INTRO

Differeorganisa commvariatio

regulatmetazoulators obvious factors to study in the investigation of the mechanis-tic basis for ontogeny, as well as in the relationship betweenontogeny and phylogeny. This paper presents a comparativestudy of the developmental patterns of homeobox geneexpression and morphogenesis along the A-P axis betweenrelated animals that have homologous regulatory genes butdifferent axial morphologies.

The participation of different numbers of segments in anygiven region of the vertebrate body and the different positionsof the appendages relative to the A-P axis, have provokedcomment and theory from morphologists for centuries. Whilecommon generative rules govern mesodermal segments withinindividual organisms (serial homology), and common ancestryaccounts for their presence in all chordates (historicalhomology), details of this segmental organization differ dra-matically between related organisms resulting in a variety ofaxial formulae, defined here as the number of vertebrae of eachmorphological type, e.g. cervical, thoracic, lumbar, etc. In1906, E. S. Goodrich proposed the term ‘transposition’ to

y crteblly lide (19y,

tranriatses

position has been an imthe establishment and radiation of different vertebrate groups(Gadow, 1933; Carroll, 1988). Mammals are a vertebrate classwith extreme morphological variation. With very few excep-tions however, they are constrained by the fixed number ofseven cervical vertebrae whether they be whales or giraffes.Birds are not constrained in this character, and vary from 13(pigeons and swifts), to 25 (swans) cervical vertebrae.Extremes among the vertebrates include the Cretaceous ple-siosaur, Elasmosaurus sp. which had as many as 76 cervicalvertebrae; snakes, with as many as 350 individual vertebrae ofequivocal types; and the modern anurans (frogs) with nevermore than 9 and as few as 6 total presacral vertebrae. Evenwhen the total number of pre-caudal vertebrae is almost thesame, as is the case between chickens and mice, the relativelength of specific regions, such as cervical versus thoracic, canvary considerably.

Members of the Hox family of homeobox genes areexpressed along the A-P axis at specific levels in the central

DevelopmPrinted in

A comtebrattributecervicapositioexperimstrated

ificatioaxis, adetermM. andpresenpatternmorphlatory

ies rmzatantposta c ofan

sic e t

ant seg

SUMM

Hox ho

Ann C

Depart 11

*Present ng 1

hanges in the number of segmentsral region. Goodrich surmised thathomologous regions behave as pliable up or down the A-P axis during10) expanded the description with a

comparing an axial structure or regionsposed up or down the scale.

ion and constraint in axial formulaeof vertebrates demonstrates that trans-portant evolutionary phenomenon in

333

of 23 Hox genes were examined in of chick, and 16 in mouse embryosion and immunolocalization tech-erior expression boundaries wereed in concert with morphologicalontributes a mechanistic level to the these regions in vertebrates. Theistic homology supports the histori-patterning mechanisms between allhese genes.

erior-posterior axis, comparativement identity, paraxial mesoderm, chick,

logy

5, USA

49, Charlestown, MA 02129, USA

ent 121, 333-346 (1995) Great Britain © The Company of Biologists Limited 1995

mon form of evolutionary variation between ver-e taxa is the different numbers of segments that con- to various regions of the anterior-posterior axis;

expression boundarthe paraxial mesodeby in situ hybridi

ARY

genes and the evolution of vertebrate axial morp

. Burke, Craig E. Nelson, Bruce A. Morgan* and Cliff Tabin

ment of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02

address: Cutaneous Biology Research Center, Massachusettes General Hospital-East, 13th Street Buildi

DUCTION

nces along the anterior-posterior (A-P) axis in manyms are set up by the spatial and temporal expression ofon set of genes early in development, despite extremen in adult morphology. The Hox genes are a family of

ory genes expressed along the A-P axis in mostans. Because homeobox genes encode transcription reg-active in pattern formation during ontogeny, they are

describe evolutionarincluded in any veserially and historicaelements that can sevolution. Lankesteruseful musical analogto a tune that can be

The pattern of vaamong different clas

l vertebrae, thoracic vertebrae, etc. The term ‘trans-n’ is used to describe this phenomenon. Genetic

ents with homeotic genes in mice have demon- that Hox genes are in part responsible for the spec-n of segmental identity along the anterior-posteriornd it has been proposed that an axial Hox codeines the morphology of individual vertebrae (Kessel, Gruss, P. (1990) Science 249, 347-379). This paper

ts a comparative study of the developmentals of homeobox gene expression and developmental

ology between animals that have homologous regu-genes but different morphologies. The axial

niques. Hox gene found to be transboundaries. This daassumed homologyrecognition of mechcal homology of baorganisms that shar

Key words: Hox genes,developmental studies,mouse

334

nervous embryo. cessivelytheir relaGraham colineariaspects otion of Drosophsible for perturbatcation oftion of vthoracic:and Grusgenes exogy of d

The linsuggeststhe evoluproposednumber oan A-P cnot varyladder ininvariantvertebralresponsespecies, to segmesitional evolutiongenes.

Anothare transthis modthe sameof the reaxial for(upstreamcluster m

The ddeletionsmodels ocould redirectly ofrom a sineeded tologeneticmorpholothe wayachievedembryonformulaeparallel segmenta

MATER

Axial forFor the p

A

system and the paraxial mesoderm in the vertebrateThey show sharp anterior boundaries proceeding suc- from anterior to posterior in an order ‘colinear’ withtive chromosomal position (Duboule and Dollé, 1989;et al. 1989). It is widely assumed that the conservedty of Hox genes reflects their primary role in somef embryonic patterning. Mis-expression and inactiva-Hox genes in mice and of their homologues inila show that Hox genes are among the factors respon-specific regional identity along the A-P axis. In mice,ion of the expression pattern of Hox genes by appli- retinoic acid can result in the homeotic transforma-ertebrae from one type to another (e.g. cervical to

Kessel and Gruss, 1990, 1991; Kessel, 1992). Kessels proposed that a ‘Hox code’ or combination of Hoxpressed in each somite specifies the unique morphol-ifferent vertebrae.

combined numbers of vertebrae that contribute to each distinct regionof the vertebral column. In tetrapods these regions include thecervical, thoracic, lumbar, sacral, and coccygeal or caudal vertebrae(Fig. 1).

The axial formula of the mouse is typical of most rodents. Thereare 7 cervical vertebrae, 13 thoracic, 6 lumbar, 4 sacral, and a variablenumber of caudals (20+).

