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Keywords:

  • Ror2;
  • Robinow syndrome;
  • somitogenesis;
  • development of limb;
  • craniofacies;
  • genital

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Robinow syndrome (RS) is a human dwarfism syndrome characterized by mesomelic limb shortening, vertebral and craniofacial malformations and small external genitals. We have analyzed Ror2-/- mice as a model for the developmental pathology of RS. Our results demonstrate that vertebral malformations in Ror2-/- mice are due to reductions in the presomitic mesoderm and defects in somitogenesis. Mesomelic limb shortening in Ror2-/- mice is a consequence of perturbed chondrocyte differentiation. Moreover, we show that the craniofacial phenotype is caused by a midline outgrowth defect. Ror2 expression in the genital tubercle and its reduced size in Ror2-/- mice makes it likely that Ror2 is involved in genital development. In conclusion, our findings suggest that Ror2 is essential at multiple sites during development. The Ror2-/- mouse provides a suitable model that may help to explain many of the underlying developmental malformations in individuals with Robinow syndrome. Developmental Dynamics 229:400–410, 2004, © 2004 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Receptor tyrosine kinases (RTKs) are a group of membrane spanning receptors that function in control of cell growth, differentiation, metabolism, cell migration, and other biological processes (Schlessinger, 2000). Human ROR1 and ROR2 belong to a small family of RTKs and have been first isolated according to their similarity to the Trk neurotrophin receptors (Masiakowski and Carroll, 1992). Subsequently, Ror homologues have been identified from Drosophila (Oishi et al., 1997; Wilson et al., 1993), partially from rat (Masiakowski and Carroll, 1992), Caenorhabditis elegans (Forrester et al., 1999), and mouse (DeChiara et al., 2000; Oishi et al., 1999). Ror genes are expressed throughout the nervous, cardiovascular, respiratory, digestive, urogenital, and skeletal system (Al-Shawi et al., 2001; Matsuda et al., 2001; Oishi et al., 1999). Similar to other RTKs, Ror genes contain an extracellular ligand-binding domain connected by means of a single transmembrane spanning region to the intracellular region harboring a conserved tyrosine kinase, a serine- and proline-rich region. The extracellular part consists of an immunoglobulin-like domain, a Frizzled-like cysteine-rich (CRD) domain and a membrane proximal kringle domain. Recent findings suggest that the Ror2 receptor is associated with the Wnt signal transduction pathway, as the Xenopus XRor2 receptor has been shown to modulate development of the axial mesoderm and neuroectoderm by means of Wnt signaling (Hikasa et al., 2002). In addition, Wnt5a and Ror2 interact physically and functionally, thereby activating the noncanonical Wnt pathway (Oishi et al., 2003).

In humans, ROR2 mutations account for autosomal recessive Robinow syndrome (Afzal et al., 2000; van Bokhoven et al., 2000; RS, MIM 268310) and autosomal dominant brachydactyly type B (Oldridge et al., 2000; Schwabe et al., 2000; BDB, MIM 113000), two distinct human malformation syndromes. Robinow syndrome is a multisystemic disease, characterized by moderate shortness of stature, mesomelic limb shortening, hemivertebrae, genital hypoplasia, and a characteristic facial appearance (for review see Patton and Afzal, 2002). The facies, also referred to as “fetal face,” is characterized by broad prominent forehead with hypertelorism, midface hypoplasia, a short upturned nose, a long philtrum, a triangular mouth with downturned angles, and micrognathia. Robinow patients exhibit characteristic oral features such as hyperplastic alveolar ridges, dental crowding, and irregular teeth within both primary and secondary dentition. Occasional midline clefting of the lower lip, ankyloglossia, and a bifid tongue indicate midline affection in RS patients. Limb malformations in RS include mesomelic shortening with or without nail hypoplasia. Spinal anomalies are characterized by hemivertebrae and fusion of ribs. A small genital is present in males, and in females, the clitoral size is reduced. Renal hydronephrosis and cystic dysplasia have been reported. In addition, congenital heart defects have been observed in approximately 15% of RS patients, and developmental delay is a feature found in a minority of patients.

For RS, both autosomal dominant and recessive mode of inheritance have been reported (Robinow, 1993). Considerable overlap can be found between the two forms. However, recessive RS seems to be more severe than the dominant form, because the shortness of stature and limb affection is more pronounced, and mortality in excess of approximately 10% has been described. The autosomal recessive form of RS is caused by homozygous missense, nonsense, and frameshift mutations in both intracellular and extracellular domains of ROR2, presumably resulting in loss of Ror2 function. For autosomal dominant RS, no gene has been assigned so far, but the phenotypic similarities of the two forms may suggest that autosomal dominant RS may be caused by a gene within the same pathway.

The function of Ror2 has been elucidated by the analysis of mice with inactivated Ror2 alleles (DeChiara et al., 2000; Takeuchi et al., 2000). Ror2-/- mice exhibit a very similar phenotype as autosomal RS patients, because they display short limbs, snout, and a shortened tail as well as segmental vertebral defects in a generally dwarfened body. They die shortly after birth, exhibiting severe cyanosis, however, the reason for the perinatal lethality remains unclear. Mice with a targeted gene disruption of Ror1 gene have no obvious skeletal or cardiac abnormalities, yet they die soon after birth due to respiratory dysfunction (Nomi et al., 2001). Ror1/Ror2 double-mutant mice show skeletal abnormalities more severe than those seen in Ror2 mutant mice (Nomi et al., 2001). So far, many aspects of Ror2 function and developmental malformations of Ror2-/- mice are not understood. In this study we present a detailed analysis of the Ror2-/- mouse as a model for the developmental pathology of human recessive RS.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Ror2 Expression Analysis

