Development 128, 3197-3207 (2001) 3197 Printed in Great Britain © The Company of Biologists Limited 2001 DEV4556

Functional specificity of the Hoxa13

Yuanxiang Zhao and S. Steven Potter* Division of Developmental Biology, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45224, USA *Author for correspondence (e-mail: [email protected])

Accepted 30 May 2001

SUMMARY

To better define Abd-B type homeodomain function, to test dominant Hoxa13 function. The uterus, in particular, models that predict functional equivalence of all Hox showed a striking homeotic transformation towards cervix/ and to initiate a search for the downstream targets of vagina, where Hoxa13 is normally expressed. chips Hoxa13, we have performed a homeobox swap by replacing were used to create a molecular portrait of this tissue the homeobox of the Hoxa11 gene with that of the Hoxa13 conversion and revealed over 100 diagnostic gene gene. The Hoxa11 and Hoxa13 genes are contiguous Abd- expression changes. This work identifies candidate B type genes located at the 5′ end of the HoxA cluster. The downstream targets of the Hoxa13 gene and demonstrates modified Hoxa11 allele (A1113hd) showed near wild-type that even contiguous Abd-B have functional function in the development of the kidneys, axial skeleton specificity. and male reproductive tract, consistent with functional equivalence models. In the limbs and female reproductive Key words: Hox, Hoxa13, Hoxa11, Homeobox, Functional tract, however, the A1113hd allele appeared to assume specificity, Abd-B, Downstream targets, Mouse

