Abstract
Skeletal formation is a fundamental element of body patterning and is strictly regulated both temporally and spatially by a variety of molecules. Among these, retinoic acid (RA) has been shown to be involved in normal skeletal development. However, its pleiotropic effects have caused difficulty in identifying its crucial target cells and molecular mechanisms for each effect. Development of cartilage primordia is an important process in defining the skeletal structures. To address the role of RA in skeletal formation, we have generated mice expressing a dominant-negative retinoic acid receptor (RAR) in chondrogenic cells by using the type II collagen ␣1 promoter, and we have analyzed their phenotypes. These mice exhibited small cartilage primordia during development and retarded skeletal formation in both embryonic and postnatal periods. They also showed selective degeneration in their cervical vertebrae combined with homeotic transformations, but not in their extremities. The cervical phenotypes are reminiscent of phenotypes involving homeobox genes. We found that the expression of Hoxa-4 was indeed reduced in the cartilage primordia of cervical vertebrae of embryonic day 12.5 embryos. These observations demonstrate that endogenous RA acts directly on chondrogenic cells to promote skeletal growth in both embryonic and growing periods, and it regulates the proper formation of cervical vertebrae. Furthermore, RA apparently specifies the identities of the cervical vertebrae through the regulation of homeobox genes in the chondrogenic cells. Great similarities of the phenotypes between our mice and reported RAR knockout mice revealed that chondrogenic cells are a principal RA target during complex cascades of skeletal development.

Retinoic acid (RA), a physiological metabolite of vitamin A, is known to affect skeletal development when applied exogenously. Administration of excess RA to pregnant mothers causes various malformations in their fetuses, such as limb defects, cleft palates, and malformed sternums and vertebrae in rodents (1) and craniofacial malformations in humans (2) . In addition to these morphological abnormalities, RA treatment induces various homeotic transformations in the embryonic skeleton, including alteration of the vertebral patterns (3, 4) . These observations have led to the prediction that endogenous RA is an important physiological regulator in development. Several lines of supporting evidence have also been obtained from the studies of dietary vitamin A deprivation (VAD) in rodents (5, 6) .
RA functions are now known to be mediated by members of the nuclear receptor superfamily that act as ligand-dependent transcription factors (7) . Receptors for RA are composed of heterodimers between retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARs and RXRs are encoded by at least three distinct genes, RAR␣, -␤, and -␥ and RXR␣, -␤, and -␥, respectively (7, 8) . Functions of endogenous RA during development have been addressed by knocking out these receptors. Mice lacking the RAR␥ gene (9) or RAR␣ and RAR␥ genes (10) had growth retardation, malformations, and homeotic transformations in their skeletons. These phenotypes have confirmed that endogenous RA is required for normal skeletal development. These results, in turn, have raised the question of what kind of cells and molecules are the RA targets in normal skeletal development.
Skeletons are developed through two distinct processes (11) . Flat bones in the skull are generated by intramembranous ossification. On the other hand, most other bones are generated through endochondral ossification: condensed mesenchymal cells first differentiate into chondrocytes to form cartilage primordia, then they are gradually replaced by osteogenic cells and calcified tissues to develop into mature bones. Many of the affected skeletons in VAD mice and RAR knockout mice are apparently made through the latter process. Furthermore, development of chondrocytes and cartilage tissues are regarded as a critical step in outlining the majority of the skeletal structures (12) . It is therefore reasonable to postulate that chondrogenic cells, the cells of a chondrocyte lineage, including prechondrocytic mesenchymal cells and mature chondrocytes, are a critical target of endogenous RA during skeletal development and growth.
Homeobox genes are well known genes involved in the specification of body patterning. In mammals, 39 homeobox genes are clustered in four different chromosomal complexes (13) . It has been documented that exogenously applied RA alters their segmental expression pattern in vivo, concomitant with the appearance of homeotic transformations (3, 4) . This strongly suggests that homeobox genes are crucial target molecules of endogenous RA, but the real relationship between endogenous RA and homeobox gene expression during mammalian development has not yet been elucidated.
