zi ng that the drosophi l i ds are hi ghl y deri ved i nsects.
Neverthel ess, the di fferences i n general cl uster organi za-
ti on between i nsects and vertebrates suggest that the evo-
l uti onary forces underl yi ng retenti on of cl ustered organi za-
ti on may al so vary between phyl a.
The temporal aspect of col i neari ty i s al so l i kel y to pl ay an
i mportant rol e i n mai ntai ni ng the organi zati on of the
vertebrate Hox cl usters. Temporal col i neari ty may depend
on chromati n accessi bi l i ty. Gene transposi ti on experi -
ments have suggested that there i s a progressi ve rel ease of a
repressi ve congurati on that al l ows Hox genes to be se-
quenti al l y acti vated i n turn, from 3 to 5, as thei r chroma-
ti n becomes accessi bl e (Kondo et al., 1998; van der Hoeven
et al., 1996). In accord wi th thi s proposal , a l ong-range
repressi ve el ement 5 to the mouse HoxD cl uster has been
i denti ed (Kondo and Duboul e, 1999). These experi ments
emphasi ze the i mportance of a genes posi ti on wi thi n the
cl uster for establ i shment of col i near expressi on; thus, del e-
ti on or transposi ti on of i ndi vi dual genes woul d have a
negati ve i mpact on the enti re cl uster and be sel ected agai nst.
Extensi ve functi onal studi es i n both i es and mi ce have
establ i shed that the basi c functi ons of Hox genes are wel l
conserved: Hox genes act as sel ectors of regi onal i denti ty
al ong the pri mary body axi s. Mutati onal anal ysi s i n Dro-
sophila has establ i shed that gai n-of-functi on mutati ons
tend to cause posteri ori zi ng homeoti c transformati ons,
where the i denti ty of a segment anteri or to the normal
expressi on domai n of the gene i s al tered to resembl e the
more posteri or segment; conversel y, l oss-of-functi on muta-
ti ons cause anteri ori zi ng transformati ons. In the tetrapod
vertebrates, mi sexpressi on and nul l mutant anal yses have
reveal ed that si mi l ar rul es appl y, al though the si tuati on i s
rendered si gni cantl y more compl ex by the exi stence of not
one but four Hox cl usters. Al so, unl i ke Drosophila, the
vertebrate Hox gene expressi on domai ns often overl ap i n
the posteri or; however, the genes tend to act at or cl ose to
thei r anteri or expressi on l i mi ts. The vertebrate Hox genes
are expressed pri mari l y i n the CNS and the paraxi al meso-
derm. Consi stent wi th these expressi on patterns, al ter-
ati ons to Hox expressi on l ead to changes i n morphol ogy of
the mesoderm-deri ved vertebrae (revi ewed by Burke, 2000),
the segmental l y organi zed neurons of the hi ndbrai n (re-
vi ewed by Lumsden and Kruml auf, 1996), and the deri va-
ti ves of the crani al neural crest (revi ewed by Trai nor and
Kruml auf, 2001).
More than one member of a vertebrate Hox paral ogue
group i s often expressed i n a gi ven l ocati on, and these
paral ogous genes tend to have parti al l y redundant func-
ti ons. For exampl e, nul l mutants of the mouse Hoxa3 gene
have defects i n neural crest-deri ved structures (Chi saka and
Capecchi , 1991; Manl ey and Capecchi , 1995). By contrast,
nul l mutants of the Hoxd3 gene show transformati ons i n
the rst two cervi cal vertebrae (Condi e and Capecchi ,
1993). Al though these phenotypes are nonoverl appi ng,
doubl e mutants of both Hoxa3 and Hoxd3 reveal redun-
dancy between the genes (Condi e and Capecchi , 1994). In
thi s parti cul ar case, the di fferences i n the phenotypes of the
i ndi vi dual mutati ons must be a consequence of di fferences
i n the cis-regul atory control of the two genes, rather than of
di fferences i n thei r codi ng sequences, as Hoxa3 and Hoxd3
are functi onal l y i nterchangeabl e i n gene-swap experi -
ments, where one codi ng sequence i s repl aced wi th the
other i n the normal genomi c context (Greer et al., 2000).
Al though the overal l expressi on patterns of the two genes
appear si mi l ar, the detai l s of thei r cis-regul ati on, i ncl udi ng
vari ati ons i n l evel of expressi on, have profound conse-
quences. Di sparate functi ons of i ndi vi dual paral ogues may
often depend l argel y on thei r cis-regul ati on; thi s i n turn
suggests that novel Hox regul ati on mechani sms must have
ari sen duri ng evol uti on of the vertebrates as Hox genes
came to pattern new features of the vertebrate body pl an.
A recent study (Manzanares et al., 2000) has begun to
address the degree of conservati on between Hox gene regu-
l ati on i n vertebrates (mouse or chi ck) and i n the cephal o-
chordate amphi oxus, whi ch as descri bed above approxi -
mates a predupl i cati on ancestral condi ti on. As amphi oxus
does not have vertebrate-speci c structures, such as neural
4 Victoria Prince
crest cel l deri vati ves and neurogeni c pl acodes, one mi ght
expect that amphi oxus Hox regul atory el ements woul d be
i ncapabl e of dri vi ng expressi on wi thi n these regi ons of a
vertebrate. However, contrary to thi s expectati on, regul a-
tory el ements from the most 5 amphi oxus Hox genes were
shown to dri ve restri cted expressi on of reporter genes i n
neural crest and pl acode deri vati ves, as wel l as i n the neural
tube of both mouse and chi ck. Thi s ndi ng i mpl i es that the
basi c machi nery for Hox expressi on i n vertebrate-speci c
ti ssues was al ready i n pl ace i n the common ancestor of
cephal ochordates and vertebrates, more than 520 mi l l i on
years ago (Mya) (based on the fossi l record; revi ewed by
Hol l and and Chen, 2001). Thi s can be expl ai ned by assum-
i ng that the vertebrates conti nue to use the same basi c set
of upstream regul ators of Hox expressi on as di d thei r
ancestors, suggesti ng that the control of expressi on of these
regul ators has been modi ed duri ng vertebrate evol uti on,
such that they are present i n vertebrate-speci c structures.
Consi stent wi th thi s model , expressi on of the amphi oxus
reporter constructs i s reti noi c aci d-dependent, and RAREs
can be recogni zed i n the amphi oxus Hox regul atory se-
quences, suggesti ng that reti noi d si gnal i ng i s i mportant for
Hox acti vati on throughout the chordate l i neage.