The chick has 14 cervical, 7 thoracic, 12-13 lumbosacral, and 5coccygeal vertebrae. The goose has 17 cervical, 9 thoracic, 13-14 lum-bosacral and 5 coccygeal vertebrae. The axial formula in birds is com-plicated by the fusion of multiple vertebrae (including the 7th thoracicin the chicken) into the ‘synsacrum’, a character unique to birds. Thearticulation of the pelvis with the vertebral column is extensive inbirds, and involves many more vertebrae than the relatively simplesacrum in other tetrapods. The vertebral fusions within the synsacrumhave resulted in some disagreement on the identity of individualvertebra (Gadow, 1933). The identity of the individual vertebrae com-prising the synsacrum in Fig. 1 have been assigned according toNickel et al. (1977). These numbers agree with our observations in

. C. Burke and others

k between gene expression and vertebral morphology

that Hox genes may have played an important role intion of specific axial variation. The axial Hox code by Kessel and Gruss (1990) could function in af ways. In principle, the Hox genes could simply marko-ordinate system with expression boundaries that do with respect to some fixed point along the somitic different species and thus would be numerically between species at each axial level. Variation in morphology would be achieved by alteration in the of downstream genes to a given Hox code. In allHox gene expression domains would map consistentlynt number within a body plan regardless of transpo-

morphology. In this case the primary events in the of axial formulae would be downstream of the Hox

er alternative is that the Hox gene expression domainsposed along the axis in register with morphology. Inel, a specific combination of Hox genes expressed in segment would determine the morphological identitysultant vertebra. The primary event in evolution ofmulae would be an alteration in the expression of) regulators of the Hox cluster, or the response of

embers to those regulators. ata from mis-expression studies and homozygous

the chick, and includes 4 lumbar, and 9 sacral vertebrae (those unitedby parapophyses and diapophyses to the ilium. They are referred tojointly as lumbosacral vertebrae.

Criteria for assignment of axial level in whole-mountembryosChicken, goose and mouse embryos, like other amniotes, have fiveoccipital somites. These are incorporated into the occipital region ofthe skull (de Beer, 1937). Fate mapping experiments on chickembryos suggest that each trunk somite contributes to two adjacentvertebrae, in keeping with the theory of Neugliederung, or reseg-mentation (Remak, 1855). Cells from the caudal portion of onesomite, and cells from the anterior part of the next posterior somitetogether form a complete vertebra (Bagnall et al., 1988; Couly et al.,1993). There is currently some debate on the reality of vertebral reseg-mentation (see Stern, 1987), as well as on the fate of the anteriorsomites (C. Ordahl and B. Christ, personal communication). In thisstudy we have defined an axial fate map as shown in Fig. 1A, anddescribed as follows: The anterior portion of the 5th sclerotome isincorporated into the occiput, while the posterior half-sclerotome ofthat somite contributes to the body of the atlas, the first cervicalvertebra. The sixth somite contributes to the atlas and the axis (2ndcervical vertebra), the 7th somite to the axis and the 3rd cervicalvertebra, and so on. Thus, for both the chick and the mouse, the firsttrunk vertebra (pv1, the atlas) forms from somite 5-6, the 10thvertebra from somite 15-16, etc. The axial, somitic level of theanterior limit of expression are therefore always designated by two

of Hox genes in mice do not distinguish between theutlined above. Experimental changes in the Hox codesult in homeotic phenotypes whether the code actsr indirectly on morphology. Moreover, data garneredngle organism do not carry evolutionary information resolve this issue. Comparative studies expose phy- variability and provide insight into how genes affectgy since the molecular genetic mechanisms constrain

s that evolutionary differences in morphology are. We therefore compared the axial Hox code inic chickens and mice, animals with different axial, to determine whether the Hox code is transposed inwith vertebral anatomy or maintains a constantl registration.

IALS AND METHODS

mulaeurposes of this study, an axial formula is defined as the

numbers.The anterior limits of expression of Hox genes are reported here

relative to somite number. At the stages used in this study (H and Hstage 19-26 in the chick; Hamburger and Hamilton, 1951, andembryonic day 9-13.5 (E9-13.5) in the mouse), precise counting ofsomites is extremely difficult. The anterior somites begin to dispersequite early, while somites are still forming posteriorly. Since anteriorsomites can not always be accurately counted, the limbs were used aslandmarks for determining somite levels (see below).We consider thesomite levels reported here to be accurate, plus-or-minus one somite.

The extent and position of the limb buds relative to the axis changesduring development. Both the fore and especially the hind limb budsbecome concentrated posteriorly as development proceeds. In thechick, the forelimb bud first appears (H and H st. 15-16) at the levelof somites 15-20. By stage 24, it extends from somite 18-19 throughsomite 21-22. These somites map to the last cervical, and first threethoracic vertebrae. The chick hindlimb first appears at the level ofsomite 25-26 to somite 31-32, and by stage 24 is at the level of somite29-30 through somite 34-35, or the last lumbar and first 5 sacralvertebrae.

The mouse forelimb bud appears at E9 extending from somite level

335

and vertebral transposition

8-9 to ssomite lthe levesiderablcenteredthe levecounting1B,C).

Whole-Embryo4% paraand stor

that of R

PreparDNA teagene) wprobes wfrom DNtated wi

Whole-The meWright stages 2fixed in 10% H2The emmM Nawith 20Xlhbox-

Fig. 1. (squares.green an(5C16 = fiVertebra

Hox genes

at room temperature overnight. Embryosr bathes of TBST followed by incubationonjugated to HRP (Jackson Labs) at a con-ernight at 4°C. The antibody detection.05 mg/ml diaminobenzidine, and 0.02%

5. The reaction was stopped with 100%leared in 2 parts benzyl benzoate: one part

e shown as ovals, the vertebrae byge 25-26 chick embryo stained with alcian by the numbers of the associated somitesreen and cleared in methyl salycilate.

omite 13-14. The hindlimb, appearing at E10, extends fromevel 23-24 to 28-29. At E13, the forelimb is still located atl of somites 8-9 to 15-16, while the hindlimb has shifted con-y and lies from the level of somite 29-30 to somite 34-35, on the first sacral vertebra. At chick stage 26 and mouse E13,ls of the limbs relative to somites can be confirmed by the vertebral bodies in cleared and stained embryos (Fig.

mount in situ hybridizations of chick, goose and mouse embryos were fixed overnight informaldehyde, rinsed in PBS, dehydrated in a methanol series

1:50 in blocking solutionwere washed in five 1-houwith goat anti-rabbit IgG ccentration of 1:1000, ovreaction was done with 0H2O2 in TBST at pH 5.methanol, embryos were cbenzyl alcohol.