We have analyzed physiological RA functions during mouse development by expressing a dominant-negative form of RAR, referred to as RAR-E, which effectively blocks RA signaling (14) . In the ligand-binding domain, RAR-E contains a point mutation originally identified at the homologous position in the thyroid hormone receptor of thyroid hormone-resistant patients (15) . We previously generated mice expressing RAR-E in the epidermis and demonstrated the requirement for RA in normal skin development (16) . Here, we report the generation of transgenic mice expressing RAR-E in chondrogenic cells and their resultant phenotypes. The significance of the chondrogenic cell-mediated RA functions in skeletal development is discussed through comparing the phenotypes of RAR and homeobox gene knockout mice.
MATERIALS AND METHODS
Generation of Transgenic Mice. The mouse type II collagen ␣1 chain gene was isolated by screening a mouse genomic library (Stratagene) with a PCR-amplified probe containing its 5Ј upstream sequence (17) . The 2.5-kb promoter region covering the 5Ј upstream region of the translation start site, the 3.8-kb enhancer region from the first intron, the rabbit ␤-globin intron, and the polyadenylation site (16) (17) (18) were ligated together with ␤-galactosidase or RAR-E cDNA, as shown in Fig. 1A . Fertilized eggs were recovered from BDF 1 females (C57BL͞6 ϫ DBA͞2) crossed with BDF 1 males and microinjected by standard procedures (19) . Copy numbers of the transgene were determined by Southern blotting with tail DNA. RAR-E offspring were generated by breeding the founder with C57BL͞6 mice. The body weights of the F 2 offspring were followed up to 77 days after birth.
Soft X-Ray Analysis. Mice were anesthetized and subjected to soft x-ray analysis (40 kV, 2.5 mA for 60 sec, HX-100, HITEX, Osaka).
Whole-Mount 5-Bromo-4-chloro-3-indolyl ␤-D-Galactoside (X-Gal) Staining. Whole-mount X-Gal staining was performed by standard procedure with some minor modifications (20) . E12.5 embryos were fixed in buffer containing 1% formaldehyde, 0.2% glutaraldehyde, and 0.02% Nonidet P-40 for 30 min at 4°C and stained with PBS containing 5 mM K 4 Fe(CN) 6 , 5 mM K 3 Fe(CN) 6 , and 1 mM X-Gal for 8 h at 30°C.
Skeletal Staining. E18.5 fetuses were skinned, eviscerated, and fixed in 95% ethanol. Intact skeletons were stained with 150 mg͞ml alcian blue 8GX in 75% (vol͞vol) ethanol͞20% acetic acid for 24-48 h, and excess tissues were removed with 1-2% KOH digestion for 2-3 days. The skeletal preparations were stained with 75 mg͞ml alizarin red S in 1% KOH for 24 h and cleared in graded glycerol solutions (21) . For adult skeletal FIG. 2 . Retardation of skeletal development in RAR-E mice. (A) Physical appearances of a male founder RAR-E mouse (Right) and its sex-matched nontransgenic littermate (Left). The RAR-E mouse is 24% lighter than the littermate. (B) Growth curves of RAR-E mice and their nontransgenic littermates. On average, transgenic males (ᮀ; n ϭ 10) weighed approximately 19% less than nontransgenic males (s; n ϭ 12), and transgenic females (E; n ϭ 18) weighed approximately 16% less than nontransgenic females (F; n ϭ 25). Statistical significance was calculated by ANOVA (P Ͻ 0.001). (C) X-ray photographs of an adult male RAR-E mouse with a conspicuous phenotype, being 22% lighter (Lower) than its sex-matched nontransgenic littermate (Upper). Bones generated through endochondral ossification appear to be preferentially shortened: the lengths of the vertebral column (from C1 to S4) and the femurs of the transgenic mouse were 19-20% shorter than those of the wild-type littermate, whereas the length of the cranium (from frontal to occipital edges) was shortened by less than 5%. analysis, tissues were digested with 2% KOH for 4-6 days. E14.5 embryos were stained with alcian blue as previously described (22) . Measurement of Bone Size. Bone sizes of the stained skeletons of the E18.5 embryos were measured; vertebral column sizes were compared between the lengths from C1 to S4; limb bone sizes were compared between the lengths along the longer axes; frontal-parietal bone sizes were compared between the lengths from the rostral edges of the frontal bones to the caudal edges of the parietal bones.
