Proc. Natl. Acad. Sci. USAVol. 93, pp. 9636-9640, September 1996Developmental Biology

Functional specificity of Hoxa-4 in vertebral patterning liesoutside of the homeodomainTADURU L. SREENATH*, ROBERT A. POLLOCK*, AND CHARLES J. BIEBERICH*tt*Department of Virology, Jerome H. Holland Laboratory, Rockville, MD 20855; and tDepartment of Biochemistry and Molecular Biology, George WashingtonUniversity Medical Center, Washington, DC 20037

Communicated by Francis Ruddle, Yale University, New Haven, CT, June 5, 1996 (received for review April 15, 1996)

ABSTRACT The Hox family of proteins plays a centralrole in establishing the body plan of a wide range of metazoanorganisms. Each member of this family of transcriptionalregulators has a distinct functional specificity, yet they bind tosimilar DNA target sequences through their conserved hom-eodomain. The mechanisms whereby Hox proteins achievetheir diverse specificities in vivo remain undefined. Using theopposing effects ofHoxa-4 and Hoxc-8 in vertebral patterning,we demonstrate by replacing the homeodomain ofHoxa-4 withthat of Hoxc-8 that the functional specificity of Hoxa-4 doesnot track with the homeodomain. These observations provideevidence that other regions of Hox proteins play an importantrole in mediating functional specificity during mammalianembryogenesis.

Many transcription factors known to regulate developmentalprocesses contain a structurally conserved 61-amino acidDNA-binding region called the homeodomain (1). Geneticand biochemical analyses of members of the Hox family ofhomeodomain proteins have indicated that their distinct de-velopmental functions (2) are mediated principally throughtheir DNA-binding regions (1). However, it has also beensuggested that Hox proteins bind to a similar core DNAsequence with only modest preferences for flanking nucleo-tides (3-8). Additionally, recent evidence suggests that phys-ical interaction with other homeodomain proteins can increasethe DNA-binding affinity of some Hox proteins but does notappear to change their binding specificities (9-12). Hence, acentral paradox remains: How can a family of transcriptionalregulatory molecules, related by virtue of a structural domainthat ensures similar target specificities in vitro achieve theirdiverse functional specificities in vivo?Attempts to define the functions of Hox proteins have relied

substantially on the use of global promoters to induce oftencomplex phenotypes in ectopic sites (for review, see ref. 13).The characterization of promoter elements that direct region-specific patterns of Hox gene expression in transgenic miceprovides a more precise method of altering Hox expression insites where these genes normally operate (for example, seerefs. 14-17). A major advantage of using restricted promotersis that it increases the likelihood, although does not ensure,that the phenotypic consequences of altered expression reflectthe action of a Hox protein on its natural targets. This"limited" gain-of-function approach could facilitate the dis-section of the basis of functional specificity of Hox proteins invivo, particularly if two proteins with opposing functions thatoperate on the same structure could be analyzed.With a view toward establishing a system to dissect func-

tional specificity of Hox proteins in vivo, we chose to examinethe effects of Hoxa-4 and Hoxc-8 on the development of ribs,which are primary morphological determinants of regional-ization within the vertebral column (18). Overexpression of

Hoxa-4 driven by its own transcriptional control elementssuppresses the development of ribs in the cervical region of thevertebral column (19). That rib suppression is a normalfunction of Hoxa-4 is supported by the analysis of null mutantsthat do not express Hoxa-4 in which cervical ribs are inducedto grow (19). Conversely, overexpression of Hoxc-8 under thecontrol of its own (unpublished data) or a heterologouspromoter induces rib growth (20). Curiously, Hoxc-8 nullmutants also show induction of rib growth (21), suggesting thata precise level of Hoxc-8 is required to support normal growthof ribs. Since overexpression of Hoxa-4 suppresses and Hoxc-8induces rib growth, the identification of these diametricallyopposed activities could create an opportunity to begin todissect the structural basis of functional specificity of mam-malian Hox proteins in vivo. For example, one could readilydetermine whether a homeodomain swap would be sufficientto convert the rib suppressor function of Hoxa-4 into a ribinduction function of Hoxc-8.