INTRODUCTION by altered cell proliferation rates (Duboule, 1995). They are viewed as the result of perturbations of cell proliferation There remain important questions concerning the functional patterns, rather than changes in segment identity. The specificity of the mammalian encoded . functional equivalence model is consistent with the observed What are their functional relationships? Do they regulate the common DNA recognition sequences of homeodomain same, overlapping, or completely distinct sets of downstream proteins identified by in vitro DNA-binding assays (Desplan et targets? The clustered homeobox (Hox) genes encode al., 1988; Hoey and Levine, 1988), and with the ability of Hox transcription factors with the DNA binding homeodomain. In genes to sometimes act as oncogenes (Blatt et al., 1988; Blatt Drosophila, mutations of these genes can result in remarkable and Sachs, 1988; Perkins and Cory, 1993; Raza-Egilmez et al., homeotic transformations of body parts, suggesting distinct 1998; Care et al., 1999). Furthermore, in several cases, Hox patterning functions. In mammals, however, Hox mutations downstream targets have now been shown to be involved in the generally result in more-subtle phenotypes, with reduced or regulation of cell proliferation (Care et al., 1996; Bromleigh absent structures. This has prompted some to propose a and Freedman, 2000; Raman et al., 2000). difference in Hox gene function in mammals and flies. It has The evidence arguing for functional equivalence of been stated that ‘even a wing to haltere transformation cannot paralogous mammalian Hox genes is particularly strong. be explained by solely differential proliferation of the same cell Paralogs reside at equivalent positions within different Hox types. Conversely, the majority of transformations observed (so clusters and are derived by evolutionary gene duplication from far) in mutant mice are unlikely to derive from the selection of a single ancestral Hox gene. They generally encode very alternative developmental programs: they may not even engage similar homeodomains, and show overlapping the regulation of qualitatively different target genes.’ (Duboule, patterns. Gene targeting experiments have shown striking 1995), and ‘a “horizontal” scheme may be at work, in which functional redundancy for paralogs. For example, mice mutant developmental decisions are taken by integrating quantitative for either Hoxa11 or Hoxd11 show no kidney defects and only inputs rather than by relying on genes’ individuality’ (Duboule, mild limb defects (Small and Potter, 1993; Davis and Capecchi, 2000). In other words, quantity of mammalian Hox gene 1994). But mice mutant for both these paralogs show absent or expression may be more important than quality. In its extreme rudimentary kidneys, and have the ulna and radius of the form, this model states that all mammalian Hox genes forearm reduced to less than one tenth of normal size (Davis regulate identical or functionally equivalent downstream cell et al., 1995). In addition, a Hoxd11-expressing transgene has proliferation target genes. According to this model, the been shown to be able to rescue Hoxa11 loss of function apparent axial segment homeotic transformations sometimes (Zakany et al., 1996). Furthermore, complete coding sequence observed in Hox mutant mice are interpreted as resulting from exchanges between the Hoxa3 and Hoxd3 paralogs have changes in the sculpted shapes of the vertebral bones caused indicated that their proteins carry out identical biological 3198 Y. Zhao and S. S. Potter functions (Greer et al., 2000). These results have been mammalian Hox genes are less pronounced than proposed by interpreted to support the functional equivalence model the mammalian Hox functional equivalence model. Perhaps the (Duboule, 2000). Drosophila HomC genes are not each functionally unique, and Although it has been argued by some that the Drosophila perhaps the mammalian Hox genes are not all functionally the paradigm may not apply to mammalian Hox gene function, it same. is nevertheless useful to consider what has been learned from To address multiple issues of homeodomain function, that system. In Drosophila there is one homeotic complex including the question of functional specificity, we performed (HomC) of genes, which represents a single split cluster. a mammalian homeobox swap experiment. The homeobox of Ectopic expression experiments have shown that different the Hoxa11 gene was precisely replaced with that of the HomC genes can induce very distinct developmental destinies. Hoxa13 gene. The Hoxa11 and Hoxa13 genes are closely For example, misexpression of Antennapedia can cause related Abd-B type genes. In a broad sense they can be imaginal disc cells that would normally form antennae to considered paralogs, as they are derived from a common instead give rise to legs, protruding from the head (Schneuwly ancestral Abd-B Hox gene. The functional equivalence model et al., 1987). And misexpression of Ultrabithorax can result in proposes that these two genes have identical or functionally the homeotic transformation of wing into haltere (Lewis, equivalent downstream targets, and therefore predicts that the 1982). The combinatorial code expression of HomC genes has swapped allele would have wild-type function. This was been proposed to determine segment identity (Lewis, 1978). indeed observed in the developing kidneys, male reproductive The apparently distinct functional specificities of different tract and axial skeleton. Striking mutant phenotypes were HomC genes have several sources. First, the encoded seen, however, in the limbs and female reproductive tract. Of homeodomains are not functionally equivalent. Homeobox particular note, in mice with the swapped allele, the uterus swap experiments in Drosophila show that developmental underwent a homeotic transformation towards cervix/vagina, function often tracks with the homeobox, suggesting the as determined by both histology and gene chip analysis of presence of in vivo target sequence binding specificity not gene expression profiles. This homeotic transformation detected by in vitro DNA-binding assays (Kuziora and indicates a patterning function for Hox genes. The altered McGinnis, 1989; Gibson et al., 1990; Mann and Hogness, gene expression profiles identify candidate Hoxa13 1990). Second, target specificity is influenced by co-factor downstream targets. The results indicate that the Hoxa11- and interactions. Different Exd-HomC heterodimers, for Hoxa13-encoded homeodomains are not functionally example, have distinct DNA-binding specificities, even when equivalent, and that in some developing tissues the Hoxa11 measured in vitro (Chan et al., 1994; Mann and Chan, 1996), allele with a swapped Hoxa13 homeobox assumed Hoxa13 and there is evidence for similar gains in specificity in developmental function. mammalian systems (Popperl et al., 1995; Chan et al., 1997). Third, different HomC proteins may possess activation or repression domains, which can determine developmental MATERIALS AND METHODS function by their distinct impacts on the same target gene expression. Fourth, according to the activity regulation model, Targeting construct binding sites for specific combinations of co-factors in a given An 11 kb SpeI-SpeI A11 genomic fragment subcloned in pBS SK was promoter would confer activation or repression effects (Li et cut with NheI, releasing a 9 kb segment with both A11 exons. The al., 1999). This is consistent with the observation that original clone, with the 9 kb NheI fragment removed, was ligated to individual HomC proteins are often able to repress some target re-circularize, and loxP-PGKNeo-loxP was subcloned into the unique genes and activate others, as measured by both genetic and FseI site 1.1 kb 3′ of the second A11 exon and herpes simplex virus transfection assays (Vachon et al., 1992; Capovilla et al., 1994; thymidine kinase (mc1HSV-tK) was subcloned into the unique Sal1 Saffman and Krasnow, 1994). In summary, a number of sequence in the multiple cloning site, giving construct I. mechanisms exist in Drosophila for providing individual The released 9 kb NheI segment was subcloned into a modified pBS, HomC proteins with functional specificity. It would seem with the multiple cloning site XhoI and BSTXI sites removed, giving reasonable to suppose that similar molecular processes might construct II. This clone had unique XhoI and BSTXI sites flanking the be used in mammals. A11 second exon with the homeobox. This XhoI-BSTXI segment was subcloned into pBS, giving the A11(XhoI-BstXI) vector. Nevertheless, even in Drosophila there is considerable Two new restriction sites, HincII and PstI were introduced at the evidence for functional overlap of HomC genes. For example, junction regions of the A11 homeobox of the XhoI-BSTXI segment by misexpression of Ubx, Abd-A or Abd-B can cause cells that PCR mutagenesis (Bi and Stambrook, 1998). The primers used were: would normally form wing to form haltere instead (Casares et XhoI primer 5′ to 3′, TCCTCTGCCACCTCCCACctcgagAGAGC- al., 1996). This is particularly informative, as Abd-A and Abd- TGG; B are normally only involved in development of the abdomen, HincII primer 5′ to 3′, TCCTTCCTTAGGTGgtcaacGCACCC- which has no wings or halteres. It has also been shown that a GCAAAA; hybrid Ubx-VP16, with enhanced transcription activation (BSTXI+PstI) primer 5′ to 3′, CCCAATTccagtaggctggAG- function, mimics Antp in developmental specificity, CCTTAGAGAAGTGGATTAGCTGAGTAGTActgcagCCGGTCTC- presumably by regulating the same set of downstream targets (TGTT.....); Lowercase letters represent restriction enzyme sites, underlines (Li and McGinnis, 1999). Furthermore, several promoter indicate silent mutation sites, bold letters represent homeobox region analysis studies suggest that the HomC genes regulate and parenthesized letters represent sequence not included in the overlapping sets of downstream target genes (Manak et al., primer sequence. 1994; Mastick et al., 1995). These observations suggest that The A13 homeobox was PCR amplified from strain 129 DNA using the differences in function between the Drosophila HomC and the following primers. The Hoxa13 homeobox 3199

