Phenotype of mice lacking region VIII
Upon visual inspection, mice homozygous for the deletion of RVIII (Hoxd-11RVIIIGe2/RVIIIGe2) were indistinguishable from their wild-type littermates. However, skeletal preparations revealed that a significant proportion of them displayed a disturbed sacral morphology, with a sacrum starting one vertebra more posterior than in wild-type animals. Instead of the L6 (six lumbar vertebrae) vertebral type observed in both normal and heterozygous mice, mutant animals showed either an abnormal S1 (first sacral vertebra) in an otherwise L6 type, or a L7 (seven lumbar vertebrae) formula (Figure 2, Table I, Hoxd-11RVIIIGe2/RVIIIGe2). Thus, RVIII-deleted mice had a recessive defect in the vertebral column reminiscent of that seen in Hoxd-11 loss-of-function alleles. In these latter cases, however, the prevalent vertebral type was L7, and defects were also reported to affect limbs and male fertility (Davis and Capecchi, 1994, 1996; Favier et al., 1995).
Figure 2. Phenotypic changes at the lumbo-sacral transition in RVIII-deleted (Hoxd-11RVIIIGe2) adult mice. (A) Vertebral type observed in wild-type mice with six lumbar vertebrae (L6) and a normal first sacral vertebra (S1), corresponding to the 27th position. (B) Most frequent phenotype (indicated as L6 in Table I) detected in RVIII-deleted homozygous mice showing an S1 partially transformed into lumbar character at its anterior part (white arrow). S3 also shows some features of a wild-type S2 vertebra, with lateral processes completely orthogonal to the main axis (arrowheads in A and B). (C) Completely transformed S1 vertebra displaying a wild-type lumbar morphology (L7, white arrow). The first sacral vertebra is at the 28th position. The 29th vertebra (S3 in wild-type) is also transformed into an S2 (28th) type. The incidence of this vertebral formula (∼10% among Hoxd-11RVIIIGe2/RVIIIGe2 animals) increased to 100% in double mutant animals Hoxd-11RVIIIGe2/RVIIIGe2; Hoxa-11Cin/Cin; see Table I for details).
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Table 1. Patterning defects in mice deleted for RVIII in various genetic backgrounds
|Genotypesa||No.||Mice with skeletal defects|
In order to confirm the allelism of this mutation with Hoxd-11 as well as to better assess the role of RVIII, we combined the Hoxd-11RVIIIGe2 allele with a variety of alleles involving either Hoxd-11 or Hoxa-11, the paralogous gene on the HoxA complex. This was deemed necessary as these two genes, as well as other posterior Hoxd genes, had been shown to act mainly through quantitative balances of their products (e.g. Davis et al., 1995; Davis and Capecchi, 1996; Kondo et al., 1996; Zákány et al., 1996). As for the case of Hoxd-11 and Hoxa-11 loss-of-function alleles (Davis et al., 1995; Zákány et al., 1996), we expected that the morphological alterations obtained upon deletion of RVIII would be more readily apparent by concurrently removing doses of the cooperating genes such as Hoxa-11 and Hoxd-11–Hoxd-13. We therefore used loss-of-function alleles for either Hoxa-11 (Hoxa-11Cin; Small and Potter, 1993) or Hoxd-11 (Hoxd-11Str; Favier et al., 1995), as well as a deficiency removing the functions of all three Hoxd-11, Hoxd-12 and Hoxd-13 genes (HoxDDel; Zakany and Duboule, 1996). The outcome of crosses involving these different alleles is summarized in Table I.