Manzanares and col l eagues (2000) further showed that,
al though the regul atory el ements for parti cul ar amphi oxus
Hox genes can target reporter gene expressi on to those
vertebrate ti ssues i n whi ch the orthol ogous vertebrate Hox
genes are expressed, these amphi oxus sequences cannot
target the reporter genes to preci sel y the correct l ocati ons;
the anteri or l i mi ts of reporter gene expressi on l i e posteri or
to the expressi on l i mi ts of the vertebrate Hox orthol ogues.
Hence, the detai l s of vertebrate Hox expressi on patterns, for
exampl e, i n speci c segments of the devel opi ng hi ndbrai n,
may wel l depend on enhancer el ements that are uni que to
the vertebrate l i neage. Thi s l atter ndi ng i s consi stent wi th
the hypothesi s that vertebrate Hox genes have evol ved
novel forms of regul ati on, and i s al so i n accord wi th the
general paradi gm that gene dupl i cati on can faci l i tate the
ari sal of novel gene regul ati on.
HOX CLUSTER DUPLICATION IN THE
RAY-FINNED FISH LINEAGE
The 4-Hox cl uster organi zati on of the mammal s was
i ni ti al l y assumed to be a general characteri sti c of al l the
jawed vertebrates (Gnathostomata). Earl y PCR screens for
tel eost Hox genes (Mi sof and Wagner, 1996; Mi sof et al.,
1996) offered hi nts that thi s mi ght not be the case, wi th 5
Hox genes i denti ed i n paral ogue group 9 of the ki l l i sh.
Ul ti matel y, detai l ed studi es of the zebrash Hox genes,
i ncl udi ng compl ete l i nkage anal ysi s, reveal ed at l east 48
Hox genes arrayed over 7 cl usters i n thi s ostari ophysan
tel eost (Amores et al., 1998). Based on both sequence and
comparati ve l i nkage anal ysi s to adjacent non-Hox genes,
the 7 cl usters (Aa, Ab, Ba, Bb, CA, Cb and D) have been
assi gned as dupl i cates of the 4 mammal i an Hox cl usters
(Fi g. 1); a dupl i cate D cl uster was ei ther l ost duri ng evol u-
ti on or mi ssed i n the i ni ti al anal ysi s. Taken together wi th
other synteny rel ati onshi p anal yses between zebrash and
mammal s, these data further suggest an addi ti onal whol e
genome dupl i cati on event i n the l i neage l eadi ng to ze-
brash (Gates et al., 1999; Postl ethwai t et al., 2000).
Studi es of other tel eost shes have shown that addi ti onal
Hox cl usters are very unl i kel y to be a zebrash-speci c
character; al l the di fferent tel eosts that have been anal yzed
appear to have passed through a cl uster dupl i cati on event.
Thus, a l i nkage map for the acanthopterygi an tel eost
medaka (Oryzias latipes) agai n reveal s seven cl usters of
Hox genes (Naruse et al., 2000). The zebrash data al so
al l owed rei nterpretati on of the descri pti on of a four cl uster
organi zati on for the tetraodonti form puffersh (Fugu ru-
bripes): Its four cl usters appear most l i kel y to compri se two
A cl usters, one B and one C, wi th the l i kel i hood of other
cl usters yet to be found (Amores et al., 1998; Apari ci o,
2000). Prel i mi nary anal ysi s of the perci form Afri can ci chl i d
(Oreochromis niloticus) has al so reveal ed at l east si x Hox
cl usters (Mal aga-Tri l l o and Meyer, 2001). Fi nal l y, al though
onl y four Hox cl usters have been recogni zed to date i n the
perci form stri ped bass (Morone saxatilis), these represent
one A, two B, and one C cl uster (Ed Stel l wag, personal
communi cati on). Taken together, the presence of dupl i cate
Hox cl usters i n these di vergent tel eost groups (Fi g. 2)
suggests that an enti re Hox compl ement dupl i cati on event,
rel ati ve to the four-cl uster state seen i n mammal s, i s a
shared feature of the tel eosts that must have occurred i n
thei r common ancestor, perhaps i n a pri mi ti ve ray-nned
sh speci es (acti nopterygi an). As tel eost morphol ogy shows
no obvi ous greater compl exi ty than that of the sarcoptery-
gi an vertebrates, whi ch have onl y four Hox cl usters, these
ndi ngs essenti al l y refute the i dea of a di rect rel ati onshi p
between number of Hox genes and compl exi ty of morphol -
ogy. Neverthel ess, tel eosts demonstrate a fasci nati ng de-
gree of vari ati on, and the avai l abi l i ty of addi ti onal Hox
genes may have pl ayed a major rol e i n al l owi ng thi s
vari ati on to ari se, thus faci l i tati ng the tel eost radi ati on.
These data may further suggest that not just the Hox
cl usters, but al so the enti re genome, was dupl i cated i n a
tel eost ancestor. Al though there i s si gni cant support for
thi s hypothesi s (e.g., Amores et al., 1998; Meyer and
Schartl , 1999; Postl ethwai t et al., 1998; Tayl or et al.,
2001a), i t has been chal l enged by Robi nson-Recharvi and
col l eagues, who propose that tel eost dupl i cate genes are the
resul t of several l ocal dupl i cati on events (Robi nson-Rechavi
and Laudet, 2001; Robi nson-Rechavi et al., 2001b,c). Di ffer-
ences over data i nterpretati on have si nce l ed to an ani mated
debate i n the l i terature (Robi nson-Rechavi et al., 2001c;
Tayl or et al., 2001c). Rather than add to thi s debate, whi ch
wi l l eventual l y be resol ved by anal ysi s of new data, I wi l l
merel y poi nt out that whether thi s dupl i cati on affected the
enti re genome of a tel eost ancestor, or merel y some sub-
component of the genome that i ncl uded the Hox cl usters,
al most al l of the i mpl i cati ons for tel eost evol uti on that I
di scuss bel ow remai n val i d.
These ndi ngs al so l eave open the questi on of when the
dupl i cati on event that produced more than 4 Hox cl usters
i n the tel eosts occurred. The major vertebrate groups, the
l obe-ned and ray-nned shes, di verged more than 400
Mya (Fi g. 2)(Carrol l , 1988); thus, the dupl i cati on must have
occurred subsequent to thi s ti me. The tel eost speci es for
whi ch Hox cl uster organi zati on i s known represent a radi a-
ti on that started more than 100 Mya (Nel son, 1994); thi s
represents the most recent date at whi ch the dupl i cati on
coul d have occurred. However, Tayl or et al. (2001a) have
recentl y esti mated that the genome dupl i cati on may have
occurred more than 300 Mya, based on anal ysi s of nucl eo-
ti de substi tuti on rates i n the 3rd codon posi ti on of 15
dupl i cated zebrash genes. More accurate pi npoi nti ng of
the ti me of the dupl i cati on wi l l requi re anal ysi s of basal
acti nopterygi an shes, such as Pol ypteri formes (bi chi r),
Aci pensi formes (sturgeon, paddl esh), or Ami i formes (bow-
n); several groups have al ready begun to expl ore Hox genes
i n these basal speci es.