MATERIALS AND METHODSThe Hoxa-4 transgene consisted of a 9.6-kb region of theHoxa-4 locus extending from a KpnI site (22) located 7.6 kbupstream of the transcriptional start site to a HindIII site 55 bpdownstream of the poly(A) addition signal (23). Promoterelements contained in the Hoxa-4 upstream region used in thisconstruct have been shown to be sufficient to direct a patternof gene expression that parallels the endogenous Hoxa-4 gene(17, 22). A 39-bp oligonucleotide tag was inserted at an NsiIsite 62 bp upstream of the poly(A) addition signal to facilitatedetection of the Hoxa-4 transgene mRNA. The same 7.6-kbHoxa-4 promoter region was used to direct expression ofHoxc-8 by adding an additional upstream 3.6-kb KpnI fragmentto p1431 (20) to create a Hoxc-8 transgene predicted to beexpressed in the same pattern as the Hoxa-4 transgene. Thechimeric Hoxa-4(c-8hd) construct was isogenic with the Hoxa-4transgene except that the 61-amino acid homeodomain ex-tending from a glutamic acid residue at position 179 throughthe histidine at position 239 were replaced with the corre-sponding residues of the Hoxc-8 homeodomain by PCR am-plification of the Hoxc-8 homeobox using primers with Hoxa-4-specific overhangs. The entire protein coding region of theresulting construct was sequenced to ensure that the predictedchimeric polypeptide was correctly encoded. The transgeneswere injected into single-cell FVB/N embryos as described (20).

Skeletons of newborns were stained with alizarin red S andalcian blue as described (20) and scored for morphologicalmarkers. Left and right sides of each vertebra were scoredindependently for bilateral markers. Cervical vertebrae weredisarticulated for analysis. Rib anlagen on C7 were identifiedas medial projections of cartilage from the transverse processthat showed evidence of ossification by alizarin red S staining.

Abbreviation: TA, tuberculum anterior.ITo whom reprint requests should be addressed at: Department ofVirology, Jerome H. Holland Laboratory, Rockville, MD 20855.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 93 (1996) 9637

Ossified rib anlagen were measured using video imaging andlengths were expressed as the percentage of the total distancebetween the transverse process and the centrum to control fordifferences in the overall size of each vertebrae. For example,a rib anlage that extended from the transverse process almostto the centrum as in Fig. 1H had a length of 80%. Specificmorphological changes were confined to C5, C6, and C7.Nonspecific morphological changes observed at varying levelsabove background in all three sets of transgenic newbornsincluded malformation of the dorsal neural arch on C2 andfusion of basioccipital and exoccipital bones. Similar changeshave been observed in several other Hox and non-Hox gain-and loss-of-function transgenic mice and will be describedelsewhere.The phenotypic effects of each transgene on costal struc-

tures were confirmed by analyzing the skeletons of newbornsrepresenting at least three independent transgene integrationsites. All three transgenes induced perinatal lethality in thefounder generation. Both independently derived founder an-imals and offspring of genetically mosaic founders died gen-erally within 24 h of birth and were retained for skeletalanalyses. Consistent with a previous report (22), it was possibleto derive, at a low frequency, hemizygous lines that expressedthe Hoxa-4 transgene. A total of 22 Hoxa-4, 18 Hoxc-8, and 18Hoxa-4(c-8hd) transgenic newborn skeletons and 30 nontrans-genic littermates, which served as controls, were scored.