In Situ Hybridization. Plasmids containing cDNAs for Hoxa-3 and Hoxa-4 were linearized and used as templates for synthesis of digoxigenin-labeled riboprobes (23) . E12.5 embryos were fixed with 4% formaldehyde in PBS for 4 h at 4°C, bleached in 5:1 (vol͞vol) methanol͞H 2 O 2 solution for 6 h at room temperature, rehydrated in a graded series of methanol͞ PBS, and embedded in 2% agarose͞PBS gel. Sections 300 m thick were obtained with a microslicer (DTK-3000, Dosaka EM, Kyoto, Japan). In situ hybridization was performed according to the described methods (23, 24) .
RESULTS

Generation of Transgenic Mice Expressing RAR-E in Chondrogenic Cells.
We utilized a mouse type II collagen ␣1 chain promoter to express transgenes in chondrogenic cells ( Fig.  1 A) . During embryogenesis, expression of the gene begins in paraxial mesenchymal cells concomitantly with the onset of their differentiation toward chondrocytes around E9.5, and the expression continues to adulthood (25) (26) (27) . Therefore, type II collagen is a useful marker for cells of the chondrocyte lineage. Strong ␤-galactosidase expression was specifically observed in cartilage primordia of vertebrae, ribs, and long bones in the E12.5 embryo (Fig. 1B and ref. 28 ). Four transgenic founders for RAR-E were identified among 59 offspring, with transgene copy numbers of 3, 4, 4, and 4 (data not shown). The integrated copy numbers for RAR-E were relatively small compared with our previous experiments (16, 29) , suggesting the influence of RAR-E on mouse viability.
Retarded Skeletal Development and Maturation in the Transgenic Mice. By visual inspection, the RAR-E founders appeared to be smaller than their littermates ( Fig. 2A) . Body weights of their F 2 offspring were monitored for several months. The difference was apparent at birth and continued until adulthood: the body weights of RAR-E mice were approximately 17% lower throughout the monitoring periods (P Ͻ 0.001) (Fig. 2B) , indicating that growth retardation in RAR-E mice occurs not only during embryonic stages but also postnatally. Consistent with the expression patterns of the transgene, x-ray analysis revealed that the vertebrae and limb bones, generated through endochondral ossification, were more prominently affected than the crania, which are generated mainly through intramembranous ossification (Fig. 2C) .
Next, we mated the F 2 offspring to generate RAR-E homozygotes from each transgenic line to obtain enhanced phenotypes through a gene-dosage effect. Unexpectedly, all homozygotes died at or shortly after birth (see below). We therefore examined their xarnold@example.net (Fig. 3A) . As in the adult, skeletons of RAR-E embryos were generally shortened, except for skulls. These effects appeared to be transgenedosage dependent, being more prominent in the homozygote than in the heterozygote: measurement of bone sizes revealed that vertebral columns and limb bones exhibited statistically significant reduction in size in an RAR-E dosage-dependent manner, whereas frontal-parietal bone sizes showed no differences (Table 1 ). Magnified views revealed that all the limb components of RAR-E mice were smaller (Fig. 3B ). In addition, the ossified regions were narrowed, especially at the distal components (Fig. 3B) . Such phenotypes were consistently observed in all four independent lines, with the homozygotes consistently exhibiting more severe phenotypes.