FATES OF DUPLICATED GENES
It has l ong been thought that gene dupl i cati on coul d pl ay
a vi tal rol e i n provi di ng new geneti c materi al for natural
sel ecti on to act upon, and mul ti pl e model s have been put
forward to predi ct the fates of dupl i cated genes. The cl as-
si cal model of gene dupl i cati on hol ds that because dupl i -
cated genes are i ni ti al l y i denti cal they can be consi dered
functi onal l y redundant, and stresses the rol e of the acqui -
si ti on of novel functi on i n the retenti on of gene dupl i cates
(Ohno, 1970). Thi s model suggests that, fol l owi ng dupl i ca-
ti on, onl y one of the two copi es needs to be mai ntai ned for
ancestral functi on to be retai ned. Thus, once one of the two
genes acqui res a strongl y del eteri ous mutati on, further
mutati ons can accumul ate i n that gene unchecked. As
del eteri ous mutati ons are far more l i kel y than beneci al
ones (Lynch and Conery, 2000), the retenti on of both
dupl i cated genes as a resul t of acqui si ti on of some key novel
functi on (neo-functi onal i zati on) i s thought to be an ex-
tremel y rare event. Accordi ng to thi s model , l oss of dupl i -
cated genes i s a common and rel ati vel y rapi d evol uti onary
event. In accordance wi th the model , the majori ty of
dupl i cated Hox genes have been l ost from the zebrash
cl usters. Furthermore, there remai ns evi dence of pseudo-
genes i n some of the l ocati ons where a dupl i cate woul d be
expected to l i e. For exampl e, Amores et al. (1998) descri bed
a pseudogene i n the l ocati on of hoxA10a. Not al l the Hox
cl uster sequence has yet been anal yzed, and so i t i s l i kel y
that other pseudogenes remai n to be di scovered. Indeed,
recentl y avai l abl e zebrash genomi c sequences (Sanger
Centre zebrash genome sequenci ng project; Khei rbek and
V.E.P., unpubl i shed data), have al l owed me to compare the
dupl i cate HoxA cl usters of zebrash wi th the HoxA cl us-
ters of both human and horn shark, to recogni ze a previ -
ousl y undescri bed pseudogene at the l ocati on of hoxA2a
(Fi g. 3).
Despi te the predi cti ons of the cl assi cal model , that many
more dupl i cates wi l l be l ost than retai ned, vertebrate ge-
nomes appear to be ri fe wi th anci ent gene dupl i cates
FIG. 3. Pi pmaker pl ot (Schwartz et al., 2000) compari ng human and horn shark HoxA cl usters wi th the hoxAa and hoxAb cl usters of
zebrash. Thi s strategy al l ows a hoxa2a pseudogene to be recogni zed. Bl ast anal yses of thi s sequence show homol ogy to previ ousl y i sol ated
Hoxa2 genes. However, there are STOPS i n al l three frames. The zebrash hoxAa sequence was pri mari l y deri ved from the zebrash
sequenci ng project at the Sanger Insti tute (hoxAa genomi c sequence at Accessi on No. AL645756; hoxAb genomi c sequence at
http://www.sanger.ac.uk/cgi -bi n/nph-getbl ast?humpub/zebrash al l dZ31B14.00422), wi th gaps l l ed by our own sequenci ng of the
HoxA1aA3a i ntergeni c sequence (Khei rbek and V.E.P., unpubl i shed data).
6 Victoria Prince
(Nadeau and Sankoff, 1997). To expl ai n thi s conundrum,
new theori es have been put forward. Gi bson and Spri ng
(1998)have suggested that changes i n mul ti domai n protei ns
are l i kel y to have domi nant negati ve effects, and thus
dupl i cate genes may be retai ned i ndeni tel y despi te thei r
functi onal redundancy, because al tered forms have negati ve
i mpact. Force and col l eagues (Force et al., 1999; Lynch and
Force, 2000) have proposed a model of subfuncti onal i za-
ti on that may be more general l y appl i cabl e. The Force
model suggests that the modul ar nature of eukaryoti c gene
enhancers may l ead to a parti ti oni ng of gene functi ons
fol l owi ng dupl i cati on, such that compl ementary expressi on
domai ns (spati al or temporal ) are l ost through degenerati on
of i ndi vi dual regul atory el ements for each dupl i cate. En-
hancers coul d al so change wi th respect to the l evel s of gene
expressi on, so that dupl i cates produce some l ower amount
of protei n than di d the ancestral , predupl i cate gene. Such
changes coul d l ead to the dupl i cates retai ni ng compl emen-
tary functi onsboth dupl i cates wi l l then be requi red to
recapi tul ate the ori gi nal gene functi on (referred to as the
dupl i cati ondegenerati oncompl ementati on, or DDC
model ; Force et al., 1999). These compl ementary mutati ons
ensure that both gene copi es are retai ned i n the genome. An
i mportant extensi on of thi s model i s that once gene func-
ti ons are di vi ded between the dupl i cates, each gene may be
freed to evol ve al ong a novel trajectory once the constrai nt
of functi oni ng i n mul ti pl e contexts i s removed.
The l i mi ted data avai l abl e suggest that di fferent tel eosts
do not al l share a common Hox cl uster archi tecture. Rather,
there appear to have been di fferent patterns of Hox gene
l osses subsequent to the genome dupl i cati on event. For
exampl e, the zebrash has a hoxC1a and a hoxC3a gene
(Amores et al., 1998), but i n the puffersh, these are
pseudogenes (Apari ci o et al., 1997). Si mi l arl y, Mal aga-Tri l l o
and Meyer (2001) have descri bed several di fferences i n the
archi tecture of the HoxA cl usters of zebrash, stri ped bass,
puffersh, and an Afri can ci chl i d. Thi s vari abi l i ty i n cl uster
organi zati on contrasts markedl y wi th our understandi ng of
a stabl e tetrapod Hox cl uster organi zati on. Neverthel ess,
rather than bei ng an excepti on wi thi n the vertebrates, thi s
vari abl e archi tecture shoul d perhaps be consi dered the rul e,
as tel eosts make up the majori ty of vertebrate speci es
(about 25,000 sh speci es have been descri bed; Nel son,
1994). Indeed, i t has been suggested that the vari abl e Hox
organi zati ons may have a di rect rel ati onshi p wi th the
di versi ty of morphol ogi es among the tel eosts (Meyer and
Schartl , 1999; Wi ttbrodt et al., 1998).