Serial sections of formalin-fixed paraffin-embedded 12.5-daygestation nontransgenic and transgenic embryos were probedwith either an 35S-labeled RNA probe specific for Hoxa-4 (23)that detects expression of endogenous Hoxa-4, as well as theHoxa-4 transgene, or with transgene-specific oligonucleotides:Hoxa-4 transgene expression alone was detected with a oligonu-cleotide probe, 5'-TGT TTCAGGTTCAGG GGGAGG TGTGGG AGG AAG CTT GCA-3', specific for the inserted tagtailed with 35S-labeled dATP as described (20). Hoxa-4(c-8hd)transgene expression was detected using the oligonucleotideprobe 5'-CTTGGTGTTGGGAAG TTTGTT CTC CTTTTTCCA CTT C-3' homologous to the 3' junction between theHoxc-8 homeobox and Hoxa-4. Italicized bases are specific for

Hoxa-4. Immunohistochemistry to detect Hoxc-8 was per-formed using anti-Hoxc-8 polyclonal antibodies essentially asdescribed (24).

RESULTS AND DISCUSSIONOur analyses of the phenotypic consequences induced by theoverexpression of Hoxa-4 driven by its own transcriptionalregulatory elements showed strong rib suppressor activity incervical vertebrae (Fig. 1). Suppression of ribs on C7 wasobserved, confirming the results of Horan et al. (18). Analysesof the costal components of other cervical vertebrae extendedthis observation and revealed suppressor activity on C6 (Fig.1) and C5 (data not shown) as well. Mammals typically do nothave articulating ribs on cervical vertebrae (19). However, ribhomologues are present and contribute to the formation of theventral border of the bilaterally symmetric transverse foraminaon C2 through C6 (25). On C6, the ventral border of thetransverse foramina bear a projection termed the tuberculumanterior (TA) (Fig. 1A). Together, the transverse foraminaand the TA play a critical role in guiding the vertebral artery,which is one of the major vessels that supplies blood to thebrain, through the cervical spinal column (26). In Hoxa-4transgenic newborns, the base of the TA was completely absentin 36% of cases (n = 44) (Fig. 1B). The lack of completepenetrance could be attributed to the variable levels of trans-gene expression among the individual Hoxa-4 transgenic ani-mals. Absence of these structures was never observed incontrol FVB animals (Fig. 1A), demonstrating that costalderivatives on C6 are the targets of Hoxa-4-induced suppres-sion. Suppression of costal growth was also seen in an adjoin-ing vertebra, C7 (Fig. 1F). Small rib anlagen appear on C7 (Fig.1E) with variable frequencies in different strains of mice: innewborn FVB mice the incidence was 48%. Such plasticity atmorphological boundaries is not uncommon and suggests thatthere may be genetic factors that can affect the developmentaloutcome. The incidence of rib anlagen in newborn Hoxa-4transgenic mice was decreased from the 48% observed incontrol FVB mice to only 6%. The phenotypic effects on C6

FIG. 1. Costal growth and suppression on C6 and C7 vertebrae. Negative images of alizarin red S- and alcian blue-stained vertebrae fromnewborn animals are shown. The posterior aspect is shown in all panels except D. (A) C6 from an FVB animal. (B) C6 from a Hoxa-4 transgenicnewborn. Note the complete absence of the tuberculum anterior on the right side (denoted by X). The small piece of floating ossified materialon the right is connected to the transverse process by a thin piece of cartilage not visible in this image. (C) C6 from a Hoxa-4(c-8hd) transgenicnewborn. Note the complete absence of the tuberculum anterior on the right side (denoted by X). (D) Anterior aspect of C6 from a Hoxc-8 transgenicnewborn. Note the rib-like appearance of the ventrolateral region of the foramen on the right side. The thick arrow parallels the region of rib-likegrowth. The anterior aspect is shown in this panel to reveal the presence of a cartilaginous rib head that articulates with the TA on the anteriorface of the vertebra (arrowhead). In this view, the cartilaginous portion of the TA points down into the plane of the figure and is, therefore, lessapparent than inA-C, where it projects upward and out of the plane of the figure. (E) C7 vertebra from an FVB newborn with a typical rib anlageattached to the transverse process on the left. (F) C7 from a Hoxa-4 transgenic newborn. Note the absence of rib anlagen. (G) C7 from aHoxa-4(c-8hd) transgenic newborn with a small rib anlage present on the left side. (H) C7 from a Hoxc-8 transgenic newborn showing extensiverib anlagen on both sides. f, Transverse foramina; r, rib anlagen; ta, tuberculum anterior.