A13 HincII primer: ACGCCAGCTCCTgtcaacGGGGGAGAAAG- kb 3′ of the A11 gene (Fig. 1C). After electroporation into ES A; cells, correct targeting was confirmed by Southern blot, PCR A13 PstI primer: CCATTAACTAGTctgcagCCGGTCTCTGATG- and sequencing (Fig. 2). The Neo gene was removed by ACTTTTTTCTCT; breeding to Cre-expressing mice, leaving the replaced The A13 homeobox PCR product was digested with HincII and PstI homeobox and the 34 bp loxP sequence 3′ of the A11 gene. To and subcloned into the A11(XhoI-BSTXI) vector, also cut with HincII test for possible effects of the loxP insertion, we also made an and PstI, replacing the A11 homeobox. This XhoI-BSTXI segment was ′ then subcloned into XhoI, BSTXI cleaved construct II, and the 9 kb A11 allele with the loxP flanked Neo inserted at the same 3 NheI segment of the modified construct II was then subcloned back position, but without the homeobox swap. Of interest, the into NheI cut construct I, making the final targeting construct. The presence of Neo, even at this 3′ position, inactivated the A11 construct was confirmed by DNA sequencing. gene. Cre mediated removal of Neo, leaving only loxP, restored full wild-type A11 function (data not shown). Gene targeting Hoxd11−/− (D11−/−) mice provided the most sensitive The targeting constructs were introduced into ES cell lines E14.1 and measure of A11 function. The paralogous A11 and D11 genes R1 (Hooper et al., 1987; Nagy et al., 1993). Targeted clones were are functionally redundant in the development of the axial enriched by positive-negative selection and identified by Southern blot skeleton, limbs, kidneys and reproductive tracts (Davis et al., and PCR analysis. DNA sequencing confirmed the precise homeobox 1995). Mice with A11−/− or D11−/− mutations, for example, swap. Four out of 100 R1 clones and 16 out of 300 E14 clones were have fairly normal development of the kidney and forelimb properly targeted, and one of each ES cell type was used to make −/− −/− chimeras. No difference in phenotypes between the two was observed. zeugopod (ulna and radius), but in A11 D11 double The Neo marker gene was removed by mating with transgenic mice homozygous mutants the kidneys are absent or severely expressing NlsCre ubiquitously at an early embryonic stage (kindly reduced in size, and the zeugopod of the forelimb is almost provided by Dr Wojteck Auerbach). entirely missing. Histology The A11 gene with an A13 homeobox provided near Tissues were fixed in 4% paraformaldehyde overnight, dehydrated and wild type function in the development of the then embedded in paraffin. Kidneys were sectioned frontally (5 µm) kidneys, male reproductive tract and axial skeleton and stained using a Periodic Acid Schiff (PAS) kit (Sigma). Testes A11−/− D11−/− double mutant mice commonly suffer perinatal were stained with Hematoxylin and Eosin. Female reproductive tracts were sectioned and also stained with Hematoxylin and Eosin. death from kidney failure (Davis et al., 1995). The presence of one wild-type allele (A11+/− D11−/− or A11−/− D11+/−) restores Alizarin staining of adult skeletons Skeletons of 4-week-old animals were prepared and stained as previously described (Selby, 1987; Small A and Potter, 1993). HoxA 3í A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A13 5í Affymetrix gene chip analysis Uterus tissue was from the uterus horn above the B N-terminal Helix 1 Helix 2 Helix 3 uterus horn joint junction and below the uterus- A11HD: TRKKRCPYTKYQIRELEREFFFSVYINKEKRLQLSRMLNLTDRQVKIWFQNRRMKEKKIN oviduct junction, and cervix tissue was collected A13HD: G----V----V-LK-----YATNKF-T-D--RRI-ATT--SE------V-D---V from the region below the uterus corpus and above C the vaginal hymen. RNA was prepared using RNAzol reagent (Tel-test). Preparation of biotinylated RNA, hybridization, washing, staining and scanning of NotI Affymetrix GeneChip probe arrays were carried out (a13) HD* LoxP neo LoxP mc1HSV1-tK according to Affymetrix protocols. Data was analyzed with Affymetrix software. Exon 1 Exon 2 (a11) HD AATAAA

P N P N RESULTS 6.1 kb a 5.5 kb Targeting construct b N P P N We performed a homeobox swap experiment to 4.1kb Cre transgenic mice examine the functional specificity of Abd-B c 7.3 kb type homeoboxes, in order to better define their normal developmental function and to search d for candidate downstream targets. The Abd-B type Hoxa11 (A11) and Hoxa13 (A13) genes are Fig. 1. Homeobox swap strategy. (A) Organization of the genes of the HoxA cluster. located at the 5′ end of the HoxA cluster and (B) Amino acid sequence comparison of the A11 and A13 homeodomains. Dashes represent sequence identity. (C) The targeting construct at the top consisted of the encode homeodomains identical at 36 of 60 A11 gene with a substituted A13 homeodomain (HD*, black rectangle), inserted loxP amino acids (Fig. 1A,B). The homeobox swap flanked Neo and HSV thymidine kinase gene. Double homologous recombination, as was made with a replacement targeting shown, results in homeobox swap and insertion of loxP flanked Neo. Mating with construct consisting of the A11 gene with a transgenic Cre mice removes Neo, leaving a single 34 bp loxP. PCR genotyping used precise A13 homeobox replacement. A Neo primers a-d, and Southern blot genotyping used restriction sites (P) PstI, (N) NheI gene flanked by loxP sequences was placed 1.1 and DNA segment probes (open lines and solid rectangles). 3200 Y. Zhao and S. S. Potter