RESULTS

A) Schematic representation of the axial formulae of the chick and the mouse. The somites ar The spinal nerves contributing to the brachial plexus are represented by black bars. (B) A stad cleared in methyl salycilate. Vertebral numbers are indicated by letter and number, flankedrst cervical, fifth and sixth somites). (C) A day 13 (E13) mouse embryo stained with alcian gl and somite numbers are designated as in B.

ed in 100% methanol at −20°C. In situ procedures followediddle et al. (1993).

ation of riboprobesmplates were made by linearizing pBluescript plasmids (Strat-

ith Hox inserts ranging from 300 bp to 2 kb (Table 1). RNAere transcribed with T3 or T7 RNA polymerase (Boehringer)A templates with digoxigenin UTP (Boehringer), precipi-

th 4 M LiCl, resuspended in TE, pH 8, and stored at −20°C.

mount immunohistochemistrythods used for immunohistochemistry were provided by C.(personal communication). Embryos of albino Xenopus at7-30 were removed from their egg capsules and membranes,Dent’s fix (20% DMSO in methanol) overnight, bleached withO2 for 24 hours and then stored in 100% methanol at −20°C.bryos where hydrated into TBST, (10 mM Tris, pH 6.8, 150Cl, 0.05% Tween-20), and then blocked for 1 hour in TBST% fetal calf serum. Immunolabeling was achieved with the1 antibody (provided by E. de Robertis) at a concentration of

We have examined the relationship between the expressiondomain of Hox genes and morphological boundaries along themain body axis of the mouse and the chick embryo with wholemount in situ hybridization. We report externally visibleboundaries in the paraxial mesoderm observed at stages whereanatomy can be unambiguously compared between differentvertebrates. Axial boundaries are assigned based on somitecounts and in relation to external landmarks (see Materials andMethods). After determining the anterior border of expressionfor 23 Hox genes in the chick, we selected 16 Hox genes thatwe expected to be particularly informative for comparison inthe mouse. The expression patterns of many Hox genes havebeen published for the mouse (reviewed by Kessel and Gruss,1990, 1991). However, in order to ensure a consistent mannerof comparison with the chick and to resolve ambiguities andconflicts in the published levels (M. Kessel, personal commu-nication), we constructed our own axial Hox map for the mousewith 16 genes. Identical in situ procedures were used for chick

336

and mouaxial bou

Table both chicdomains as the ph

A hers

Tab use genes used in this study

A. Chick

Gene name Restriction sites Description

a-4 Pst-Xho 5′+B+3′a-9 linker-Sma 5′a-10 Hinf1-Hinf1 5′+Ba-11 linker-Nco1 5′+B+3′a-13 Sac1-BamH1 5′+Bb-4 EcoR1-EcoR1 5′+B+3′b-8 EcoR1-linker B+3′b-9 linker-linker 5′+B+3′c-4 linker-Kpn1 5′+Bc-5 Sac1-Sac1 B+3′c-6 linker-Not1 5′+Bc-8 linker-Bgl2 5′+Bc-9 linker-Sma 5′c-10 linker-Sac1 5′+Bc-11 linker-Hinc2 5′d-4 PCR 5′+Bd-8 Bg12-linker B+3′d-9d-10d-11d-12d-13

B. Mouse

Gene name

a-4a-9b-4b-7b-8b-9c-4c-5c-6c-8c-9d-8d-9d-10d-11d-12

(A) Chictemplates utemplate remade fromincludes thChick Hoxdescribed iidentity wapublished cwas assignclones, andpublished ccluster wassequence, cfield southeencoding thprobes used

Table 2. Summary of anterior somitic Hox expression boundaries

Gene Mouse level Chick level Anatomical region

Hoxa-4 7-8 10-11 CervicalHoxb-4 6-7 7-8 CervicalHoxc-4 7-8 10-11 CervicalHoxd-4 − 7-8 CervicalHoxc-5 10-11 17-18 CervicalHoxc-6 12-13 19-20 ThoracicHoxb-7 15-16 − ThoracicHoxb-8 Amb. Amb. ThoracicHoxc-8 17-18 23-24 ThoracicHoxd-8 18-19 Amb. ThoracicHoxa-9 23-24 25-26 ThoracicHoxb-9 Amb. 25-26 ThoracicHoxc-9 23-24 25-26 ThoracicHoxd-9 29-30 29-30 LumbarHoxa-10 − 29-30 LumbosacralHoxc-10 − 30-31 LumbosacralHoxd-10 31-32 30-31 Lumbosacral

. C. Burke and ot

le 1. Chick and mo

Size (kb)

0.90.90.51.40.71.30.70.40.60.61.41.20.451.10.40.50.7

olinearity (Duboule and Dollé, four Hox clusters (Hoxa, Hoxb, represent the products of twole ancestral gene cluster during (Krumlauf, 1992; Kappen andfferent clusters that are homol-e ancestral cluster (united bycalled paralogues. There are 13x clusters, though every clustere member of every paralogueill be described below in order

4-35 Lumbosacral7-38 Lumbosacral9-40 Lumbosacral/caudal40+ Caudal40+ Caudal40+ Caudal

s have been previously reported; see

clusters, a phenomenon called c1989; Graham et al. 1989). TheHoxc, and Hoxd) are thought tosequential duplications of a singthe evolution of the vertebratesRuddle, 1993). Hox genes in diogous to the same gene in thstrong sequence similarity) are paralogue groups in the four Hodoes not have a representativ(Fig. 2). Expression patterns wof paralogue groups.

0.4 linker-Sma1 5′1.7 linker-linker 5′+B+3′0.6 linker-Nar1 5′0.55 linker-EcoR1 5′0.5 Pst1-Xho1 5′+B

Size (kb) Source

0.9 R. Krumlauf0.7 H. Haack/P. Gruss0.3 R. Krumlauf0.8 J. Deschamps0.9 J. Deschamps2.2 R. Krumlauf1.0 P. Sharpe0.3 P. Sharpe0.5 K. Bentley0.45 K. Bentley0.6 M. Capecchi2.0 D. Duboule1.1 D. Duboule0.45 D. Duboule1.1 D. Duboule1.1 D. Duboule

k Hox genes The size and defining restriction sites of the DNAsed to generate riboprobes are listed, as well as the position of thelative to the homeobox. For instance, the probe for Hoxa-4 was

Hoxa-11 − 3Hoxc-11 − 3Hoxd-11 34-35 3Hoxd-12 35-36Hoxa-13 −Hoxd-13 −

−, not done. Amb, Ambiguous level.Many of the murine expression level

text for details.

se embryos, and identical criteria were used to assignndaries relative to somite number.2 gives an overview of the Hox genes examined forken and mouse in this study. The gene expressionin the paraxial mesoderm follow the same sequenceysical order of the genes within each of the four Hox

Paralogue groups 1, 2 and 3 were not examined in this study.These genes have expression boundaries in the hindbrain andthe occipital somites. The chick and mouse have the samenumber of occipital somites, and the axial expression of Hoxgenes in this region are in register (R. Krumlauf, personal com-munication).

Paralogue groups 4 and 5Representatives of the fourth and fifth Hox paralogue groupshave anterior boundaries of expression within the cervicalregion of the chick and the mouse. The cervical region of thechick comprises fourteen vertebrae derived from somites 5-19.The mouse cervical region has only seven vertebrae, derivedfrom somites 5-12 (Fig. 1).