Viability of the Transgenic Mice. Genotypes of offspring from transgenic parents were examined at various stages. They were in accordance with the mendelian rules up to birth: the number of wild types, heterozygotes, and homozygotes were, respectively, 32, 88, and xarnold@example.net; 21, 35, and xarnold@example.net; and 39, 66, and xarnold@example.net. Therefore, the viability in utero was not considerably affected by RAR-E in the established transgenic lines. However, , and an RAR-E homozygote (Bottom). In transgenic mice, the entire limb was decreased in size and bony portions (red) of the scapula, humerus, ulna, radius, femur, tibia, fibula, metacarpal͞metatarsal, and phalangeal bones were all shortened in an RAR-E gene dosage-dependent manner. as described above, almost all the homozygotes (24͞26) died within several hours after birth, apparently because of respiratory distress. Although the precise cause of death remains to be determined, these results indicate the requirement for proper skeletal development for survival. Degenerative Malformations of Cervical Vertebrae. In E18.5 RAR-E fetuses, the cervical vertebrae were found to be particularly affected (Fig. 4 A-C) . Cervical vertebral columns were hypoplastic in a transgene-dosage-dependent manner (Fig. 4 B and C) . In addition, their neural arches failed to fuse to the vertebral bodies (dyssymphysis) transgene dosagedependently: only C1 and C1 to C4 dyssymphyses were observed in the heterozygote and the homozygote, respectively (Fig. 4 B and C) . These defects were not observed in thoracic and lumbar vertebrae. Similar defects were xarnold@example.net, when chondrogenesis is actively occurring: C1 to C2 and C1 to C6 defects appeared in the heterozygotes and the homozygotes, respectively (Fig. 4 D-F) . Note also xarnold@example.net the cartilage primordia of the limbs were shortened in an RAR-E gene-dosage-dependent manner (Fig. 4 D-F) , showing that generalized retardations of skeletal development already occurred at the period of active chondrogenesis.
We next examined the cervix of adult RAR-E heterozygotes (Fig. 5) , as we could not obtain adult homozygotes. Vertebral deformities were observed through C1 to C7 (Fig. 5 B and C) . C1-C2 dyssymphysis frequently occurred. In severely affected lines, the adjacent vertebrae were fused and ossified as a mass (Fig. 5 C, E, and F) . Such degenerative changes were not seen in the more caudal vertebrae of T1 and below. In summary, degeneration of skeletons was restricted to the cervical region through developmental stages and adult periods.
Homeotic Transformations in the RAR-E Cervical Vertebrae. Analysis of our RAR-E mice revealed a significant incidence of homeotic transformations in the cervical vertebrae (Fig. 5 B, D , E, and F, see legend for details). We observed both anterior and posterior transformations; anterior transformations included C3 to C2 and C7 to C6; posterior transformations, C5 to C6, C7 to T1, and T1 to T2 (Table 2) . These transformations were observed in independent RAR-E lines. No additional transformations were observed in any other parts of the vertebrae or in the digits, or in any of the 34 nontransgenic littermates (data not shown).
Decreased Expression of Hoxa-4 in the RAR-E Cervical Prevertebrae. We noticed that homeotic changes in our mice resembled those reported in Hoxa-4 knockout mice (30) (31) (32) . By in situ hybridization, we actually found a reduction in Hoxa-4 expression in the cervical prevertebrae of RAR-E homozygous xarnold@example.net (Fig. 6 ). In the nontransgenic animal, strong hybridizing signals were observed in the prevertebrae (pv) from pv3 to pv9 and weak signals in pv2 and caudal to pv10 (Fig. 6 A and B) . In the RAR-E mouse, hybridizing signals in the cervical prevertebrae were significantly reduced (Fig. 6 E and F) . The expression boundary (1998) appeared to stay at pv2 (Fig. 6 E and F) as in nontransgenic mice ( Fig. 6 A and B and ref. 33) . Note that the expression was not affected in the neural tube, where RAR-E was not targeted for expression. In contrast, the Hoxa-3 expression level was not affected in the same prevertebrae; the expression boundary of Hoxa-3 was constantly observed at pv1 (Fig. 6 C, D, G , and H, and ref. 34 ). This observation indicates that the Hoxa-4 reduction is specific and not the result of suppressive effects of RAR-E on the general transcription machinery or the abnormal prevertebra structures. These data strongly indicated that RA specifies identities of the cervical vertebrae through the regulation of homeobox genes in chondrogenic cells.