The homeobox swapped A1113hd allele also provided wild- type function in the development of the male reproductive tract. A11−/− D11+/+ males are generally sterile with undescended testes and a ductus deferens that is anteriorized to resemble the epidydimis (Hsieh-Li et al., 1995). A1113hd/13hd D11+/+ and A1113hd/− D11+/− males were fertile, with descended testes and mature sperm in seminiferous tubules (Fig. 4A), and their ductus deferens lacked the tortuosity typical of the anteriorized ductus deferens of the A11−/− D11+/+ mouse (Hsieh-Li et al., 1995; Fig. 4B). A11−/− D11+/+ mutants show posteriorization of the 13th thoracic segment into the first lumbar, and anteriorization of the first sacral segment to a sixth lumbar (Small and Potter, 1993). Interestingly, in all six A1113hd/13hd D11+/+ mice examined the axial skeleton was normal, without anteriorization or posteriorization (data not shown). Fig. 2. Southern blot and PCR analysis. (A) Southern blot analysis of In summary, the A1113hd allele provided apparent wild-type targeted ES cell lines. The positions of external probe and internal function in the development of the kidney, male reproductive probe used for screening targeted ES cell lines are indicated in Fig. tract and axial skeleton, confirming that the targeted allele 1c. Targeted cell lines were confirmed by two different restriction digestions (P) PstI, and (N) NheI, generating a 6.1 kb wild-type band produces functional mRNA and protein. These observations and 4.1 kb targeted band, and 5.5 kb wild-type band and 7.3 kb are consistent with models predicting that all Hox-encoded targeted band, respectively. (B) PCR identification of the swapped proteins bind identical or functionally equivalent downstream homeobox encoding the A13 homeodomain (HD) using primers a targets. However, in examining the limbs and female and b (indicated in Fig. 1C). PCR amplified fragments were digested reproductive tracts, quite different results were obtained. with PstI, HincII (HcII) and EcoRV (RV). Only the swapped A13 PCR fragment was cut by all three enzymes to give distinguishable The A1113hd allele provided antagonizing function in smaller sized bands. (PstI, 480, 354 and 126 bp; HincII, 480, 323 and the development of the limbs 157 bp; EcoRV, 480, 260 and 220 bp). The presence of the precise In the developing hindlimb, the A1113hd allele antagonized homeobox swap was confirmed by sequencing. (C) PCR identification of Cre recombination. Cre-expressing transgenic mice normal A11, D11 function. The A11 gene is expressed in the were used to remove the Neo gene in the targeted A11 locus. A11 zeugopod (tibia and fibula in the hindlimb), while the A13 gene specific primers c and d (indicated in Fig. 1C) did not amplify the 2 is expressed more distally, in the autopod (paw; Yokouchi et kb (loxpNeoloxp) fragment under the PCR conditions used, but gave al., 1991; Haack and Gruss, 1993; Small and Potter, 1993). At a 317 bp fragment with loxp, or a 285 bp fragment without loxp least five mice were examined for each of the following (wild type). wt, wild type; M, pBR322 DNA-MspI marker. genotypes: A1113hd/+ D11+/+, A1113hd/+ D11+/−, A1113hd/+ D11−/−, A1113hd/− D11+/+, A1113hd/− D11+/−, A1113hd/− D11−/− and A1113hd/13hd D11+/+. The A1113hd allele gave more severe near normal kidney development and survival. To define the − +/− +/+ function of A1113hd in kidney development, survival rates of phenotypes than A11 . The A11 D11 hindlimb was the A1113hd/− D11−/− mice were determined and kidney histology same as wild type on the genetic background used in these studies. In the zeugopod region of the hindlimb, the A1113hd/+ examined. A control cross between double heterozygous +/+ +/− +/− D11 mutants showed a distinct separation of the distal tibia A11 D11 mice gave 278 progeny, of which only eight −/− +/+ −/− −/− and fibula, similar to that seen in the A11 D11 mice, and (2.88%) A11 D11 pups were found. Predicted additional 13hd/13hd +/+ double mutants presumably died shortly after birth and were the A11 D11 hindlimbs showed an even more −/− −/− pronounced separation (Fig. 5A). In additional allele eaten. Only two (0.72%) A11 D11 mice survived to − postnatal day (P) 30. By contrast, a cross between A1113hd/+ combination genotypes, a substitution of the A11 allele with 13hd D11+/− and A11+/− D11+/− produced 280 pups of which 19 the A11 allele consistently resulted in more severe (6.79%) were A1113hd/− D11−/−, with 14 (5%) surviving to P30. separation of the distal tibia and fibula (Fig. 5B and data not shown). In the autopod of the hindlimb, the talus and calcaneus This compared well with the Mendelian predicted percentage − − of 6.25%. The A1113hd allele therefore restored near normal bones of A11 / D11+/+ mice appeared normal, whereas for survival. Furthermore, the A1113hd/− D11−/− kidneys appeared A1113hd/13hd D11+/+ mice the talus was malformed (not shown) grossly normal, except for the reproducible presence of an and the calcaneus truncated to the length of the talus (Fig. 5C). indentation in the anterior region of the left kidney (Fig. 3A), All allele combinations with at least one A1113hd showed and with one pair of kidneys, of eight pairs examined, having calcaneus truncation, whereas in the absence of A1113hd the small cysts visible on the surface (data not shown). calcaneus appeared normal in all allele combinations, except − − − − Histologically the A1113hd/− D11−/− kidneys were also near for A11 / D11 / , in which the calcaneus was shortened and wild type, with fewer dilated distal tubules and more distinct also fused with the fibula (data not shown). The penetrance of proximal tubule lumens than observed in A11−/− D11−/− the truncated calcaneus phenotype increased with increasing kidneys (Fig. 3B). However, the medulla layer of kidney was dosage of A1113hd and to a lesser extent, A11− and D11− alleles. as severely reduced in size and disorganized in the A1113hd/− For example, the truncated calcaneus was seen in 1/14 (7%) of D11−/− mutant as in the A11−/− D11−/− mutant when compared A1113hd/+ D11+/+, in 4/12 (33%) of A1113hd/− D11+/+, in 8/11 with wild type (Fig. 3B). (72%) of A1113hd/− D11+/− and in 10/10 (100%) of A1113hd/− The Hoxa13 homeobox 3201