Developmental Biology: Sreenath et al.

9638 Developmental Biology: Sreenath et al.

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FIG. 2. Expression of the transgenes in cervical and upper thoracic regions of 12.5-day gestation embryos. Brightfield (A) and darkfield (B) viewsof a control FVB embryo hybridized with an RNA probe specific for Hoxa-4 (29). Brightfield (C) and darkfield (D) views of a Hoxa-4 transgenicembryo hybridized with the same probe as in A and B. Brightfield (E) and darkfield (F) views of a Hoxa-4(c-8hd) transgenic embryo hybridizedwith an oligonucleotide probe specific for the Hoxa-4(c-8hd) transgene mRNA. (G) Wild-type embryo stained with the same anti-Hoxc-8 antibody.(H) Hoxc-8 transgenic embryo stained with an anti-Hoxc-8 polyclonal antibody and visualized using diaminobenzidine as a substrate for horseradishperoxidase as in G. C6, neural arch of sixth cervical prevertebra; C7, neural arch of seventh cervical prevertebra; lu, lung; rb, rib blastema; Ti,neural arch of first thoracic prevertebra; T5, T6, and T7, fifth, sixth, and seventh thoracic prevertebra, respectively.

and C7 perfectly correlate with the sites of highest prevertebralexpression of the Hoxa-4 transgene in C6 and C7 rib blastemas(Fig. 2 C and D). Since the rib blastemas give rise to the costalstructures (27), these results suggest that the observed effectsare a direct consequence of overexpression of Hoxa-4.Having confirmed that Hoxa-4 could suppress rib growth on

C7 (18) and established that it could have a similar effect onC6, we asked whether the Hoxc-8 rib-inducing function (20)could operate on the same vertebrae by using the same Hoxa-4transcriptional regulatory elements to drive the expression ofthe Hoxc-8 coding region. Using a combination of in situhybridization (data not shown) and immunohistochemicalanalyses (Fig. 2H), the expression pattern of the Hoxc-8transgene was shown to be indistinguishable from that of theHoxa-4 transgene. Although Hoxc-8 expression is not normallyseen in cervical prevertebrae but is detected in developingthoracic ribs, we reasoned that moving its anterior expressionboundary to include cervical rib blastemas would be likely tohave a phenotypic consequence. Examination of skeletonsfrom Hoxc-8 transgenic newborns revealed that the rib growth-

promoting activity of Hoxc-8 can function well in the cervicalregion of the axial skeleton. In the Hoxc-8 transgenic new-borns, 31% (n = 36) of the C6 transverse foramina werealtered in appearance as a result of distinctively rib-likecharacter of the costal component (Fig. 1D). In the majorityof cases, the rib-like structure developed a cartilaginous headon either proximal or distal end or both, strongly suggestingthat the part of a differentiation program typical of thoracicribs had been imposed on costal derivatives on C6 (Fig. 1D).A similar effect-was also seen in C5 transverse foramina (datanot shown) although at a lower frequency, consistent with thelower level of transgene expression observed in C5 comparedwith C6 rib blastemas (Fig. 2). Analyses of C7 vertebrae fromHoxc-8 transgenic newborns further substantiated the rib-inducing activity of Hoxc-8. The incidence of rib anlagen innewborn Hoxc-8 transgenic mice was increased from the back-ground level of48% to 91%. In addition, the average length oftheribs was also increased more than 3-fold (Fig. 1H). Thus, theeffects on C6 and C7 demonstrated that the Hoxc-8 transgenecould induce rib growth in this cervical region.