The most anterior, or 3′, paralogue group examined wasparalogue four, which has a gene in each cluster (Fig. 2). Allof these were examined in the chick. Hoxa-4, Hoxb-4, andHoxc-4 were examined in the mouse (Fig. 3). The expressionboundaries of these genes have similar relationships to eachother in each animal, Hoxb-4 is the most anteriorly expressed

a template of 900 base pairs between Pst and Xho sites thate homeobox (B) and sequence both 5′ and 3′ to the box (5′+B+3′).genes were isolated from stage 24 chick limb cDNA libraries, andn detail elsewhere (Nelson et al., unpublished data). In brief,s determined for the majority of the genes by comparison withhick cDNA fragments. Identity of Hoxa-10, Hoxa-13, and Hoxb-9

ed based on sequence identity with published mouse and human also, in the case of Hoxa-10 and Hoxa-13, on comparison withhick expression patterns. Identity of the genes in the chick Hoxc based upon comparison with published mouse and humanonsistency of expression patterns within the cluster, and pulse-rn blots that are consistent with physical linkage of the genese cloned cDNAs. (B) The size of the templates for the mouse Hox in this study and the source of the DNA.

337Hox genes and vertebral transposition

membelevel 6Hoxc-411), an

A 2 A 1A 7 A 6 A 5

B5B6

C

A 3

B3 B2 B1

D3 D1

Vertebrate Hox Clusters

A 4

B4

C5 C4

B8 B7

C6

A 1 1 A 9

B9

C9

D11

C8

D8D9D10

A

C10C11

D4

A 1 0

5 ' 3 'Fig. 2. Schematicrepresentation of thefour vertebrate Hoxclusters. Theindividual genes arerepresented by boxes.Filled boxes indicategenes examined inboth the chick and themouse. Half filledboxes indicate genesexamined in either thechick (top) or themouse (bottom).

extends anteriorKrumlaupeak exp10-11. Shigh ovesimilar t22 embrGaunt aHoxa-4somite lfading oreportedthe axis,species,

D13

C1213

D12

1 3

r-7

d

Fig. 3. Whole-mount in situhybridizations with membersof paralogue 4. Arrowsindicate the anterior limit ofgene expression in theparaxial mesoderm. (A) ChickHoxa-4 (st. 26) with a sharpanterior boundary in themesoderm at somite level 10-11. Labeling is stronger inanterior-half somites and

p he base of the tail. (B) Chick Hoxb-4 (st. 26), with a sharp anterior boundary at somite level 7-8, several segments t

iroynheu s

osteriorly to t

of group 4 (at somite level 7-8 in the chick and somite in the mouse). Slightly more posterior Hoxa-4 and

share the same boundary in the chick (somite level 10- in the mouse (somite level 7-8). Anatomically these

levels are similar in the two species; Hoxb-4 is associated withanterior cervical vertebrae, and Hoxa-4 and Hoxc-4 map tovertebrae towards the middle of the cervical series of bothanimals.

o the boundary for Hoxa-4, in agreement with data from stage 10 embryos that show the 7th as the first Hoxb-4 labeled somite (R.f, personal communication). Labeling quality is similar to Hoxa-4, but shows more uniform intensity dorsally across each somite, withression across seven or eight segments, fading posteriorly. (C) Chick Hoxc-4 (st. 25), is faint with a paraxial boundary at somite levelgnal covers fewer than ten segments across the brachial region, and fades below detection by mid trunk. (D) Chick (st. 22) Hoxd-4 hasall background, but there is clear paraxial labeling that is not present in the sense control (data not shown). The tissue distribution is Hoxb-4, only much weaker at all stages examined. The paraxial expression extends 11 segments anterior to the forelimb bud in stageos, which places the anterior expression boundary at somite level 7-8, aligned with the boundary of Hoxb-4. This is in agreement withd Strachan (1994) who found somite 7 to be the first labeled somite at both stage 11-12 and stage 25-26 chicks. (E) Mouse (E12)as an anterior expression boundary in the paraxial mesoderm at somite 7-8. (F) Mouse (E11.5) Hoxb-4 is slightly anterior to Hoxa-4 atvel 6-7. The somitic label extends clearly for only seven segments for both genes, extending over the axial level of the forelimb, andt in the trunk. (G) Mouse (E13) Hoxc-4 is expressed with a clean paraxial boundary at somite level 7-8, in agreement with databy Geada et al. (1992). As in the chick, labeling for Hoxc-4 is weak, but in the mouse Hoxc-4 RNA extends further posteriorly alongcontinuing weakly into the tail. It is not known if these variations in labeling intensity and posterior extent of expression reflectstage, or probe differences.

338

Hoxc-studied heregion, buchick andlevel 10-1anatomicasecond to

ParaloguHoxc-6, examinedof the firs(Fig. 5A,B19-20, twthe middexpressioHoxc-5 ethe forelim

ParaloguAll mem

xamined have anterior expression boundaries in the thoracicegion of both chick and mouse. Hoxb-7 expression was onlyxamined in the mouse. The anterior-most somite with positiveabeling is somite 15-16, at the position of the forelimb’sosterior boundary with the body wall, mapping to the 4thhoracic vertebrae (Fig. 6B).

All three members of the eighth paralogue group, Hoxb-8,oxc-8 and Hoxd-8, were examined in both the chick and theouse (Fig. 6). Hoxb-8 labeling is clearly stronger in the mid-

runk mesoderm than the cervical region of both animals, buto specific somite number could be assigned in either animaldata not shown). Hoxc-8 axial expression borders in both thehick and the mouse lie in the trunk region, posterior to theorelimb. The anterior limit of Hoxc-8 expression in the chickorresponds to somite level 23-24, or the fifth thoracic vertebraFig. 6A). In the mouse, the anterior-most region to showoxc-8 expression is somite level 17-18, corresponding to the

A

erelpt

Hmtn(cfc(H

. C. Burke and others

Fig. 4. Whole-mount insitu hybridizations withmembers of paralogue 5.

sixth thoracic vertebra. Hoxd-8 expression in the paraxial

5 is the only member of the 5th paralogue group

mesoderm of the chick does not resolve into a clear A-Pboundary. Signal comes up gradually behind the forelimb andincreases in the trunk but no discrete axial level could beassigned (data not shown). In situ hybridizations with Hoxd-8in the mouse however, show a positive signal in the paraxialmesoderm that comes on at somite level 18-19, or the sevenththoracic vertebra (Fig. 6D).

Paralogue 9The complete ninth paralogue set was studied for both thechick and the mouse, and gives a similar picture in each animal(Fig. 7). Hoxa-9, Hoxb-9 and Hoxc-9 map near the end of thethoracic series in both animals. In the chick, their anteriorexpression boundaries lie four or five segments anterior to thehind limb and four segments posterior to the fore limb. Thiscorresponds to somite level 25-26, or the last thoracic vertebra(T7). In the mouse, Hoxa-9, Hoxc-9 have anterior expressionboundaries four or five segments anterior to the hindlimb,which in the mouse is nine or ten segments behind theforelimb. This maps to somite level 23-24, representing thesecond to last thoracic vertebra (T12). Hoxb-9 labeling isstronger in the posterior trunk, but does not form a discreteboundary, and no somite number was assigned.