RESOLUTION OF ZEBRAFISH HOX
GENE DUPLICATES
The zebrash provi des a tractabl e model system to exam-
i ne the functi onal si gni cance of Hox gene dupl i cati ons.
Usi ng comparati ve sequence, expressi on, and functi onal
studi es, we can begi n to i nvesti gate what events have
al l owed retenti on of sel ect pai rs of zebrash Hox gene
dupl i cates (al though many dupl i cate genes have been l ost,
at l east 10 dupl i cates have been mai ntai ned). If novel
functi ons coul d be uncovered for zebrash Hox genes, thi s
woul d be consi stent wi th the hypothesi s that the avai l abi l -
i ty of dupl i cate Hox genes was i mportant i n faci l i tati ng the
tel eost radi ati on. Ideal l y, we woul d compare zebrash Hox
genes to those of a speci es that approxi mates the ancestral ,
predupl i cati on condi ti on. Unfortunatel y, i nformati on on
the Hox genes of the basal acti nopterygi ans, whi ch are most
l i kel y to provi de such a compari son group, has not yet
reached the l i terature.
However, Hox genes are unusual l y conserved i n thei r
sequence, cl ustered organi zati on, and regul ati on, whi ch
permi ts (even requi res) compari sons to be made over wi de
evol uti onary di stances. Indeed, Hox genes are so conserved
at the l evel of protei n functi on that, i n some cases, they can
be functi onal l y substi tuted for one another between di ffer-
ent phyl a (e.g., Lutz et al., 1996). Thus, i nformati ve com-
pari sons can be made between zebrash and such phyl oge-
neti cal l y di stant ostei chthyans as mi ce, al l owi ng us to take
advantage of the weal th of data concerni ng mouse Hox gene
expressi on and functi on. Thi s approach rel i es on the as-
sumpti on that the four-cl uster organi zati on, seemi ngl y
wi despread i n sarcopterygi ans, reects the ancestral os-
tei chthyan condi ti on. Whi l e thi s assumpti on al ready seems
reasonabl e, i t woul d be even more strongl y supported were
two addi ti onal Hox cl usters to be found i n the chondri ch-
thyan horn shark, bri ngi ng the total number of Hox cl usters
to four i n the si ster group to Ostei cthyes.
There are a total of 48 Hox genes descri bed for the
zebrash (compared wi th 39 for mouse and human), yet
despi te thi s di fference i n gene number, the majori ty of
zebrash Hox genes show expressi on patterns that are
essenti al l y si mi l ar to those of thei r muri ne orthol ogues
(Pri nce et al., 1998a,b). One i nteresti ng excepti on to thi s
rul e i s the zebrash hoxA1a gene (McCl i ntock et al., 2001),
whi ch i s di scussed i n more detai l bel ow. Al though the
zebrash i s wel l known for i ts tractabi l i ty as a geneti c
model system, no homeoti c mutants have been uncovered
i n l arge-scal e forward geneti c screens. Zebrash Hox mu-
tants woul d be expected, based on our knowl edge of mouse,
to cause al terati ons i n vertebral morphol ogy and hi ndbrai n
segmental i denti ty. The l arge-scal e zebrash mutagenesi s
screens were not desi gned to i denti fy such phenotypes, and
thus i t i s unsurpri si ng that homeoti c mutants have not yet
been found.
Neverthel ess, other types of mutant have provi ded useful
i nformati on about zebrash Hox gene functi on. In parti cu-
l ar, the phenotype of the lazarus (lzr) mutant has suggested
that zebrash Hox genes must pl ay very si mi l ar functi onal
rol es to mammal i an Hox genes (Popperl et al., 2000). The
lzr mutant affects a Pbx gene, zebrash pbx4; Pbx protei ns
are Hox cofactors, bi ndi ng together wi th Hox protei ns on
thei r target sequences to provi de proper speci ci ty to regu-
l ati on of the downstream targets (revi ewed by Mann and
Affol ter, 1998). The zebrash pbx4 gene provi des the major
Pbx cofactor acti ng duri ng earl y devel opment, and i n i ts
absence there are mul ti pl e defects wi thi n the devel opi ng
hi ndbrai n regi on. Al l of the phenotypes have been i nter-
preted as correspondi ng to l osses of Hox gene functi on, by
anal ogy to known mouse Hox mutants (Popperl et al.,
2000). Thi s i nterpretati on has been further supported by our
anal ysi s of Hox gene knock-down phenotypes, where
Hox protei n transl ati on i s bl ocked by usi ng anti sense re-
agents (Hunter and Pri nce, 2002; McCl i ntock et al., 2002,
see bel ow).
Compari son of the lzr mutant phenotype wi th the phe-
notypes of mouse Hox mutants does not reveal any major
zebrash-speci c Hox gene functi ons duri ng earl y devel op-
ment: The lzr phenotype l argel y phenocopi es nul l mutants
of mouse Hox genes. It shoul d be remembered, however,
that any novel l ate functi ons of these Hox genes woul d not
be recogni zed due to the l ethal nature of the lzr mutant.
Furthermore, zebrash Hox genes may have evol ved Pbx-
i ndependent functi ons that woul d be unaffected by the lzr
mutant. Whether or not the zebrash Hox genes have taken
on novel functi ons (a questi on that remai ns wi de open at
present), we do know that some dupl i cate genes were
retai ned. A comparati ve approach can be used to try to
establ i sh the mechani sms underl yi ng these retenti ons.
Bruce et al. (2001)performed the rst study to i nvesti gate
why a pai r of zebrash Hox genes have been retai ned rather
than one gene bei ng l ost from the genome. In thi s study, the
expressi on patterns of zebrash hoxB5a and hoxB5b were
compared to that of the si ngl e mouse Hoxb5 gene, and
found to recapi tul ate i ts overal l expressi on. The zebrash
hoxb5 dupl i cates have di fferent, but overl appi ng, expres-
si on patterns, yet appear to share i denti cal bi ochemi cal
functi ons as assessed by a gai n-of-functi on approach. Thus,
i t seems that i n thi s case, zebrash hoxb5a and hoxb5b
represent a parti ti oni ng of expressi on domai ns wi th respect
to the muri ne Hoxb5 gene. Assumi ng that the muri ne
Hoxb5 gene reects the ancestral ostei chthyan state, the
Hox dupl i cati on i n the tel eost l i neage appears to have l ead
to a subfuncti onal i zati on for these zebrash hoxB5 dupl i -
cates i n accordance wi th the DDC model . Further tests of
the model woul d i ncl ude demonstrati ng that these two
zebrash genes are abl e to functi onal l y substi tute for one
another, al though i t shoul d be remembered that, even when
the DDC model i s i nvoked to expl ai n the xati on of gene
dupl i cati ons, thi s does not rul e out subsequent al terati ons
that mi ght obscure i ni ti al functi onal equi val ence. It woul d
al so be of i nterest to expl ore the regul atory sequences of the
zebrash hoxB5a and hoxB5b genes, to attempt to i denti fy
degenerati ve changes i n the zebrash sequences that under-
l i e the presumed parti ti oni ng of the ancestral expressi on
domai n.