In the presence of two normal doses of Hoxa-11, more than half of Hoxd-11RVIIIGe2/RVIIIGe2 animals had an abnormal L6, two of which showed an L7 formula. In wild-type control and heterozygous animals, only one out of 60 specimens showed a malformation of the first sacral vertebra, while no L7 specimens were observed (Table I). The expressivity of this phenotype dramatically increased when one or both copies of Hoxa-11 were removed, such that seven out of 11 animals of the Hoxd-11RVIIIGe2/RVIIIGe2; Hoxa-11Cin/+ genotype had a transformation of S1 into L7, while all double homozygotes were L7 (Table I; Figure 2). Consistently, whenever the homozygous Hoxa-11Cin/Cin background was used, deletion of one copy of RVIII led to full penetrance and expressivity of the L7 phenotype (six out of seven; Table I). These results were confirmed by the incidences of supernumerary accessory processes on L4 (Table I, L4). In summary, the phenotype of RVIII-deleted mice in the vertebral column mirrored that obtained upon inactivation of Hoxd-11 (Favier et al., 1995), though being somewhat hypomorph since double Hoxd-11/Hoxa-11 mutant mice were of the L8 type (Davies et al., 1995) while Hoxd-11RVIIIGe2/RVIIIGe2/Hoxa-11Cin/Cin were L7.
This resemblance to the Hoxd-11 knock-out phenotype was not found when limb skeletons were analysed. In contrast to Hoxd-11 loss-of-function alleles, which led to severe forelimb defects when combined with either one or two Hoxa-11 mutant alleles (Davis et al., 1995), the Hoxd-11 RVIII-deficient allele induced no limb defects when combined with Hoxa-11Cin/Cin mice, other than those known to derive from Hoxa-11 inactivation alone (Figure 3; Table I). The fact that Hoxd-11 function in developing limbs was independent of the RVIII regulatory sequence was demonstrated further by using HoxDDel mice, i.e. mice lacking Hoxd-13, Hoxd-12 and Hoxd-11 functions (Zákány and Duboule, 1996). While all animals trans-heterozygous for the deficiency and the Hoxd-11 null allele (HoxDDel/Hoxd-11Str) displayed strong alterations in digits and carpal bones, the latter being characteristic of Hoxd-11 inactivation (essentially an abnormal fusion within the proximal row of carpal bones; see Davis and Capecchi, 1995; Favier et al., 1995), none of the HoxDDel/Hoxd-11RVIIIGe mice exhibited these alterations. Meanwhile, full penetrance and strong expressivity were recovered in the vertebral phenotype (Figure 3; Table I). Taken together, these genetic analyses demonstrated that: (i) RVIII deletion is allelic to Hoxd-11 and (ii) RVIII is selectively involved in the specification of the vertebral column whereas it takes no part in the function of Hoxd-11 during the development of the limbs and the urogenital apparatus. This latter point confirmed that the Hoxd-11RVIIIGe2 allele resulted from a regulatory, rather than structural, mutation.
Figure 3. Hoxd-11 function in limb development is independent of region VIII control. Skeletal preparations of adult forelimbs. (A) Mice homozygous for RVIII deletion (Hoxd-11RVIIIGe2/RVIIIGe2). These hands were indistinguishable from those of wild-type animals. (B) Double homozygous Hoxd-11RVIIIGe2/RVIIIGe2; Hoxa-11Cin/Cin animals. The pisiform (pi) and pyramidal (py) bones were fused (arrowhead) and attached to the navicular-lunate (nl, arrow). The distal ends of both radius (r) and ulna (u) were abnormally broad. All these defects are characteristic of the Hoxa-11Cin/Cin genotype alone (Table I). (C) Animals trans-heterozygous for RVIII deletion and a HoxD deficiency inactivating Hoxd-13, Hoxd-12 and Hoxd-11 functions (Hoxd-11RVIIIGe2/Del). Size reductions of the second phalanges (P2) of digits II and V were scored (arrowheads), typical of the defects routinely found in Hoxd-11Del/+ allele alone. (D) For comparison, an animal trans-heterozygous for both the same HoxD deficiency and a loss-of-function allele is shown (Hoxd-11Del/Str). In such hands, the complete loss of the second phalanges of digits II and V (arrowheads) were combined with defects in the P2s of other digits, as well as with fusions of proximal carpal elements.
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Hoxd gene expression in RVIII-deleted mice
In wild-type mid-gestation fetuses, Hoxd-11 transcripts are normally detected mostly in limbs and in the genital bud, as well as along the main body axis. In the trunk, expression starts rather posteriorly since the anterior limit of expression in the spinal cord matches the 25th pre-vertebra while in paraxial mesoderm transcription starts at the 27th pre-vertebra (Dollé and Duboule, 1989; Dollé et al., 1989, 1991; Izpisúa-Belmonte et al., 1991).