D11−/− mice. All mice with two A1113hd alleles showed the forelimb development. Although the A1113hd/+ D11+/+ truncated calcaneus phenotype. forelimb was normal, the A1113hd/13hd D11+/+ forelimb was The A1113hd allele also had antagonizing effects on more severely malformed than the A11−/− D11+/+ forelimb. In the zeugopod region, the ulna and radius were about one half of normal length, somewhat resembling the three allele null mutant A11−/− D11+/−, although with distinctive shapes (Fig. 6A). The styloid apophyses, which were only very mildly affected in A11−/− D11+/+ mutants were reduced and/or fused to the ulna and radius in A1113hd/13hd D11+/+ mice (Fig. 6B, small arrows), approaching the severity of the A1113hd/− D11−/− or A11−/− D11−/− mutants (Fig. 6B, large arrows). In addition, similar to the hindlimb zeugopod, a substitution of the A11− allele with the A1113hd allele in other allele combinations generally resulted in more severely shortened and malformed forelimb zeugopod (data not shown), with the exception of the A1113hd/− D11−/− mice, which had relatively more normal forelimb development than the A11−/− D11−/− mice (Fig. 6A). This exception could reflect some normal limb development function of the A1113hd allele, which remained hidden in the presence of wild- type A11 or D11 alleles. Alternatively, the null A11 mutation, with a deletion and an insertion of a PGK-Neo, may have produced subtle alterations in the expression patterns of flanking Hox genes not present with the A1113hd allele during limb development. It is interesting to note that ectopic expression of Hoxa13 or Hoxd13 in the chick zeugopod (Yokouchi et al., 1995; Goff and Tabin, 1997), or ectopic expression of Hoxd13 in the mouse zeugopod (van der Hoeven et al., 1996; Herault et al., 1997; Peichel et al., 1997) also results in shortening of this limb element, similar to the effect observed for A1113hd. The A1113hd allele assumes A13 function in development of the female reproductive tract The A1113hd allele caused a partial homeotic transformation of the uterus to the more posterior cervix/vagina. The A11 gene is expressed in the developing uterus and cervix, while expression of A13 is more posteriorly restricted, to the cervix and vagina (Taylor et al., 1997; Post and Fig. 3. The A1113hd allele provided near normal function in kidney development. Innis, 1999). Mice examined were 4.5 weeks of (A) Gross appearances of wild type, A11−/− D11−/− and A1113hd/− D11−/− kidneys. age, and estrous cycle matched (Laboratory, The A11−/− D11−/− kidneys shown are among the least affected, coming from one 1966). The lining of the wild-type uterus consists of the two mice that survived to P30. One kidney was severely reduced in size, with − − − of a single layer of columnar epithelial cells, the other more normal. The A1113hd/ D11 / kidneys appeared grossly normal while the lining of the wild-type cervix is many except for a reproducible indentation on the left kidney (arrow). (B) Kidney 13hd/− −/− cells thick, making a squamous epithelium. In histology. Top and middle panels: the A11 D11 kidneys appeared relatively −/− +/+ −/− −/− contrast to A11 D11 mice, in the normal compared with the A11 D11 kidneys, which showed many occluded 13hd/13hd +/+ proximal tubule (P) lumens (long arrows) and severely dilated distal (D) tubules A11 D11 mutants the uterine lining (arrowheads). Short arrows point to glomeruli. Bottom panels: the medulla layer of resembled that of the wild-type cervix (Fig. 7, both the A11−/− D11−/− kidney and the A1113hd/− D11−/− kidney (not shown) was arrows). This transformation extended severely reduced in size and disrupted by cysts (asterisk) when compared with the throughout most of the uterus, with the columnar wild type. C, cortex; M, medulla. to squamous transition normally present at the 3202 Y. Zhao and S. S. Potter

Fig. 4. Wild-type function of A1113hd in the male reproductive tract. (A) Sperm were present in the lumens of wild-type and A1113hd/13hd D11+/+ seminiferous tubules (arrows), but not in A11−/− D11+/+ testis. (B) The A1113hd/13hd D11+/+ ductus deferens showed a wild-type morphology, while A11−/− D11+/+ mutants showed a coiled configuration resulting from an anteriorization towards epidydimis (arrows). E, epididymis; T, testis; V, vas deferens. uterus-cervix junction in wild-type mice shifted anterior to The homeotic transformation of the A1113hd/13hd D11+/+ near the uterus-oviduct junction in A1113hd/13hd D11+/+ uterus was confirmed at the molecular level with Affymetrix mutants. The stromal layer of the mutant uterus also approximated that of the wild-type cervix, with lower cell density and more fibrous tissue (Fig. 7, asterisks). The A1113hd/13hd D11+/+ mutants also lacked uterine glands and were sterile. While A11+/− D11+/+ mice are fertile, females with even a single A1113hd allele were missing uterine glands and sterile. It was therefore necessary to carry the homeobox swapped A11 gene with the Neo insertion, which gave a null recessive phenotype, and then to remove the Neo by germline Cre activity in the last step of breeding. Because of A1113hd/+ female infertility, it was extremely difficult to make mice with the A1113hd/13hd D11−/− genotype.