Chi u et al. (2002) have recentl y i nvesti gated the mol ecu-
l ar evol uti on of HoxA cl usters across the major gnathos-
tome l i neages: They compared compl ete HoxA cl uster
sequences of zebrash, human, and horn shark. Dupl i cate
genes have been retai ned for three of the more 5-l ocated
zebrash HoxA genes, yet the dupl i cated zebrash cl usters
di d not show evi dence for the ki nd of compl ementary
degenerati ve changes i n cis-regul atory el ements that the
DDC model predi cts. Instead, the two zebrash HoxA
cl usters, as wel l as the one reported stri ped bass HoxA
cl uster, showed a conspi cuous l oss of putati ve cis-regul a-
tory el ements that are conserved between human and horn
shark. The authors concl ude that the changes they have
found i n the zebrash sequences are consi stent wi th adap-
ti ve modi cati on rather than the more passi ve mechani sms
associ ated wi th subfuncti onal i zati on. By contrast, com-
parati ve sequence anal ysi s of the i ntergeni c regi on between
Hoxb2 and Hoxb3 of human, mouse, zebrash, fugu, and
stri ped bass has reveal ed extensi ve conservati on of tran-
scri pti on factor bi ndi ng si tes (Scemama et al., i n press). The
conserved si tes have been shown to be i mportant for proper
expressi on of mouse Hoxb2, and consi stent wi th conserved
functi on of these el ements, the expressi on patterns of the
vertebrate Hoxb2 orthol ogues are l argel y conserved. Inter-
esti ngl y, i n several cases, the bi ndi ng si tes occur i n di fferent
orders i n di fferent speci es, and such reorgani zati on of smal l
cis-regul atory el ements may make i t di fcul t for l arge-scal e
al i gnment techni ques to pi ck up functi onal homol ogy.
Recent studi es i n my own l ab have al so focused on the
questi on of why some Hox dupl i cates have been retai ned i n
the zebrash genome. We have concentrated on the four
zebrash Hox genes compri si ng paral ogue group (PG) 1,
whi ch i ncl ude a pai r of dupl i cates wi th respect to the
four-cl uster state, hoxB1aand hoxB1b. In thi s case, we have
found good evi dence for an anci ent subfuncti onal i zati on
between the dupl i cates. However, we addi ti onal l y nd
evi dence for a subsequent more compl ex si tuati on of
functi on shufi ng among the members of the paral ogue
group.
FUNCTION SHUFFLING AMONG
PG1 GENES
The PG1 genes are a parti cul arl y good system i n whi ch to
i nvesti gate potenti al subfuncti onal i zati on because two of
the three mouse genes have had both gene functi on and
regul ati on studi ed i n great detai l . These experi ments have
shown that mouse Hoxa1 and Hoxb1 are necessary for
proper devel opment of the hi ndbrai n. In zebrash, as i n
mouse and chi ck, hi ndbrai n morphol ogy i s conceptual l y
si mpl e, wi th overt segmentati on di vi di ng the hi ndbrai n
i nto seven l i neage-restri cted compartments termed rhom-
bomeres (r1r7 from A to P). Thi s basi c organi zati on i s
conserved across the vertebrates, and there are a weal th of
mol ecul ar and neuroanatomi cal markers that al l ow the
i denti ty of i ndi vi dual rhombomeres to be unambi guousl y
recogni zed (revi ewed by Moens and Pri nce, 2002).
Hoxa1 and Hoxb1 are coexpressed i n the mouse hi nd-
brai n from the earl y stages of gastrul ati on, wi th an i denti cal
anteri or expressi on l i mi t at the presumpti ve boundary
between r3 and r4 (Barrow et al., 2000; Frohman et al., 1990;
Murphy and Hi l l , 1991; Wi l ki nson et al., 1989). Thi s
8 Victoria Prince
expressi on i s dependent on reti noi c aci d response el ements
(RAREs) that l i e 3 of each gene. Hoxa1 expressi on i s very
transi ent i n r4, retracti ng posteri orl y out of the hi ndbrai n
duri ng earl y somi te stages. In contrast, Hoxb1expressi on i s
stabl y mai ntai ned i n r4, whi l e expressi on i s gradual l y l ost
from r5 and r6 to l eave an r4 stri pe of Hoxb1 expressi on.
Thi s r4 Hoxb1 domai n i s mai ntai ned by an autoregul atory
posi ti ve feedback mechani sm, whi ch i s dependent on three
dened Hox/Pbx bi ndi ng si tes upstream of Hoxb1 (Popperl
et al., 1995).
Mutant anal ysi s of mouse Hoxa1and Hoxb1has reveal ed
that these two paral ogues pl ay di vergent, but parti al l y
redundant, rol es i n patterni ng the hi ndbrai n. The pri me
functi on of the Hoxb1 gene i s to confer proper r4 i denti ty,
as l oss of Hoxb1functi on resul ts i n major al terati ons to the
r4-deri ved faci al motor neurons, whi ch no l onger undergo
thei r normal mi grati on behavi or (Gaufo et al., 2000; God-
dard et al., 1996; Studer et al., 1996). By contrast, l oss of
Hoxa1 functi on causes a radi cal reducti on i n the AP extent
of r4 and r5, wi th an accompanyi ng reducti on i n the si ze of
the adjacent oti c vesi cl e (Carpenter et al., 1993; Chi saka et
al., 1992; Lufki n et al., 1991; Mark et al., 1993); thus, Hoxa1
i s rather unusual as i t i s i mportant for setti ng up proper
segmental organi zati on of the hi ndbrai n, not just for con-
ferral of segmental i denti ty. Doubl e knockouts of both
Hoxb1 and Hoxa1 show synergi sti c phenotypes (Barrow et
al., 2000; Gaval as et al., 1998; Rossel and Capecchi , 1999;
Studer et al., 1998) reveal i ng redundancy of functi on be-
tween the paral ogues.