Unexpectedly, the expression boundary of Hoxd-9 does not

Arrows indicate theanterior limit of geneexpression in the paraxialmesoderm. The overalllabeling quality in bothanimals is very similar tothe quality of Hoxc-4.(A) Chick (st. 25) Hoxc-5paraxial expression startsseven segments posteriorto chick Hoxc-4expression, at somitelevel 17-18, or thethirteenth cervicalvertebra. (B) Mouse(E13) Hoxc-5 expressionstarts only two segmentsbehind mouse Hoxc-4expression, at somitelevel 10-11, the sixthcervical vertebra. Thisaxial level is consistentwith observations ofGaunt et al. (1990).

re (Fig. 4). Hoxc-5 is also expressed in the cervicalt it is widely separated by somite number between mouse (somite level 17-18: chick, versus somite1: mouse). The expression boundary marks the samel level, however; the somites contributing to the

last cervical vertebrae.

e group 6the only member of the sixth paralogue group, has an anterior boundary of expression at the levelt thoracic vertebrae in both the chick and the mouse). In the chick Hoxc-6 labeling begins at somite level

o vertebrae behind Hoxc-5 expression, aligned withle of the forelimb bud. In the mouse Hoxc-6n begins at somite level 12-13, two vertebrae behindxpression and in axial alignment with the middle of

b bud.

e groups 7 and 8 bers of the seventh and eighth paralogue groups

conform to the axial level observed for its paralogues in eitheranimal, but rather are shifted posteriorly towards the lum-bosacral transition. In the chick Hoxd-9 expression is three orfour segments behind the expression of Hoxa-9, Hoxb-9 andHoxc-9. It lies one segment anterior to the hindlimb, at somitelevel 29-30, representing the last lumbar vertebra (L4). In themouse, the anterior boundary of expression of Hoxd-9 lies atthe same numbered somite, 29-30, which in the mouse corre-sponds to the penultimate lumbar vertebra (L5).

Paralogue 10All three of the Hox 10 paralogues, Hoxa-10, Hoxc-10, andHoxd-10 were examined in the chick and Hoxd-10 wasexamined in the mouse (Fig. 8). All the tenth paralogue genesare expressed close to the lumbosacral transition. Hoxa-10 hasthe most anterior expression in the chick, starting one segmentanterior to the hindlimb at somite level 29-30. This axial levelrepresents the last lumbar vertebra (L4), and is aligned withthe expression of chick Hoxd-9. Boundaries for Hoxc-10 and

Hoxd-10first sacanteriormidline vertebra

ParalogThe Hohave a mgroup. Achick anmouse aries ofchick amemberalthougharies wHoxd-11chick hboundarping wispondinThe antesomite lof the hsacral posteriothe hindexpressiat the lalevel 34limb.

Hoxdand the 13 werparaxialthese gesomite nshown).in the chfrom thedomain mouse somite vertebraand Hoxdistal en

Expresthe domOur anaand theexpressimorphospecificaboundarin the cthis travertebrathis anastability

339Hox genes and vertebral transposition

lie just behind Hoxa-10 at somite level 30-31, or theral vertebra. In the mouse, Hoxd-10 labeling shows an boundary at somite level 31-32, aligned with the

of the hindlimb, and mapping to the first sacral, as in the chick (Fig. 8D).

ues 11, 12 and 13xa, Hoxc, and Hoxd clusters each

ember in the eleventh paraloguell three were examined in the

d Hoxd-11 was examined in the(Fig. 9). The expression bound- paralogue eleven genes in there not as close together ass of other paralogue groups, they all have expression bound-

ithin the sacral series, as does

6 and the cervical-thoracic transition, we examined theexpression of Hoxc-6 in two additional species with verydifferent cervical lengths: the domestic goose and the frogXenopus (Fig. 5).

in the mouse. Hoxa-11 in theas the most anterior expressiony, at somite level 33-34, overlap-th the hindlimb bud and corre-g to the fourth sacral vertebra.rior boundary of Hoxc-11 lies atevel 36-37, at the posterior edgeindlimb, mapping to the seventhvertebrae. Hoxd-11 lies morer still, about two segments behindlimb, probably at somite 40. Theon of Hoxd-11 in the mouse fallsst sacral vertebra (S4) at somite-35, immediately behind the hind

-12 was examined in the chickmouse, and Hoxa-13 and Hoxd-

e examined in the chick. The mesoderm expression patterns ofnes are limited to the tail and aumber was not assigned (data not The anterior extent of Hoxd-12ick is a distance of 4-5 segments rear border of the hind limb. Theof Hoxd-12 expression in the

is posterior to all the others, atlevel 35-36, the first caudal. The expression of both Hoxa-13d-13 in the chick is found in thed of the tail.

sion boundary of Hoxc-6 inestic goose and Xenopus

lysis of Hox genes in the chick mouse suggest that Hox geneon is transposed in concert withlogy. For example, Hoxc-6lly marks a key morphologicaly, the cervical-thoracic transitionhick and the mouse even thoughnsition is transposed by sevene in these two species. To extendlysis and test the phylogenetic of the correlation between Hoxc-

Fig. 5. Whole-mount in situ hybridizations with members of paralogue 6. Arrows indicate theanterior limit of gene expression in the paraxial mesoderm. (A) Chick (st. 25) Hoxc-6expression boundary lies at somite level 19-20, aligned with the middle of the forelimb, at thefirst thoracic vertebra (T1). The paraxial mesoderm is labeled in an ‘arcade’ pattern along theflank: intersegmental labeling forms arches that meet dorsally in mid-segment, defining eachsomite. Somite labeling continues posteriorly into the tail. Laterally in the mid-flank there is aband of nearly continuous expression running anterior-posterior along the original boundarybetween somitic and lateral plate mesoderm. Lateral to this, in the body wall are streaks ofinter segmental expression that trace the myosepta. (B) Mouse (E12) Hoxc-6 expression isaligned with the middle of the forelimb at somite level 12-13, the first thoracic vertebra,consistent with data reported by Jegalian et al. (1992). The mouse does not show an ‘arcade’pattern of expression but the label is very strong in a near-continuous region of trunkmesoderm that covers approximately eight segments and extends laterally to the horizontalseptum. Ventrally, the label is inter segmental, tracing the myosepta and rib positions in thebody wall. (C) Goose at the morphological equivalent of a stage 25-26 chick embryohybridized with chick the Hoxc-6 probe. Two different probes were used, both giving positiveresults with a variable degree of background that is comparable in chick and goose embryos.The labeling pattern is very similar to that of the chick including anterior-proximal limblabeling reported elsewhere (Sharp et al. 1988: Nelson et al. unpublished data), and a partialarcade pattern of somitic labeling. The paraxial mesoderm labeling starts at an axial level thataligns with the middle of the forelimb, at somite level 22-23, the first thoracic vertebra. (D) Whole-mount immunohistochemistry with XlhBox-1 antibody on stage 35 Xenopusembryos. Labeling begins at the border between somite 3 and somite 4, forming a continuousborder across the neural tube and the mesoderm in agreement with labeling reported byOliver et al. (1988).

340

The riboprobe for chicken Hoxc-6 was used to examineexpression in the domestic goose, following the same generalin situ protocol used for the chick. The paraxial mesodermlabeling starts at an axial level that aligns with the middle ofthe forelimb. In the goose, with 17 cervical vertebrae, this issomite level 22-23, the first thoracic vertebra.