DISCUSSION
There have long been many unsolved questions regarding the physiological functions of RA, especially in skeletal formation in developmental biology. Accordingly, tremendous efforts have been made to generate mice lacking RAR genes to elucidate their functions (9, 10, 35, 36) . We have chosen an alternative strategy using dominant-negative RAR (RAR-E) and have examined the chondrogenic cell-mediated RA functions in skeletal formation. The skeletal formation involves several major types of cells, including chondrogenic cells, osteoblasts, osteoclasts, etc. As RA has been shown to act not only on chondrogenic cells but also on many other cell types (37), we initially supposed that we would obtain a portion of the RAR knockout mice phenotypes reflecting RA action on chondrogenic cells. However, a lot more of the RAR knockout phenotypes were reproduced in our mice than expected, revealing that chondrogenic cells are critical RA target cells in skeletal development. Our mice exhibited generalized growth retardation of the skeletons. Although small skeletons have long been observed in not only RAR knockout but also VAD mice (6, 9, 10) , this phenotype has been estimated to result from the defects and malfunctions of other affected organs. Using RAR-E, we were able to assess RA functions in the skeleton by minimizing such secondary effects. The direct effect of RA on skeletal growth is also evident from small cartilage xarnold@example.net under active chondrogenesis (Fig. 4) . Given that hormonal control of embryonic growth has long remained to be elucidated (38) , RA could be highlighted as a soluble factor that directly acts on chondrogenic cells and promotes skeletal growth during embryogenesis and also after birth.
Another unexpected replication between RAR-E mice and RAR knockout mice is the manifestation of lethality. Anatomical and histological examinations of the dead RAR-E mice revealed only poorly ventilated lungs; no apparent abnormality was observed in other vital organs (not shown). Such respiratory failure could be caused by a smaller thoracic cage and hypoplastic cartilage lining the respiratory tract, as observed in chondrodysplasia mutant mice (39, 40) . Severe cervical hypoplasia, as shown in Fig. 4 , may cause fatal cervical dislocations and͞or compression of the cervical spinal cord at or after birth. Although the definite causes of death remain to be determined in further analysis, our data indicate that cartilage and skeleton are vital RA targets and that lethality reported in RAR knockout mice (10) may be attributed in part to the defect in RA signaling in chondrogenic cells, a finding that was totally unexpected.
Vertebral abnormalities are also replicated phenotypes. An exception is the C2 to C1 transformation (neural arch thickening and an ectopic anterior arch on C2), observed only in partial RAR knockout mice (10) . However, we do not deny the possibility of C2 to C1 transformation in our mice, because we were unable to fully examine this point due to severe degenerative change as is the case with complete RAR␣ and -␥ knockout mice (10) . Other phenotypes, fusions of the neural arch, dyssymphyses in C1 and C2, and homeotic transformations including C7 to C6 and C7 to T1, were commonly observed (9, 10) . These results again indicate that chondrogenic cells are a principal RA target during cervical development.
A major difference in the skeletal phenotypes between RAR knockout mice and RAR-E mice is the absence of front limb deformities in RAR-E mice. A possible explanation is that chondrogenic cells might not be the direct RA target for limb patterning. Although RA was proposed as a morphogen that produces positional information in the limb buds (41) , subsequent experiments showed that RA itself is not a morphogen but generates a secondary morphogenic signaling molecule(s) that determines the cell identities in the limb-bud mesenchyme (42) .