In the zebrash, the hoxB1 dupl i cates, hoxB1a and
hoxB1b, have expressi on prol es that are i ntri gui ngl y si mi -
l ar to those of mouse Hoxb1 and Hoxa1, respecti vel y
(McCl i ntock et al., 2001), al though zebrash hoxB1a l acks
the earl y gastrul a-stage expressi on shown by mouse Hoxb1.
By contrast, the zebrash orthol ogue of mouse Hoxa1,
zebrash hoxA1a, i s not expressed i n presumpti ve r4, and
thus cannot pl ay a rol e i n earl y patterni ng of thi s hi ndbrai n
terri tory (McCl i ntock et al., 2001; Shi h et al., 2001). Hence,
expressi on data suggest the hypothesi s that zebrash
hoxB1a and hoxB1b are the functi onal equi val ents of
mouse Hoxb1 and Hoxa1, respecti vel y.
A new knock-down technol ogy, usi ng stabi l i zed anti -
sense morphol i nos, has al l owed us to test di rectl y the
functi ons of the zebrash hoxB1 dupl i cates (McCl i ntock et
al., 2002). We have demonstrated that zebrash hoxB1aand
hoxB1b do i ndeed pl ay si mi l ar rol es to mouse Hoxb1 and
Hoxa1. Thus, the zebrash hoxB1a gene, l i ke mouse
Hoxb1, i s requi red for proper mi grati on of faci al nerve
neurons from r4 and for i ts own posi ti ve regul ati on. The
hoxB1b gene, l i ke mouse Hoxa1, i s requi red for proper
segmental organi zati on of the hi ndbrai n, and for devel op-
ment of a normal l y si zed oti c vesi cl e. How can our ndi ng
that a zebrash HoxB dupl i cate gene and a mouse HoxA
gene are functi onal l y equi val ent be reconci l ed wi th the
DDC subfuncti onal i zati on model ? Data emergi ng from the
zebrash sequenci ng project have hel ped us to devel op a
model to expl ai n our ndi ngs.
Accordi ng to the DDC model , the dupl i cates woul d be
expected to di vi de out the ancestral expressi on domai n. In
accord wi th the model , the hoxB1b gene has an expressi on
pattern resembl i ng the gastrul ati on phase of muri ne Hoxb1
expressi on, whi l e the hoxB1a gene has an expressi on pat-
tern resembl i ng the l ater r4 stri pe phase of mouse Hoxb1
expressi on. The DDC model al so predi cts the degenerati on
of di screte compl ementary cis-regul atory el ements i n the
two dupl i cates. We nd that, al though hoxB1b possesses a
3 RARE wi th a two-nucl eoti de spacer between the hal f
si tes, si mi l ar to the one whi ch i n mouse Hoxb1 confers
gastrul ati on stage expressi on, we are unabl e to detect such
an el ement 3 of hoxB1a, consi stent wi th i ts l ack of an earl y
expressi on phase. Si mi l arl y, zebrash hoxB1a retai ns per-
fect copi es of al l three Hox/Pbx bi ndi ng si tes, whi ch i n
mouse Hoxb1 confer autoregul ati on i n r4, yet hoxB1b has
poi nt changes i n each of the i ndi vi dual si tes, consi stent
wi th the absence of a l ate r4 expressi on domai n for thi s
gene. Thi s degenerati on of di fferent regul atory modul es i n
each of the two dupl i cates i s l i kel y to have been sufci ent
to al l ow preservati on of the two genes as postul ated by the
DDC model (Fi g. 4), but l eaves open the questi on of how the
functi on of a HoxA gene coul d have shi fted to a HoxB gene.
Our model for how hoxB1b came to take on the rol e that
i n mouse i s pl ayed by Hoxa1 has been i nuenced by our
expressi on anal yses of vertebrate Hoxa1 orthol ogues. We
have shown that the zebrash hoxA1a gene i s expressed at
l ate neurul ati on stages i n a smal l , bi l ateral l y l ocated group
of neurons i n the ventral mi dbrai n (McCl i ntock et al., 2001;
Shi h et al., 2001). As mi dbrai n expressi on has not general l y
been descri bed for Hox genes, thi s domai n seems at rst
observati on to reect a potenti al neofuncti onal i zati on
event. However, our comparati ve anal yses have demon-
strated that mi dbrai n expressi on i s more l i kel y a pri mi ti ve
characteri sti c of the vertebrate PG1 genes. Thus, we nd
expressi on of Hoxa1 orthol ogues i n a si mi l ar group of cel l s
not onl y i n another tel eost, medaka, but al so i n the sarcop-
terygi an chi ck (C. Jozefowi cz. and V.E.P., unpubl i shed ob-
servati ons). Furthermore, we have conrmed a previ ous
descri pti on of mi dbrai n expressi on for Xenopus Hoxa1
(Kol m and Si ve, 1995). As Xenopus and chi ck combi ne both
hi ndbrai n and mi dbrai n expressi on domai ns of Hoxa1, we
hypothesi ze that these two separate expressi on domai ns
represent the ancestral condi ti on. In the zebrash, hoxB1b
has taken on the hi ndbrai n patterni ng rol e of tetrapod
Hoxa1, whi ch may have freed hoxA1a to l ose i ts hi ndbrai n
expressi on domai n whi l e retai ni ng the ancestral mi dbrai n
patterni ng rol e (Fi g. 4). We have termed thi s phenomenon
functi on shufi ng (McCl i ntock et al., 2001, 2002), and i t
rel i es upon a phase of parti al functi onal redundancy be-
tween nonorthol ogous genes, i n thi s case the paral ogous
hoxA1a and hoxB1b. These data reveal that i t may be
essenti al to study an enti re group of rel ated genes to ful l y
understand the consequences of a parti cul ar dupl i cati on
event.
We have al so been abl e to combi ne the morphol i no
knock-downs wi th mRNA mi sexpressi on to test the degree
of i nterchangeabi l i ty of Hox PG1 codi ng sequences (Mc-
Cl i ntock et al., 2002). In these experi ments, we attempted
rescue of knock-down phenotypes wi th di fferent mRNAs.
We found that mouse Hoxb1 can functi onal l y substi tute for
ei ther zebrash hoxB1aor hoxB1b, consi stent wi th the model
that the two zebrash dupl i cates have subfuncti onal i zed the
ancestral rol es that i n mouse conti nue to be pl ayed by the
si ngl e Hoxb1 gene. However, we al so found that, al though
hoxB1acan functi onal l y substi tute for hoxB1b, the reci procal
i s not true. Thus, hoxB1bhas l ost the capaci ty to al l ow proper
mi grati on of faci al nerve neurons. Once agai n, thi s i s consi s-
tent wi th the model of Force and col l eagues (Force et al., 1999;
Lynch and Force, 2000): Thei r DDC model states that, al -
though compl ementary degenerati on of cis-regul atory el e-
ments i s what i ni ti al l y al l ows mai ntenance of a pai r of
dupl i cates, i t does not prevent the i ndi vi dual genes from then
becomi ng ne-tuned to thei r separate functi ons or eventu-
al l y taki ng on novel functi ons.