Fig. 5. A1113hd hindlimb phenotype. (A) The arrowhead points to the normal tibia and fibula fusion point and broad arrow points to the calcaneus bone in wild-type hindlimb. Mice of A1113hd/+ D11+/+ or A11−/− D11+/+ genotypes showed similar more distal separation of tibia and fibula, while A1113hd/13hd D11+/+ mice showed more extreme tibia, fibula separation (thin arrows) when compared with wild type. (B) Substitution of the A11− allele by an A1113hd allele in either A11+/− D11−/− or A11+/− D11+/− mutants resulted in greater separation of tibia and fibula (arrows). (C) Ventral views of isolated autopods of hindlimbs. A1113hd/13hd D11+/+ mutants had a severely truncated calcaneus compared with wild type (arrows). A11−/− D11+/+ mutants were normal. c, calcaneus; F, fibula; T, tibia. The Hoxa13 homeobox 3203

Fig. 6. The A1113hd allele antagonizes A11 and D11 function in forelimb development. (A) In A1113hd/13hd D11+/+ mice, the zeugopod region was shortened to near half of normal length, resembling A11−/− D11+/− mice. R, radius; U, ulna. A central bulge was observed in the shortened radii of A1113hd/13hd D11+/+ and A11−/− D11−/− mice (arrowheads). The olecranon of the ulna, however, was truncated and replaced with a floating sesamoid bone in A11−/− D11−/− mice and was more wild type in appearance in A1113hd/− D11−/− mice (arrows). (B) Wrist region. The styloid apophyses of A1113hd/13hd D11+/+ mice were reduced and/or fused with the ulna and radius, while they were only very mildly affected in A11−/− D11+/+ mutants (thin arrows). The styloid apophyses were also reduced and/or fused in A11−/− D11−/− and A1113hd/− D11−/− mutants (thick arrows). gene chip probe arrays. Murine genome U74A gene chips, comparison of mutant uterus versus wild-type uterus gave 108 with approximately 12,000 genes, were used to measure gene genes with over tenfold difference. The two lists of differently expression levels in the 4.5-week-old wild-type uterus, wild- expressed genes shared 54 genes, or about half, consistent type cervix and A1113hd/13hd D11+/+ uterus. The resulting molecular fingerprints were then used to determine if the A1113hd/13hd D11+/+ uterus was shifted towards the cervix in character. The transcript profile of the mutant uterus created a detailed molecular portrait showing clear posteriorization. Over 30 genes normally expressed in the cervix but not the uterus were found transcribed in the A1113hd/13hd D11+/+ uterus. This list contained several keratin genes associated with squamous epithelium, including K16, K6α, K6β, K14 and, notably, K13, a marker of the ectocervical epithelium in the female reproductive tract (Gorodeski et al., 1990), and several other genes of interest (see Appendix). Other genes, normally expressed in the wild-type uterus and absent in cervix, were inactive in the mutant uterus, again consistent with posteriorization to cervix. This list included KAP and calbindin-28, both of which show estrogen responsive expression in the normal uterus (Meseguer et al., 1989; Gill and Christakos, 1995; Runic et al., 1996), and the decysin gene, which encodes a metalloprotease (Mueller et al., 1997). In addition to these qualitative on/off differences there were many quantitative changes in the mutant Fig. 7. Histological comparison of female reproductive tracts. (A) Wild-type uterus showed single columnar epithelial cell layer (arrow), and contained endometrial glands uterus gene expression levels diagnostic of −/− +/+ posteriorization (Appendix). Hoxa13 (G) in the stromal cell layer (asterisk). (B) The A11 D11 mutant uterus had fewer glands and a thinner stromal cell layer than wild type, but its epithelium and stromal tissue expression was not detected in either the resembled the wild-type uterus morphologically. (C) The A1113hd/13hd D11+/+ mutant mutant uterus or wild-type uterus. In total, uterus showed an absence of glands, a multi-layered epithelium and a more fibrous comparison of wild-type cervix versus wild- stromal layer, all consistent with a posteriorization to cervix. (D) The wild type cervical type uterus gave 106 genes with a tenfold or canal showed a multi-layered squamous epithelium and a fibrous stromal layer with a greater expression level difference, while lower cell density than seen in the uterus. 3204 Y. Zhao and S. S. Potter with the incomplete homeotic transformation of mutant uterus also possible that expression of the entire Hoxd13 protein to cervix observed at the level of histology. gives different developmental consequences in the kidney than expression of the A11 protein with a group 13 swapped homeodomain. DISCUSSION The homeotic transformation of uterus towards cervix/ vagina clearly indicated that in this tissue the A1113hd allele In this study, we precisely replaced the homeobox of the assumed A13 function. The Hox code model of Lewis (Lewis, Hoxa11 gene with the homeobox from the Hoxa13 gene. The 1978) predicts that Hox null mutations will result in major conclusion is that even for these two closely related Abd- anteriorizations and Hox ectopic expression will drive B type genes, the homeoboxes were not functionally posteriorizations. Null mutations of A11 and A10 have been equivalent. previously reported to anteriorize the uterus towards oviduct In the kidney, male reproductive tract and axial skeleton, (Satokata et al., 1995; Gendron et al., 1997), and mutation of the A1113hd allele provided near wild-type function, A13 