Frogs have highly specialized vertebral morphology,without a clear distinction between cervical and thoracicvertebrae. The axial level of innervation for the forelimbhowever can be used as a landmark, and derives from segmentsnumber 2, 3 and 4. The Xlhbox-1 antibody generated againstthe Hoxc-6 protein of Xenopus (Oliver et al., 1988; Wright etal., 1989) was used to visualize the anterior expressionboundary in the paraxial mesoderm of the chick embryo andin Xenopus. Consistent with the in situ data, the anteriorboundary in the chick marks the cervical-thoracic transition(data not shown). In stage 35 Xenopus larvae (Nieuwkoop and

Faber, 1967), the labeling begins at the border between somite3 and somite 4 (Fig. 5D). This axial level corresponds to spinalnerves that innervate the forelimb in frogs.

DISCUSSION

The evolution of the tetrapod vertebral column into specializedregions has allowed a great deal of functional diversity andadaptation. This diversity arose from an ancestral conditionexemplified by the rhipidistian fishes. All fishes exhibit adegree of axial regionalization effected primarily by the extentof the coelom, the position of the median fins, and the mor-phology of the tail. Tetrapod axial regionalization has appar-ently evolved from this ancestral condition primarily throughadaptations for weight bearing locomotion (Goodrich, 1930;Gadow, 1933; Panchen, 1980).

A. C. Burke and others

Fig. 6. Whole-mount in situ hybridizations with members of paralogues 7 and 8. Arrows indicate the anterior limit of gene expression in theparaxial mesoderm. (A) Chick (st. 25) Hoxc-8 with an expression boundary at somite level 23-24, the fifth thoracic vertebra. In this specimen,the expression is very strong in the first three or four labeled segments, and fades posteriorly. Other embryos show uniformly strong labelingfrom somite level 23-24 into the tail. In stage 19-23 chick embryos, the labeling has a strong posterior-half somite bias, but by stage 25 this isnot as obvious, and the labeling takes on the arcade pattern. (B) Mouse (E11.5) Hoxb-7 shows an expression boundary at somite level 15-16,4th thoracic vertebra. Kessel and Gruss (1991) give the anterior boundary of Hoxb-7 as the 4th thoracic vertebra. However, Vogels et al. (1990,1993) looking at sectioned material place the anterior boundary in the paraxial mesoderm at somite 11, which maps to the 6th and 7th cervicalvertebrae. With our probe, signal is gone except in the CNS by E13. (C) Mouse (E13.5) Hoxc-8, expression at somite level 17-18,corresponding to the sixth thoracic vertebra (in agreement with Gaunt, 1988). (D) Mouse (E13.5) Hoxd-8 gives a positive signal in the paraxialmesoderm at somite level 18-19, the seventh thoracic vertebra in contrast to the report of Izpisua-Belmonte et al. (1990) who found a weaksignal in the mouse at the tenth thoracic vertebra (somite level 21-22) with increasing intensity posteriorly. Labeling is also strongly positive inthe nephric tissue and limb mesoderm.

341Hox genes and vertebral transposition

Fig. 7. Whole-mount in situ hybridizations with members of paralogue 9. Arrows indicate the anterior limit of gene expression in the paraxialmesoderm. Chick (st. 24-25) Hoxa-9 (A) Hoxb-9 (B) and Hoxc-9 (C), all show expression at somite level 25-26, or the last thoracic vertebra.Dorsal root ganglia are strongly labeled for Hoxa-9 and Hoxb-9 in more anterior segments. Myotomal extensions from the Hoxc-9-labeledsomites can be clearly seen in the body wall. (D) Chick (st. 23-24) Hoxd-9 expressed at somite level 29-30, the last lumber vertebra. (E) MouseHoxa-9 (E13.5) and (G) Hoxc-9 (E11) both show expression at somite level 23-24, or the last thoracic vertebra. The dorsomedial portion ofthese somites is negative, and label decreases in the base of the tail. Segmental labeling with Hoxa-9 is also apparent in the leading edge of thebody wall closing around the viscera but not visibly continuous with the somites. (F) Hoxb-9 (E11.5) did not resolve to a distinct somite level,but is stronger in the posterior thoracic region. (H) Mouse (E13) Hoxd-9 with a boundary at somite level 29-30.

Fig. 8. Whole-mount in situ hybridizations with members of paralogue 10. Arrows indicate the anterior limit of gene expression in the paraxialmesoderm. (A) Chick (st. 24) Hoxa-10, is expressed at somite level 29-30, the last lumbar vertebra. Labeled somites show a posterior bias andstrongly positive dorsal ‘caps’. (B) Hoxc-10 (st. 26) and (C) Hoxd-10 (st. 25) share a boundary behind Hoxa-10 at somite level 30-31, or thefirst sacral vertebra. Somites labeled with Hoxc-10 have the arcade character, like the other chick Hoxc genes, and the labeling with Hoxd-10resembles that of Hoxa-10 without the ‘caps’. (D) Mouse (E12.5) Hoxd-10 shows an anterior boundary at somite level 31-32, mapping to thefirst sacral vertebra. There is an intersegmental bias resulting in a striped effect.

342

Verteachievedthe highgiraffes’The eamesoderstantiallycializatiocharacte

A

Fig. 9. W s with members of paralogue 11. (A) Chick (st. 24) Hoxa-11 at somite level 33-34. (B) Chick (st. 24)Hoxc-11 (st. 24) Hoxd-11 at somite 40. (D) Mouse (E12.5) Hoxd-11 at somite level 34-35. Arrows indicate theanterior l raxial mesoderm.

Mou

Ch

A

B

. C. Burke and others

hole-mount in situ hybridizationat somite level 36-37. (C) Chickimit of gene expression in the pa

b

CaudalSacralLumbar

9 10 11 12

9 10 11

9

ThoracicCervical

A

B

C

4

4

4 6 85

ick

ral diversity in tetrapods ranges from the secondarily uniformity of snakes and other limbless tetrapods, toly specialized morphology of the turtle’s shell, the long neck and the prehensile tail of certain monkeys.rly developmental pattern from unsegmentedm, through somites, into vertebrae, has not altered sub- between fishes and tetrapods. The subsequent spe-ns and subtleties of structure are, however, significant

rs in the distinctions between vertebrate groups.