Functi on shufi ng may prove to be common among
zebrash paral ogues. For exampl e, i t has recentl y been
shown usi ng morphol i no-based knock-down that the
zebrash eng2 and eng3 genes have earl y devel opmental
rol es equi val ent to that of the nonorthol ogous mouse
EN1 gene (Schol pp and Brand, 2001). Furthermore, func-
ti on shufi ng may not be l i mi ted to transcri pti on factor
genes: The secreted si gnal i ng mol ecul e bmp2a from ze-
brash appears to pl ay an equi val ent functi onal rol e to
the nonorthol ogous Xenopus Bmp4 duri ng dorsoventral
patterni ng of gastrul a-stage embryos (Nguyen et al.,
1998). On a practi cal note, these ndi ngs suggest that, i n
cases where orthol ogy rel ati onshi ps are uncl ear, i t may
not hel p to assume that common functi on can hel p wi th
FIG. 4. Model outl i ni ng the evol uti onary mechani sm of Hox PG1 gene functi on shufi ng. The cis-regul atory el ements characteri zed
for the mouse and human Hoxa1and Hoxb1genes [3 RAREs (bl ue), Hox/Pbx bi ndi ng si tes (red)] are assumed to be present i n the ancestral ,
pre- thi rd -dupl i cati on, condi ti on. We al so postul ate the presence of a regul atory domai n di recti ng mi dbrai n expressi on of Hoxa1(mauve),
al though no such domai n has yet been characteri zed. The dupl i cati on event i n the l i neage l eadi ng to tel eosts produced redundant copi es
of both Hoxa1 and Hoxb1 i n an ancestor of the zebrash. The hoxA1b dupl i cate was eventual l y l ost by accumul ati on of del eteri ous
mutati ons ( nonfuncti onal i zati on ) as predi cted by cl assi cal model s. In contrast, the hoxB1a and hoxB1b genes accumul ated compl emen-
tary degenerati ve changes i n thei r cis-regul atory el ements such that hoxB1a l ost earl y, RARE-medi ated expressi on, and hoxB1b l ost
autoregul ati on. Thi s l ed to retenti on of the dupl i cate genes, as both were requi red to mai ntai n the expressi on pattern and functi on of the
si ngl e Hoxb1 ancestral gene (subfuncti onal i zati on), as predi cted by the DDC model . As hoxA1a and hoxB1b shared si mi l ar codi ng
sequences and expressi on patterns, these two genes were now functi onal l y redundant wi th respect to a rol e duri ng gastrul ati on i n setti ng
up segmental organi zati on of the hi ndbrai n. These nonorthol ogous genes were thus abl e to go through another subfuncti onal i zati on
event, such that hoxA1a l ost i ts earl y RARE-medi ated expressi on, whi ch was retai ned by hoxB1b. Thus, hoxB1b became essenti al for
proper hi ndbrai n segmentati on, the rol e pl ayed i n the ancestral state by Hoxa1. Retenti on of the hoxA1a gene i n the l i neage l eadi ng to
zebrash was presumabl y dependent on a functi on that was not redundant wi th hoxB1b, possi bl y a rol e i n mi dbrai n patterni ng. We term
thi s rearrangement of PG1 gene rol es functi on shufi ng.
10 Victoria Prince
assi gnmentssynteny rel ati onshi ps are more l i kel y to be
a rel i abl e tool .
CONCLUSION AND FUTURE DIRECTIONS
There i s no doubt that Hox gene functi ons are i nti matel y
associ ated wi th axi al patterni ng, and therefore changes i n
Hox genes are l i kel y to pl ay a key rol e i n the evol uti on of
new body pl ans. Many researchers have emphasi zed the
i mportance of al terati ons i n cis-regul ati on of Hox and other
devel opmental control genes for al l owi ng di fferi ng mor-
phol ogi es to ari se duri ng evol uti on (revi ewed by Carrol l ,
2000). Studi es i n i nvertebrates have tended to support the
noti on that cis-regul ati on can be ti nkered wi th more
easi l y than protei n codi ng sequences, presumabl y because
detri mental effects are l ess l i kel y to resul t from sequence
changes. However, recent work has reveal ed that al ter-
ati ons to Hox protei ns, as opposed to al terati ons i n regul a-
ti on of Hox expressi on, can underl i e major morphol ogi cal
transi ti ons. Two studi es (Gal ant and Carrol l , 2002; Ron-
shaugen et al., 2002) have demonstrated that i nsects l ost
thei r abdomi nal l i mbs, such that they have onl y si x l egs, as
a resul t of functi onal changes i n the Hox protei n Ubx.
These reports underscore the i mportance of consi deri ng
both gene regul ati on and protei n functi on as we try to
unravel how changes i n Hox genes have i nuenced verte-
brate evol uti on.
Consi stent wi th the i dea that changes to Hox genes can
underl i e new morphol ogi es, the l arge-scal e gene dupl i ca-
ti ons i n the vertebrate stem l i neage provi ded many addi -
ti onal Hox genes, whi ch correl ate wi th the i nnovati ons that
characteri ze the vertebrates (revi ewed by Hol l and et al.,
1994). It has been suggested that the addi ti onal dupl i cati on
event i n the l i neage l eadi ng to tel eosts such as the zebrash
provi ded yet more raw geneti c materi al for sel ecti on to act
upon, and that thi s may have faci l i tated the broad radi ati on
of tel eosts (Meyer and Schartl , 1999). Al though the radi a-
ti on of the tel eosts has been underway for about 200 My,
thi s ti me frame i s rel ati vel y short i n compari son wi th the
di stant ori gi n of vertebrates, more than 520 Mya. Thus,
studi es of tel eost shes hol d si gni cant promi se for al l ow-
i ng us to test the i mportance of changes i n Hox genes for
the generati on of new forms. In order to pursue these
studi es, i t wi l l be i mportant for the dupl i cati on event that
has l ed to addi ti onal Hox cl usters i n tel eosts to be more
accuratel y dated. Thi s wi l l al l ow us to recogni ze the l ast
common ancestor of ani mal s wi th and wi thout the extra
dupl i cati on, and provi de an appropri ate compari son poi nt
for al l future studi es. To thi s end, upcomi ng new data on
basal tel eosts and ray-nned shes wi l l be i nval uabl e.