anteriorizes the cervix/vagina towards uterus (Post and consistent with models predicting identical or functionally Innis, 1999). The A1113hd allele appeared to effectively give equivalent downstream target genes for all Hox encoded ectopic expression of A13 function in the uterus, causing it proteins. In the developing limbs, however, the homeobox to posteriorize to cervix/vagina, where the A13 gene is swapped A11 gene gave antagonizing function. This could normally expressed. These results suggest patterning function result from a dominant negative effect, with the A1113hd for Hox genes in the development of the female reproductive encoded protein binding to the same downstream gene tract. targets as for A11/D11, but with opposite effect (e.g. Distinct segment identity functions have also been defined repression versus activation). Alternatively, the A1113hd for Hox genes in the developing rhombomeres of the protein could bind to different targets, perhaps those of A13, mammalian hindbrain. Both misexpression (Alexandre et al., leading to distinct developmental outcome. Of interest, 1996; Bell et al., 1999) and targeted mutation (Chisaka et al., according to the ‘posterior prevalence’ rule in Drosophila, 1992; Carpenter et al., 1993; Goddard et al., 1996; Studer et Hox genes located at more 5′ positions in the Hox clusters, al., 1996; Gavalas et al., 1997; Gavalas et al., 1998; Rossel and and expressed in more posterior domains, are dominant over Capecchi, 1999) studies support a Hox code model. For more 3′ genes. This rule predicts that the more 5′ A13 gene, example, ectopic expression of Hoxb1 results in the homeotic or a homeobox swapped A11 gene with A13 function, would transformation of rhombomere 2 to rhombomere 4 (Bell et al., be dominant over a wild-type A11 gene. It has been shown 1999). These results are consistent with those described in this that ectopic expression of Hoxa13 or Hoxd13 (but not report, and are again difficult to reconcile with models Hoxa4) in the zeugopod region in the chick resulted in predicting that Hox gene function is restricted to the regulation zeugopod truncation (Yokouchi et al., 1995; Goff and Tabin, of cell proliferation. 1997). In addition, ectopic expression of Hoxd13 in the The genes altered in expression in the mutant uterus are developing zeugopod in mice also resulted in reduction of candidate downstream targets of the A13 gene. The single this limb element (van der Hoeven et al., 1996; Herault et initial difference between wild type and mutant developing al., 1997; Peichel et al., 1997), similar to what we observed uteri was the presence of the A1113hd allele. Expression of this in mutants with the swapped A11 gene. This suggests that swapped homeobox gene dramatically shifted the gene the A1113hd allele assumed Hoxa13 function in the limb and expression profile of the mutant uterus towards that of the antagonized function of the group 11 Hox genes, probably cervix/vagina. The differently expressed genes therefore using the same mechanism that controls the posterior represent the combination of direct and indirect targets of the prevalence phenomenon in normal development. It is notable A1113hd allele. Moreover, as the identity of the mutant uterus that severe zeugopod truncation was observed in the is shifted towards the cervix/vagina, where A13 is normally forelimbs but not in the hindlimbs in our mutants. This could expressed and as the A1113hd and A13 alleles encode identical be due to a quantitative insufficiency of antagonizing activity homeodomains, these genes are also excellent downstream in the hindlimb, as an additional group 11 Hox gene, target candidates for A13 itself. Many of the target genes Hoxc11, is normally expressed in the hindlimb, but not appear to have functions not related to the regulation of cell forelimb (Peterson et al., 1994; Hostikka and Capecchi, proliferation. 1998). It is interesting to note that mutation of Hoxc13, a It is interesting to note that possible antagonizing paralog of A13, gives a hairless mouse (Godwin and interactions between the paralogous group 13 and 11 Hox Capecchi, 1998). It has been suggested that ‘Hoxc13 could genes were also previously observed in kidney development. directly control transcription of hair keratin genes’ (Godwin Insertion of a Hoxd9/lacZ construct into the 5′ region of the and Capecchi, 1998). The A13 and Hoxc13 genes encode HoxD complex causes ectopic expression of Hoxd13, homeodomains identical in 55 out of 60 amino acids. resulting in kidney agenesis that resembles the agenesis found The observed increased expression of a number of keratin in mice without A11 and D11 function (Kmita et al., 2000). genes in the A1113hd/13hd uterus further indicates that the This result contrasts with our observation that the A1113hd Hox genes of the 13 paralogous group can regulate keratin allele drives near normal kidney development. The most likely genes. explanation is that the induced misexpression of Hoxd13 does It has previously been reported that the Hoxa3 and Hoxd3 not properly recapitulate normal Hox group 11 expression in encoded proteins are functionally equivalent (Greer et al., terms of cell type, timing and expression levels, therefore 2000). This added to evidence indicating strong functional perturbing rather than promoting kidney development. It is redundancy between Hox paralogs (Condie and Capecchi, The Hoxa13 homeobox 3205