We have evaluated the correlation between morphologicalregions of the vertebral column and the anterior-mostexpression of Hox genes as determined by whole-mount in situhybridization (Fig. 10). It should be noted that this techniquelacks the sensitivity and resolution to draw specific mechanis-tic conclusions about the roles of Hox genes at a given axiallevel. This is exemplified by the fact that in mice carryinghomozygous deletions of certain Hox genes, phenotypic effectsare observed anterior to the detectable limits of Hox gene

CaudalSacralLumbarThoracicCervical

A

B

C

D

94

119 10 12

4

4 5 6 8 9

7

se

D 4 10 119 12Fig. 10. (A) Schematic representation of the axialformulae and somite levels of the chick and themouse. The Hox genes discussed in the text arelisted in register with the axial level of their anteriorboundary of expression in the paraxial mesoderm ofthe chick (above) and the mouse (below). (B) Schematic representation of the axial anatomyin chicks and mice with the anterior boundary ofHox gene expression indicated by region.

expressRamireCapeccare expboundahybridinecessacases. Hcomparciated wanimalsHox gethe axisphologevoluti

Hox gregionThe Hooccipitathe momunicaregion the hinsomitesregion phologremain

In coposed bgene extransporegion,chick alevel 10level ogenes othe fore

The indicateanatomtransitiexpressgoose hposed cthe antefirst thsegmenthe fore

The based omusclenerves the limbridgesand protion in frogs). brachiaThe antis the fi

343Hox genes and vertebral transposition

ion (e.g. Hoxa-4, Kostic and Capecchi, 1994; Hoxb-4,z-Solis et al., 1993). As pointed out by Kostic andhi (1994), one explanation for this is that these genesressed at a significantly lower level anterior to thery of expression observed in the whole-mount in situzations. The limits of expression we define here are notrily the absolute anterior limits of expression in allowever, in every Hox gene examined in cross-species

ison, the expression boundaries are consistently asso-ith morphology, not with absolute somite number, in

with different axial formulae. The relative shifts inne expression that we observe in different areas along reflect the relative expansion and contraction of mor-

ical regions. This implies a role for the Hox genes in theon of axial variation.

ene expression conforms to anatomicals along the axis

The expression border of Hoxc-6 is located at the somitesthat map to the first thoracic vertebra (that carries the last spinalnerve to the brachical plexus) in the chick, the goose and themouse (Fig. 11). The significance of this association isextended by the pattern of Hoxc-6 expression found in Xenopusand in the zebrafish. In stage 30 Xenopus larvae, labeling withthe Xlhbox-1 antibody against the Hoxc-6 protein begins at theborder between somite 3 and somite 4 (Fig. 5C). The forelimbbud in metamorphosing Xenopus tadpoles arises just behindthe gills, and is innervated from spinal nerves 2, 3 and 4, whichbridge the cervical-thoracic transition of adult frogs.

In many modern bony fishes, the pectoral fin is locatedimmediately behind the jaw, and no distinction is madebetween cervical and thoracic vertebrae. (The extreme anteriorpositioning of the pelvic fin and its migration from its level ofinnervation, was the major feature behind Goodrich’s 1906description of transposition). Transplantation experiments inamphibians and chicks demonstrate that the segmentation and

x expression boundaries that have been examined in thel region of the chick conform exactly to those found in

use (e.g. Guthrie et al., 1992; Krumlauf, personal com-tion). There is no transposition of segments in thisbetween the two species since the segmental pattern ofdbrain rhombomeres and the number of occipital do not differ between these two species. Thus, in theof the amniote axial skeleton that does not show mor-ical transposition, Hox gene expression boundaries constant between species.ntrast, in regions of the axial skeleton that are trans-etween the chick and the mouse, the borders of Hoxpression are shifted in concert with the morphologicalsition. For example, Hoxc-5 is expressed in the cervical but at an axial level separated by 7 somites betweennd mouse (somite level 17-18: chick, versus somite-11: mouse). This transposition is in concert with the

f the forelimb and the brachial plexus, suggesting thatf the fifth paralogue are causally linked to the level oflimb.

expression boundaries of Hoxc-6 in chick and mice also a causal connection to the position of the limb and theical boundary between the neck and the thorax (C-Ton). This correlation was expanded by examining theion of Hoxc-6 in the domestic goose. The domesticas 17 cervical vertebrae, and thus the forelimb is trans-audally by 3 segments relative to the chick. In the gooserior limit of expression falls at somite level 22-23, the

oracic vertebra, and thus is also transposed by threets relative to the chick, in concert with the position oflimb. forelimbs of tetrapods can be assigned an axial leveln the number of the spinal nerves that innervate the

s of the limb. The ventral rami of several different spinalconverge together and are concentrated at the base ofb. The series of nerves that form the brachial plexus the transition between cervical and thoracic vertebrae,vides a landmark to locate the cervical-thoracic transi-animals with highly derived vertebral morphology (e.g.In amniotes (which include birds and mammals), thel plexus usually comprises four or five spinal nerves.erior three or four of these are cervical, and the last onerst thoracic.

outgrowth of the spinal nerves is dependenmal somites, which in turn are influenced by(Detwiler, 1934; Keynes et al., 1987). Thenerve roots in the limb plexus reflects the nuthat contribute to the limb musculature. Mo

Chick GooseMouse Xen

1 1 1 1

5 5 5 5

10

11

12

13

10

18

19

20 20

21

22

23

24

25

26

10

2

3

4

6

7

8

Fig. 11. Schematic representation of the somite lecervical-thoracic transition in mouse, chick, gooszebrafish. Black bars represent the spinal nerves plexus, and the level of the limb or fin bud is indiline. The shaded somite levels indicate the level oexpression as determined by whole-mount in situimmunohistochemistry. The level for the zebrafisMolven et al. (1990).

t on the mesoder- lateral mesoderm number of spinalmber of myotomeslven et al. (1990)

opus Zebrafish

1

5

2

3

4

6

vels bridging thee, Xenopus,of the brachialcated with a curvedf Hoxc-6 hybridization, orh is taken from

344

used the Xembryos oexpressionsomites 4somites twHoxc-6, ation for tfrom spinexpressionoverlap wposterior-in the zebamniotes vertebra.

Additio6 membehomozygoCapecchi,vertebra, cervical v

The othwith morseventh aanterior eboth chiccomparedobvious mof this gexpressedwhich it cervical r

The exare foundtion fromIn the luabsolute lspecies. Tfollowed mammaliParalogueaxial exprthree cogbosacral banatomicasomite.

Membegrouped paraloguecontrast texpressionboundariethe chicksacral serthe chick.somites anPotter, 19in the motion of Hextensive expressionfound at th

A.

lhbox-1 antibody to localize the Hoxc-6 protein inf the zebrafish, Brachydanio rerio. They report the boundary in the paraxial mesoderm to fall between

and 5. In the embryo the finbud appears adjacent too and three, and has anterior-proximal expression of

s seen in the limbs of chicks and mice. The innerva-he pectoral fin (the piscine brachial plexus) comesal nerves 3, 4 and 5 (Myers, 1985). Thus, while the of Hoxc-6 in the paraxial mesoderm does not

ith the placement of the finbud, it does align with themost segment that innervates the pectoral appendagerafish, as it does in an amphibian and three different(Fig. 11). In the tetrapods this is the first thoracic

nal evidence for the culpability of paralogue grouprs in the cervical-thoracic transition comes fromus deletions of Hoxa-6 in the mouse (Kostic and

members of paralogue 13 were found further posterior in thetail.