To further i nvesti gate the rol es of Hox genes wi thi n the
radi ati ng tel eosts, i t wi l l be vi tal to study speci es wi thi n a
phyl ogeneti c framework. In parti cul ar, i t wi l l be i mportant
to correl ate known morphol ogi cal vari ati on wi th di ffer-
ences i n Hox organi zati on and functi on. Speci es sui tabl e for
such studi es mi ght i ncl ude the members of the tetraodon-
ti fomes, whi ch have a remarkabl y vari ant morphol ogy
(Santi ni and Stel l wag, 2002; Tyl er and Sorbi ni , 1996). Thi s
wi l l entai l much hard work i n determi ni ng detai l s of Hox
cl uster archi tecture for a range of speci es, and thus i t wi l l be
i mportant to choose speci es wi sel y. It wi l l al so be useful to
have a rel i abl e means of di srupti ng gene functi on, and
conveni entl y, the new morphol i no technol ogy for gene
knock-down shoul d be equal l y appl i cabl e to any system
where earl y embryos can be mi croi njected. As the majori ty
of sh speci es have embryos that devel op external l y, thi s
opens up the prospect of broad comparati ve functi onal
anal yses.
Another approach to understandi ng the geneti c basi s of
morphol ogi cal evol uti on i s to use vari ati on among cl osel y
rel ated speci es to i denti fy l oci that contri bute to the ob-
served vari ati on. Pei chel et al. (2001)have used quanti tati ve
trai t l ocus (QTL) mappi ng to i nvesti gate vari ati on i n skel -
etal armor and feedi ng morphol ogi es of the threespi ned
sti ckl ebacks, wel l -studi ed tel eosts that have undergone
rapi d di vergence and speci ati on over the l ast 15,000 years.
Thi s work has i denti ed a l arge number of QTL associ ated
wi th the di fferi ng morphol ogi es. It wi l l be i nteresti ng to
know whether these QTL correl ate wi th known devel op-
mental control genes, i ncl udi ng Hox genes, al though tar-
geted studi es of the expressi on patterns of speci c Hox
genes i n morphol ogi cal l y di sti nct popul ati ons of sti ckl e-
backs have not yet reveal ed any correl ati ons wi th the
di fferent morphol ogi es (Ahn and Gi bson, 1999).
It i s i mportant to note that gene dupl i cati on events may
be i mportant for al l owi ng speci ati on to occur vi a mecha-
ni sms that are separabl e from the generati on of new
morphol ogi es. Lynch and col l eagues have postul ated that
di vergent resol uti on of dupl i cate genes coul d cause spe-
ci ati on wi thi n popul ati ons that are temporari l y geographi -
cal l y i sol ated (Lynch and Conery, 2000; Lynch and Force,
2000; revi ewed by Tayl or et al., 2001a,b). Thi s woul d rel y
upon speci c pai rs of dupl i cated genes undergoi ng di fferent
fates i n di fferent popul ati ons, for exampl e, l oss of di fferent
dupl i cate genes, or subfuncti onal i zati on versus nonfunc-
ti onal i zati on. Such events woul d reduce the fecundi ty of
future hybri ds once the separated popul ati ons become re-
uni ted. Consi stent wi th thi s hypothesi s, the sal moni d
shes, whi ch have gone through a recent genome dupl i ca-
ti on event, are si gni cantl y more speci ose than a si ster
taxon that has not (revi ewed by Tayl or et al., 2001b). The
more di vergentl y resol ved l oci present, the more effecti ve
such an i sol ati on mechani sm woul d be, thus i n the case of
the radi ati ng tel eosts thi s model i s more rel evant to a whol e
genome dupl i cati on event than to a more l i mi ted, Hox-
speci c, dupl i cati on event.
What then can we l earn from the Hox genes of the
zebrash, the tel eost that i s currentl y best understood at
both the mol ecul ar geneti c l evel , and i n terms of i ts earl y
devel opment? It has been establ i shed that zebrash has
retai ned at l east 10 dupl i cated Hox genes, openi ng up the
possi bi l i ty that, i n some cases, dupl i cates were xed be-
cause one of them attai ned a novel functi on. In the two
cases that have been i nvesti gated i n detai l , thi s appears not
to be the case. The hoxB5 dupl i cates have subfuncti onal -
i zed i n accordance wi th the DDC model (Force et al., 1999),
whereas the PG1 genes have gone through an i nteresti ng
functi on shufi ng, whi l e sti l l not undergoi ng any obvi ous
neofuncti onal i zati on. However, i t shoul d be noted that
neofuncti onal i zati on may prove di fcul t to recogni ze, es-
peci al l y i n the absence of a compl ete knowl edge of the
pri mi ti ve condi ti on. Important changes coul d be subtl e
for exampl e, mi nor but cri ti cal changes i n ti mi ng of gene
expressi on, concentrati on of gene product, or ori gi n of a
new l ate expressi on pattern that woul d not be detected
wi thi n the usual ti me frame of devel opmental expressi on
studi es. Furthermore, the comparati ve sequence anal ysi s of
Chi u et al. (2002) has provi ded evi dence for adapti ve modi -
cati on i n tel eost Hox regul atory el ements, suggesti ng that
new expressi on domai ns may wel l have ari sen fol l owi ng
dupl i cati on. Al ternati vel y, the 10 retai ned dupl i cate Hox
genes may al l prove to have undergone some vari ati on on
the subfuncti onal i zati on theme. Neverthel ess, thi s woul d
not undermi ne Ohnos hypothesi s that gene dupl i cati on
faci l i tates evol uti on by provi di ng new geneti c materi al and
al l owi ng genes to take on new functi ons. Rather, there may
be other devel opmental control genes that have gai ned
i mportant novel functi ons subsequent to dupl i cati on, and
very good candi dates for such genes woul d be the down-
stream effectors of Hox functi on.
ACKNOWLEDGMENTS
I thank James McCl i ntock, Chri s Jozefowi cz, Ed Stel l wag, and
Anni e Burke for many hel pful di scussi ons and comments on the
manuscri pt. I am grateful to Ed Stel l wag for shari ng unpubl i shed
data and to Mazen Khei rbek for sequenci ng a l arge part of the
hoxA3ahoxA1a i ntergeni c el ement. The work di scussed here
from my l ab was funded by NSF Grant IBN 0091101 and by MOD
Grant #FY00-336.
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Recei ved for publ i cati on March 8, 2002
Revi sed May 28, 2002
Accepted May 28, 2002
Publ i shed onl i ne August 7, 2002

Prince V - The Hox Paradox - 2002

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TheHox Paradox: MoreComplex(es) Than Imagined

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