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Proc. Natl. Acad. Sci. USA 93 (1996) 9639

The opposing effects on ribs induced by overexpression ofHoxa-4 and Hoxc-8 using the same promoter in transgenic miceprovided an opportunity to ask whether the homeodomainplays a central role in providing the specificity. To address thisquestion, a chimeric Hoxa-4 gene was constructed in which the183-bp homeobox encoding the homeodomain within theHoxa-4 transgene was precisely replaced with that of Hoxc-8.The chimeric transgene, called Hoxa-4(c-8hd), was expressed inthe same pattern as the Hoxa-4 and Hoxc-8 transgenes (Fig. 2E and F). If the homeodomain is the principal mediator offunctional specificity, one might expect a chimeric Hoxa-4protein with a Hoxc-8 homeodomain to behave like Hoxc-8and induce rib growth. Surprisingly, skeletal analyses of new-born Hoxa-4(c-8hd) transgenic animals revealed a pattern ofcostal suppression on C6 and C7 similar to that seen in Hoxa-4transgenic newborns. The ossified base of the TA on C6 wascompletely absent in 39% (n = 36) of cases in newbornscarrying the chimeric gene (Fig. 1C). Loss of this structure wasnot observed in control animals (Fig. 1A). The incidence of C7rib anlagen in mice carrying the chimeric transgene wasdecreased by 31% relative to control animals, and the averagelength of the anlagen was decreased by 47% (Fig. 1G).Although the rib suppression effect of the chimeric gene on C7was not as strong as that of the Hoxa-4 transgene, the reductionin both incidence and length is clear and is directly opposite tothe rib inducing effect of the Hoxc-8 transgene.Our observations demonstrate that although the homeodo-

mains of Hoxa-4 and Hoxc-8 diverge substantially, differing at21 of 61 amino acid positions (1), the Hoxc-8 homeodomaincan functionally substitute for that of Hoxa-4 in vivo. Regula-tory specificity of Hoxa-4 in vertebral patterning must then belargely dictated by regions other than the homeodomain.Extending from this observation, one would predict that thecomplementary chimera, in which the Hoxc-8 homeodomainwas precisely replaced with that of Hoxa-4, would retainHoxc-8 activity. A truly reciprocal experiment would involveexpression of such a Hoxc-8(a4hd) chimeric protein in thenormal domain of Hoxc-8 action and to compare its effectswith those induced by overexpression of wild-type Hoxc-8 andHoxa-4. This approach may now be feasible, given the recentidentification of cis-acting elements capable of directing apattern of gene expression that faithfully mimics the complexbiphasic pattern of the endogenous Hoxc-8 gene (28).Although several analogous swap experiments in fly Hox

genes have indicated that regulation of segment identity trackswith the homeodomain (29-32), results that conflict with thisgeneral conclusion have also been reported (33). For example,a chimeric protein in which the homeodomain of Antennape-dia (Antp) has been replaced with the corresponding regionfrom Sex Combs Reduced (Scr) no longer behaves like theparental Antp protein but instead causes changes in segmentidentity characteristic of Scr (31, 32). On the other hand, achimeric protein in which the Ultrabithorax (Ubx) homeodo-main has been replaced with that of Antp alters segmentidentity like Ubx and not like Antp (33). This apparentdiscrepancy is complicated by the fact that analyses of thefunctional specificity of fly homeodomain proteins have beenbased almost exclusively on the interpretation of the effects oftransient expression of heat shock-induced wild-type andmutant proteins in ectopic body sites, acting through mecha-nisms that are sometimes known to be indirect. Whether thephenotypic changes induced by such transient ectopic expres-sion reflects the utilization of normal developmental pathwaysis not entirely clear. A case in point involves the pair-rule genefuzi tarazu (ftz), where a homeodomain deletion mutant has beensuggested to be capable of carrying out several regulatory func-tions of the wild-type protein (34, 35). However, unlike wild-typeFtz, the homeodomain-deleted protein completely fails to rescueftz null mutants when expressed under the control of its ownpromoter (36), demonstrating that the homeodomain is indis-