1994; Davis et al., 1995; Horan et al., 1995; Fromental-Ramain Quantitative change in wild-type cervix and et al., 1996a; Fromental-Ramain et al., 1996b). In this report, A1113hd/13hd D11+/+ uterus compared with however, we show that even the homeoboxes of two contiguous expression in wild-type uterus Abd-b type Hox genes are not functionally interchangeable in Wild-type Mutant all developing tissues. cervix uterus Annexin 8 +42-fold +88-fold Krt1.10 (keratin, type I) +108-fold +56-fold APPENDIX Cellular retinoic acid binding Protein II +31-fold +55-fold Proteins present in A1113hd/13hd D11+/+ uterus and Minopontin (osteopontin like) Absent −19-fold wild-type cervix, but absent from wild-type uterus Matrix metalloproteinease 7 −55-fold −34-fold Plasminogen activator inhibitor 2 +10-fold +14-fold Keratins Osteoglycin +6-fold +29-fold K1.13, K2.6α, K2.6β, K1.14, K1.16, K2.1,K2.4 Peptidylarginine deiminase type IV +8-fold +24-fold Cornified cell envelope components Procollagen XV +5-fold +17-fold SPRR1b protein, SPRP 3, Repetin, Loricrin Complement component factor I Absent −13-fold Glucocortoid-regulated Transcription factors inflammatory prostaglandin − Sine oculis-related homeobox 1 () G/H synthase (GriPGHS) Absent 11-fold HNF-3/forkhead homolog 1 like (HFH-1l) Eotaxin precursor (Scya11) +7-fold +10-fold − − E74-like factor 5 (Elf5) Ceruroplasmin 12-fold 10-fold 14-3-3 protein sigma +11-fold +10-fold Desmosomal adhering junction proteins Small inducible cytokine subfamily B, 5 +7-fold +9-fold Small inducible cytokine subfamily D, 1 −6-fold −6-fold Plakophilin 1, Desmocollin 2 G6pd-2 +5-fold +6-fold Others Accession numbers p73H ( related protein) AA727291 +37-fold +28-fold Maspin (tumor suppressor) M32486 +6-fold +14-fold Apoptosis signal-regulating kinase 2 (ASK2) AW228162 +15-fold +13-fold Neuropsin (extracellular protease) Al854029 +17-fold +11-fold 7-dehydrocholesterol reductase (DHCR7) Al844853 Absent −11-fold Squalene epoxidase Al181346 +10-fold +8-fold Connexin 30 (gap junction protein) Al85344 −4-fold −7-fold Vinculin (cytoskeletal protein) Galectin 7 (β-galactoside-binding protein) Insulin-like growth factor binding protein (IGFBP-2) We thank Wojteck Auerbach for kindly providing nlsCre transgenic Intracellular Ca2+ binding protein (MRP8) mice. We also thank Sheila Bell, Jun Ma, Kathy Molyneaux, Larry Intracellular Ca2+ binding protein (MRP14) Patterson, William Scott and Chris Wylie for their comments on Neural visinin-like Ca2+-binding protein, type I (NVP-1) preparing this manuscript. This work was supported by a grant from Interleukin 1 , type II NIH. MHL class III regions Hsc70t gene Pgp-1 (CD44 antigen) β defensin 1 (mBD-1, host defense) REFERENCES LIM and SH3 protein 1 (Lasp-1) Squamous cell carcinoma antigen 2 (Scca2) Alexandre, D., Clarke, J. D., Oxtoby, E., Yan, Y. L., Jowett, T. and Holder, Friend virus susceptibility 1 (Fv1) N. (1996). Ectopic expression of Hoxa-1 in the zebrafish alters the fate of the mandibular arch neural crest and phenocopies a retinoic acid-induced Accession numbers phenotype. Development 122, 735-746. Al604345, Al119347, Al173973, AW259538, AA716963, Bell, E., Wingate, R. J. and Lumsden, A. (1999). Homeotic transformation AA726579, Al118078, AV233274, Al060798, AW123650, of rhombomere identity after localized Hoxb1 misexpression. Science 284, AA794189 2168-2171. Bi, W. and Stambrook, P. J. (1998). Site-directed mutagenesis by combined chain reaction. Anal. Biochem. 256, 137-140. Proteins present in wild-type uterus, but absent Blatt, C., Aberdam, D., Schwartz, R. and Sachs, L. (1988). DNA 13hd/13hd +/+ from wild-type cervix and A11 D11 uterus rearrangement of a homeobox gene in myeloid leukaemic cells. EMBO. J. Kidney androgen regulated protein (KAP) 7, 4283-4290. Calbindin-28 (Ca2+-binding protein) Blatt, C. and Sachs, L. (1988). Deletion of a homeobox gene in myeloid Decysin (disintegrin metalloproteinase) leukemias with a deletion in 2. Biochem. Biophys. Res. Melanoma antigen (Accession Number, D10049) Commun. 156, 1265-1270. Bromleigh, V. C. and Freedman, L. P. (2000). p21 is a transcriptional target B-cell leukemia/lymphoma 3 (Bcl3) of HOXA10 in differentiating myelomonocytic cells. Genes Dev. 14, 2581- Aldo-keto reductase (AKR1C13) 2586. L-Histidine decarboxylase (HDC) Capovilla, M., Brandt, M. and Botas, J. (1994). Direct regulation of KvLQT1 (K+ channel subunit) decapentaplegic by Ultrabithorax and its role in Drosophila midgut Interleukin-18 binding protein . Cell 76, 461-475. MAP kinase kinase 6c (MKK6) Care, A., Silvani, A., Meccia, E., Mattia, G., Stoppacciaro, A., Parmiani, 3206 Y. Zhao and S. S. Potter

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