From the above genetic analysis, we expected Hoxd-11 transcripts to be importantly down-regulated in the developing trunks of Hoxd-11RVIIIGe2 mutant animals. Surprisingly, whole-mount in situ analyses of Hoxd-11 transcript accumulation at E10 and subsequent stages in trunk paraxial mesoderm and neuro-ectoderm of mutant animals gave expression patterns identical to those of wild-type specimens. Examination of earlier developmental stages, however, led to a different conclusion: at E9, the mutant fetuses showed very reduced levels of Hoxd-11-specific signal at the anterior part of the expression domain, i.e. at the level of the 25–27th somites (Figure 4). Strikingly, at the 28th somite stage, no signal was detected between the 25th and 27th somites, while expression in the spinal cord was observed at the level of somite 26 and posteriorly. Expression in the emerging hindlimb buds and in the tail bud region was present in RVIII mutant embryos, even at these early stages (Figure 4), and no visible change of expression in limbs and genitalia of mutant animals were observed subsequently.
Figure 4. Hoxd-11 transcript accumulation at E9 in RVIII-deleted mice, as detected by whole-mount in situ hybridization. (A) Hoxd-11 expression in 20, 22 and 24 somite stage wild-type embryos. (B) Hoxd-11 expression in corresponding Hoxd-11RVIIIGe2/RVIIIGe2 embryos. In mutant embryos, the signal was more restricted at any given stage, and is absent from dorsal structures (arrowheads). (C) Hoxd-11 expression in 20 somite stage wild-type (left) and Hoxd-11RVIIIGe2/RVIIIGe2 (right) embryos. Note the strong hybridization signal in the entire tail bud and non-segmented mesoderm region, in wild-type embryo, while the signal was absent from most of the tail bud and from the entire paraxial mesoderm in mutant embryos. The signal in mutant tail bud was localized to the ventral mesoderm lining the posterior pole of the coelom. (D) Hoxd-11 expression in 27 somite stage wild-type (right) and mutant (left) embryos. Note the absence of signal in the 25–27 somite domain (arrowheads). (E) Hoxd-10 expression in 26 somite stage wild-type (right) and mutant (left) embryos. Note the reduced signal in the 26–27 somite domain (brackets). In the embryos shown in (D), the signals in forelimb buds were indistinguishable between mutants and wild-type. Likewise, mutant and wild-type embryos showed equally strong hybridization signals with both probes in the respective expression domains at E10 and later (not shown).
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Since region VIII is located between Hoxd-11 and Hoxd-10 (Figure 1), and because the 3′ immediately adjacent region IX has been shown to be shared by these two neighbouring genes (Gérard et al., 1996), we analysed Hoxd-10 expression in Hoxd-11RVIIIGe2 mice. In early wild-type E9 embryos (<27 somites), expression of Hoxd-10 was weakly detected in the 24th pair of somites and became progressively stronger at the 25th, 26th and 27th somite levels. The anterior limit of expression was the same in both spinal cord and paraxial mesoderm, and extended posteriorly into the tail bud region. The incipient forelimb field in lateral plate mesoderm was also strongly positive for Hoxd-10. RVIII mutant fetuses from this stage, as well as from earlier stages, displayed dramatically reduced accumulation of Hoxd-10 transcripts in the limb field and tail bud (Figure 4). However, as seen with Hoxd-11, the Hoxd-10 expression pattern at E10 was resumed and appeared identical when homozygous RVIII-deficient mice were compared with normal littermates.