pensable for normal ftz function. Studies of Hox homeodomaindeletion mutants in flies have also demonstrated that a home-odomain is essential for activity in vivo (32).We have studied the significance of the homeodomain in

functional specificity by employing a limited gain of functionapproach to express a chimeric Hox protein within the normalspatial and temporal domains of action of the parent protein.This is an important and distinguishing feature of our ap-proach, since it provides the chimeric protein the opportunityto interact with the same targets and, ultimately, to modify thesame structures that are regulated by the parent protein.Furthermore, the opposing effects of Hoxa-4 and Hoxc-8 onrib growth simplifies the interpretation of the effects of thechimeric protein. Although our observations demonstrate thatthe homeodomain of Hoxc-8, in the context of the remainderof the Hoxa-4 protein, can substitute for the homeodomain ofHoxa-4 in mediating rib suppression, it is possible that thechimeric protein may be unable to perform other functionsmediated by the Hoxa-4 homeodomain that do not have ameasurable phenotypic outcome.Although it has been suggested that some homeodomain

proteins bind to DNA as monomers (37), a growing body ofevidence points to the interaction of these proteins with eachother as well as with nonhomeodomain partners as a means ofmodulating DNA-binding (for review, see ref. 38). In partic-ular, recent studies have suggested that interactions of Hoxproteins with a divergent homeodomain protein called Pbxl inmammals and Extradenticle (Exd) in Drosophila could providea mechanism to alter the affinity of Hox proteins for certaintargets (38). These proteins appear to increase the DNA-binding affinity of Hox proteins by inducing cooperativebinding in vitro. Protein-protein interactions mediated in partby a conserved hexapeptide motif that lies just N-terminal tothe homeodomain have been implicated in this cooperativity(11, 12,39). However, while interaction with Pbxl may alter theDNA-binding affinity of Hox proteins that bear the conservedmotif, it may not be a principal means of achieving targetspecificity among the 38 members of the mammalian Hoxfamily since 23 genes, including Hoxa-4 and Hoxc-8, encode aconserved motif. It is therefore likely that other molecularpartners interacting in nonconserved regions outside of thehomeodomain will play a critical role in modulating functionalspecificity in vivo.The problem of achieving functional specificity in mammals

is particularly vexing, given that two duplication events haveincreased the number of Hox gene clusters from one ininvertebrates to four in mammals (for review, see ref. 40).Within this expanded gene family, 13 distinct paralogousgroups can be distinguished largely on the basis of the con-served amino acid sequences of their homeodomains (41).Comparison of gain of function effects of two paralogs hasidentified unique activities for each gene although their ho-meodomains are 98% identical in sequence (42). Loss offunction studies comparing the functions of paralogs have ledto similar conclusions (43, 44). Thus, with the results reportedhere, these observations raise the possibility that mechanismsfor generating functional specificity of Hox proteins that areless dependent on the homeodomain have evolved. The utilityof new genes that arose by duplication of Hox clusters inproviding the developmental plasticity to evolve more complexbody plans may have depended on their ability to acquire newregulatory specificities and hence new functions. This could beachieved, at least in part, by retaining similar DNA bindingspecificities by conserving the homeodomain, while allowingother regions of the protein to diverge to the point where theycould interact with different cofactors to achieve distinctdevelopmental outcomes. The results presented here demon-strate that specificity of Hox proteins in mammals can lieoutside of the homeodomain and provides an opportunity to

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9640 Developmental Biology: Sreenath et al.

systematically dissect the regions required to mediate theireffects in vivo.

We gratefully acknowledge the encouragement and support of Dr.Gilbert Jay. We thank Lien Ngo for expert technical assistance and Dr.Alexander Awgulewitsch for providing the Hoxc-8 antibodies. Thisresearch was supported by a National Institutes of Health grant toC.J.B. (HD27943) and a National Research Service Award to R.A.P.

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