Hox genes and the evolution of axial diversity invertebratesThe full set of Hox genes appears to have been present in thecommon ancestor of tetrapods and modern fishes (F. Vander-Hoeven and D. Duboule, personal communication). Thisancestral animal lacked the distinct axial regions present intetrapods, but it is virtually certain that its Hox genes wereexpressed in an A-P, colinear progression. Primitive Hoxexpression patterns presumably served to define units that havesubsequently been elaborated into specialized regions, facili-tating the evolution of axial structures along separate, discretemorphological paths in fishes and tetrapods.

The anatomical distribution of Hox gene expression bound-aries is remarkably consistent between the chick and mouse.

C. Burke and others

1994). These mice have a rib on the last cervical The association of individual genes with specific anatomical

a partial posterior transformation of the seventhertebra towards a thoracic morphology.er Hox genes studied are similarly transposed alongphological regions. Briefly, all members of thend eighth paralogue groups examined here havexpression boundaries within the thoracic region ink and mouse. The expression boundaries of Hoxc-8 between the two species do not correspond to anyorphological landmarks. However, the transposition

ene expression boundary is clear. If Hoxc-8 were in the chick at the same absolute somite number atis expressed in the mouse, it would be in the chickegion rather than the thoracic region.pression boundaries of Hoxa-9, Hoxb-9 and Hoxc-9 in close association with the morphological transi- thoracic to lumbar vertebrae in both chicks and mice.mbar region the expression boundaries differ inocation by only one or two somites between the twohis is because the long neck of the chicken isby a short trunk, while the relatively short

an neck is followed by a long trunk in the mouse. group nine also shows a significant disparity in theession of one of its members, Hoxd-9, relative to itsnates. Hoxd-9 is expressed just anterior to the lum-

boundaries is quite clear. Particularly striking is the associa-tion of Hoxc-5 and Hoxc-6 with the cervical-thoracic transi-tion; the association of Hoxa-9, Hoxb-9 and Hoxc-9 with theend of the thoracic series; the posterior displacement of thecognate Hoxd-9 to the end of the lumbar series; followed bythe Hox-10 members in the initial segments of the sacrum. Inthe case of Hoxc-6, we have extended the comparison toinclude an amphibian and a fish. The cervical-thoracic transi-tion, and the innervation of the pectoral appendage are furtherlinked by the expression of Hoxc-6, even in animals where amorphological transition is not perceived (e.g. fishes).

The axial expression of Hoxd-9 is anomalous in that thisexpression level is significantly out of register with the othermembers of the ninth paralogue group. The significance of theshift in the expression boundary of Hoxd-9 may have traces inthe history of vertebrate anatomy. Based on fossil evidence,the advent of a pelvis attached to the vertebral column (sacrum)was a major transition between tetrapods and their fish-likeancestors. Also, neither fossil or recent amphibians subdividethe trunk into thoracic and lumbar regions, so a distinctionbetween thoracic and lumbar vertebrae is not a universalcharacter of tetrapods, and birds and mammals are derived inthis regard (Goodrich, 1930; Gadow, 1933). Hoxa-9, Hoxb-9and Hoxc-9 are associated with the transition from thoracic to

order in both the chick and the mouse, again anl level that differs in absolute number by only one

rs of the tenth paralogue group examined areat the lumbosacral transition, and members of eleven are expressed at levels within the sacrum. Ino members of the other paralogue groups, whose borders are relatively close together, the expression

s of Hox-11 genes are spread over seven somites in. We found Hoxd-11 expression at the end of theies in the mouse, similar to the anatomical level in Published reports for Hoxa-11 in the mouse are fourterior to this at the lumbosacral boundary (Small and93), indicating that expression of the Hox-11 genesuse extend over the full sacral series. The distribu-ox-11 gene expression in the chick mimics theexpansion of the sacral region in birds. The boundaries of the Hox-12 genes examined weree beginning of the caudal series of both species, and

lumbar vertebrae, and Hoxd-9 is apparently anchored to thepresacral-sacral transition in chicks and mice. It is not unrea-sonable to suppose that all the genes in the ninth paraloguegroup shared the same anterior expression boundary at onetime. The shift of Hoxd-9 expression away from its paralogues,resulting in a novel gene expression boundary, may have beeninstrumental in the evolutionary transition from fish totetrapod. Alternatively (or subsequently), expression shiftswithin the Hox-9 paralogue may have been influential in theevolution of the thoracic-lumbar transition from a moreuniform amphibian-type trunk. This hypothesis suggests thatthe generation of novel gene expression relationships by trans-position of expression boundaries could result in the genera-tion of new morphological regions, as well as positionalchanges in pre-existing regions.

Hox genes as characters or units of biologicalhomologyIn the 19th century, the German school of Naturphilosophie

developinvarianaturalof morpunits thorganisof BaupThe regused asof animet al., 1

The in any biologidata disfor axiexistinggene exwhich providethat haFor inssnakes betweeanimalshomolopholog

The becomedirectiomoleculogicallthe dowresult iphyla sbeen chomolomental using msegmenmechanmationMoreovif we acpods hbasis ohas beeconsidechordatPax-6 icating amentall1994).

The genes aphologdetermregionsmorphothe absbetwee

345Hox genes and vertebral transposition

ed the concept of Bauplan (body plan), to describe thent aspects of morphology that unite organisms into groups (see Russell, 1916 for review). Invariant aspectshology indicate homology, and homology identifies theat can be compared meaningfully between any twoms. Originally a purely morphological concept, the idealan has been expanded today to include molecular data.ulated A-P expression of Homeobox genes has been

the primitive character uniting widely divergent groupsals into a common Bauplan, called the Zootype (Slack993). usefulness of the Bauplan concept is explicitly manifestphylogenetic study, and is employed implicitly by allsts searching for universal patterns or mechanisms. Thecussed here explores a degree of molecular homology

al regions among amniotes that strengthens the pre- assessment of homology based on morphology. Hoxpression constitutes an additional set of characters with

by evolutionarily changes in expression domains of Hox genesalong the anterior-posterior axis.

We are grateful to the following people for mouse Hox probe con-structs: R. Krumlauf (Hoxa-4, Hoxb-4, Hoxb-9); H. Haake and P.Gruss (Hoxa-9); M. Capecchi (Hoxc-9); J. Deschamps (Hoxb-7 Hoxb-8); D.Duboule (Hoxd-8, Hoxd-9, Hoxd-10, Hoxd-11, Hoxd-12); P.Sharp (Hoxc-4 and Hoxc-5); K. Bentley (Hoxc-6, Hoxc-8). E. DeR-obertis provided the Xlhbox-1 antibody. E. DiMambro assisted in thecharacterization of chick Hox clones. D. Roberts provided gooseembryos. J. Khorana assembled the figures. We thank R. Krumlauf,E. Gilland, and M. Scott for critical reading of the manuscript, and ananonymous reviewer for helpful comments. A. C. B. is supported bya National Institutes of Health, NRSA postdoctoral fellowship. Thiswork was supported by a grant from the National Institutes of Healthto C. T.

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vtan

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(Accepted 21 October 1994)

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he expression of Hoxb-3,hick somites and reached

d here.

ox code during morphological