A couple of alternative mechanisms may make it possible to explain the phenotypic differences between RAR knockout and RAR-E mice. First, RAR-E might affect other unknown nuclear receptor signalings whose response elements are highly related to RARs'. Second, RAR-E expression might cause repression of basal transcription activities through binding to the corepressor (43) (44) (45) , instead of a failure of the transcriptional activation that occurs in the RAR knockout mice. It is difficult to completely rule out these alternative possibilities, but the data that the observed skeletal RAR-E phenotypes are mostly, if not all, included in the RAR knockout phenotypes are well fit for the idea that additionally observed phenotypes of RAR knockout mice are derived from the different cell origins, and therefore are presently consistent with our presupposition that tissue-specific expression of RAR-E would reveal physiological RAR functions in the tissues concerned. FIG. 6 . In situ hybridization analysis of Hoxa-4 and Hoxa-3 gene expression. Hoxa-4 (A, B, E, and F) and Hoxa-3 (C, D, G, and H) expression was detected in E12.5 embryos by using digoxigenin-UTPlabeled riboprobes in nontransgenic (A-D) and RAR-E homozygous (E-H) embryos. Sagittal (A and E) and parasagittal (B and F) sections reveal the reduction in the level of Hoxa-4 expression in the cervical prevertebrae of the RAR-E homozygote. Hoxa-4 expression extends from the second prevertebra down to lower prevertebrae in both nontransgenic and RAR-E homozygotes. Note that the Hoxa-4 expression in the neural tube is indistinguishable in the two mice. In contrast, Hoxa-3 showed no difference in either the expression level or the boundary, in sagittal (C and G) and parasagittal (D and H) sections: it extends from the first prevertebra to the lower in both mice. pv1, the first prevertebra; pv2, the second prevertebra; pv3, the third prevertebra; pv9, the ninth prevertebra; nt, neural tube.
Much experimental evidence has suggested that the expressions of certain homeobox genes are under the regulation of endogenous RA; RAR␣-or ␥-deficient EC cells showed aberrant expression of homeobox genes in vitro (46) ; RAresponsive elements in the Hoxb-1 gene are essential for its proper expression in vivo (47, 48) . In addition to such circumstantial evidence, our data offer strong evidence for a direct link among physiological RA signaling, homeobox gene expression, and body specification in mammalian development. Our RAR-E phenotypes are closest to those of Hoxa-4 knockout mice; Horan et al. (30) reported C3 to C2 transformation and C7 to T1 transformation with penetrance of 71% and 48%, respectively; Kostic and Capecchi (31) reported 100% transformation of C3 to C2. In addition, combined null mutants for Hoxa-4 paralogues showed C7 to C6 transformation with the penetrance of 85-100% (32) . We therefore examined the expression of Hoxa-4 and observed its reduction in cervical prevertebrae. Identification of a functional RA-responsive element in the 5Ј upstream regulatory region of human HOXA-4 gene (49) further supports the idea that Hoxa-4 is a direct target gene of RA in cervical development. Relatively low penetrance of each transformation in our mice could be explained by the low but detectable level of Hoxa-4 expression. It is, of course, easily imaginable that homeobox genes other than Hoxa-4 are also affected in our transgenic mice: some of the transformations, e.g., T1 to T2, were not reported in Hoxa-4 mutants but were observed in Hoxa-6 knockout mice (31) .
These observations convincingly demonstrate that specification of cervical vertebrae is physiologically determined by RA signaling through chondrogenic cells and that this determination is mediated through the regulation of homeobox gene expression. Furthermore, the present analyses reveal the importance of chondrogenic cells in RAR-mediated body patterning and skeletal development, and they advance our understanding of RA physiology as well as the roles of these cells in body formation.

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