Insertional regulatory mutagenesis at the Hoxd-11 locus
The phenotypic analysis of Hoxd-11RVIIIGe1 homozygous mice, i.e. mice without RVIII but still containing the PGKneo selection cassette (before exposure to the Cre enzyme; Figure 1), gave a related though clearly different result. Homozygous mice of this configuration displayed vertebral defects also at the lumbo-sacral transition, but both the expressivity and penetrance largely exceeded the figures obtained with the PGKneo excised allele. This difference was accentuated by the presence of a highly penetrant abnormal phenotype in the carpus, where proximal carpal bone fusions were scored in most cases (Figure 5; Table I). Nevertheless, in contrast to the homeodomain disruption alleles (Davis and Capecchi, 1994; Favier et al., 1995), involvement of the pisiform bone in these fusions was rare, and more distal structures remained unaffected. Likewise, RVIII mutant homozygous males containing the PGKneo cassette (Hoxd-11RVIIIGe1) were fully fertile while Hoxd-11 mutant mice were hypofertile. Both the vertebral and limb defects were recovered in Hoxd-11RVIIIGe1/Str trans-heterozygote animals (Figure 5, Table I), suggesting allelism with the Hoxd-11 locus. This recessive phenotype was thus hypomorphic and showed a large subset of the defects observed in mice lacking Hoxd-11 function. These genetic studies suggested that the PGKneo transcription unit probably interfered with the proper regulation of Hoxd-11.
Figure 5. Alterations in the limbs of Hoxd-11RVIIIGe1/RVIIIGe1 mice (containing the PGKneo cassette). (A) Skeletal preparations of a juvenile Hoxd-11RVIIIGe1/RVIIIGe1 forelimb showing fusion of cartilage and ossification centres between the pyramidal and navicular-lunate (arrow in left panel). In the middle, a trans-heterozygous Hoxd-11RVIIIGe1/Str paw which shows similar (though partial) alterations, i.e. cartilage fusion of the same proximal carpal elements (arrow); for comparison, a homozygous Hoxd-11RVIIIGe2/RVIIIGe2 paw is shown on the right (indistinguishable from wild-type). (B) Hoxd-11 down-regulation in presumptive carpal structures. Whole-mount in situ hybridization of Hoxd-11 transcripts in E13 forelimbs of various genotypes. Wild-type (left), Hoxd-11RVIIIGe1/+ (middle) and Hoxd-11RVIIIGe1/RVIIIGe1 (right). In the proximal autopod domain (arrowheads), transcript accumulation is reduced in the heterozygotes and is not detectable in homozygous animals. (C) neo gene expression is detected concomitantly with Hoxd-11 down-regulation. Whole-mount in situ hybridization of neo-specific transcripts in a E13 heterozygous Hoxd-11RVIIIGe1/+ animal. A clear signal is detected in the same proximal autopod domain where Hoxd-11 transcription is silenced (arrowhead). In fact, down-regulation of Hoxd-11 is observed wherever strong neo expression appears.
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To verify this point, we analysed the expression of Hoxd-11 in Hoxd-11RVIIIGe1 homozygous mice by whole-mount in situ hybridization. RNA accumulation showed a strong difference with respect to either the wild-type or the RVIII-deficient, PGKneo-less, mice. In the trunk, Hoxd-11 transcript accumulation was severely delayed (for longer than in PGKneo-deleted mice; not shown). An important reduction in transcript accumulation was observed also in limb buds at E10 (not shown), as well as in a region immediately proximal to the forelimb autopod at E13 (Figure 5). In this prospective wrist area, Hoxd-11 transcripts were not detected in the expected anterior proximal domain (Figure 5, arrowhead). In contrast, hybridization using a neo-specific probe indicated that the PGK promoter was particularly active in this domain, as judged by accumulation of neo gene transcripts (Figure 5, arrowhead). In this case, the overall expression pattern of the neo gene was clearly reminiscent of that shown by the neighbouring Hoxd-11 gene, further indicating that the PGK promoter responded to the adjacent Hox regulatory signals (Rijli et al., 1994; van der Hoeven et al., 1996). A precise correspondence was established in this domain between a strong expression of the neo gene, a down-regulation of Hoxd-11 and the occurrence of a Hoxd-11-specific carpal phenotype. This suggested that part of the regulatory interactions leading to the expression of Hoxd-11 in this particular part of the carpus had been shifted towards the PGK promoter complex. Therefore, in the lumbo-sacral region, the penetrance and expressivity of the RVIII-deleted phenotype substantially increased in mice with the PGKneo cassette, due to the combined effects of the two mutagenic mechanisms, i.e. the deletion of an activating element and promoter competition or interference caused by the insertion of the selective marker. In the forelimb, the latter component induced a transcriptional suppression and consistent limb defect.