Ror2 exhibits a widespread expression in a variety of organ systems (Fig. 1). We have analyzed the Ror2 expression pattern by using whole-mount in situ analysis for mouse embryonic stages E10.5 and E11.5 and 33P-labeled in situ hybridization analysis of sections of stages E12.5, E13.5, E14.5, and E16.5. Particular emphasis was put on the sites/organs that are involved in RS, i.e., craniofacial structures, axial skeleton, limb skeleton, and genitals. At E10.5 and E11.5 Ror2 is strongly expressed in the white matter substance of the forebrain, the orofacial region, and the first and second pharyngeal arch (Fig. 1A,B). Expression is also seen in the presomitic mesoderm (PSM), the primitive streak, the somitomeres of the presomitic mesoderm, the epithelium of the differentiating somites, but not in the forming somite (Fig. 1C–F).

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Figure 1. Ror2 expression pattern. A–F: Whole-mount in situ hybridization. G–P: 33P-labeled in situ hybridization showing darkfield and brightfield images. A:Ror2 expression at embryonic day (E) 10.5 in the forebrain, limbs (arrow indicates the forelimb), and somites. B: Frontal view of E10.5 embryo demonstrates Ror2 expression in the pharyngeal arches (arrow). C,D:Ror2 expression at E10.5 in the primitive streak, somitomeres, and the epithelium of the somites. There is no Ror2 expression in the forming somite (arrow). E: Sagittal section of somites showing Ror2 expression in the epithelium covering the somite at E11.5. F: Frontal section of somites of at E12.5. G–L:Ror2 expression in craniofacial structures: around Meckel's cartilage at E14.5 (G), and at E16.5 (H–L) around the malleus (arrow) and incus and in the ganglion trigeminale (H), in the nasal conchae (I), the epithelium of the frontonasal septum (J), the pituitary gland (K), and the tooth germs (L). M,N:Ror2 expression in the forelimb of embryos at stages E12.5 (M) and E14.5 (N). The arrow indicates chondrogenic condensations. O,P:Ror2 expression in the renal cortex at E14.5 (O) and the genital tubercle at E13.5 (P). FNP, frontonasal process; MC, Meckel's cartilage; OC, oral cavity; OP, os petrosum; PA, palate; PI, pituitary gland; TG, tongue.

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A detailed Ror2 expression analysis of the craniofacial region of wild-type (wt) embryos was performed at different stages of development. At E14.5 Ror2 is strongly expressed around Meckel's cartilage (Fig. 1G) and later on at E16.5 around the derivatives of Meckel's cartilage, the malleus (Fig. 1H), and incus of the inner ear. There is marked Ror2 expression in the secondary palate, the mesenchyme surrounding the nasal conchae (Fig. 1I), and the epithelium of the frontonasal septum (Fig. 1J). In addition, Ror2 expression can be found in the pituitary gland (Fig. 1K) and in the tooth germs of the molars (Fig. 1L).

Ror2 is strongly expressed in the epithelium of the developing limb bud (Fig. 1A). Later on during limb development, its expression is confined to chondrogenic condensations, the interdigital mesenchyme, and the epithelium of the forming limb (Fig. 1M,N). In addition, Ror2 is expressed in the cortex of the developing kidney and in the genital tubercle at E13.5 (Fig. 1O,P).

Craniofacial Phenotype

To further elucidate the role of Ror2 and the developmental pathology of RS, Ror2-/- mice generated by Takeuchi and coworkers (2000) were analyzed in detail. Skeletal preparations were performed as previously described (Mundlos, 2000). Homozygous mutants died a few hours after birth, they were significantly smaller than the wild-type or heterozygous littermates.

Ror2-/- mice displayed a craniofacial outgrowth defect, hypertelorism, midface hypoplasia, and oral abnormalities. We compared bone and cartilage staining of homozygous mutants with wild-type littermates from stages E14.5, E16.5, E18.5, and newborns. At E14.5 and E16.5, the skull was broadened, and there was truncation of Meckel's cartilage. The rostral process of Meckel's cartilage in the mutant embryo failed to form at the distal tips (symphysis; Fig. 2A,B). There was thickening of the tip, that was indistinct from the rest of Meckel's cartilage in the mutant, pointing downward, compared with an upward orientation in the control animal (Fig. 2C,D). The nasal capsule was truncated and thickened in the mutant, often only on one side of the embryo, and ossification of the maxilla was delayed (Fig. 2E,F). At the skull of the Ror2-/- embryo, an ectopic cartilage was present, extending out from the anterolateral process of the ala temporalis (Fig. 2G,H). It was fused with the tegmen tympani of the otic capsule above the incus and malleus bones of the middle ear of the ala temporalis. The trabecular basal plate was shortened, reducing the size of the fenestra sphenoparietalis on each side. Close examination of mandibles of E18.5 mutant animals showed that the angle between the angular process and the condylar process of the jaw point were disrupted, and the two processes lie over each other rather than the angular being clearly on the outside (not shown). Again this defect was observed on one side of the embryo only. As seen at earlier stages, the rostral process of Meckel's cartilage was disrupted in the mutant and partially unfused (Fig. 2I,J). In addition, the mutant exhibited a cleft palate due to impaired fusion of palatine bones in the Ror2-/- mouse (Fig. 2M,N). In the newborn mutant, defects in the middle ear were observed. The caudal part of the malleus formed, with the manubrium inserting into the tympanic membrane, but this part of the malleus was cut off from the main body (Fig. 2K,L).

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Figure 2. Craniofacial phenotype of Ror2-/- mutant. Alizarin red/Alcian blue staining of skulls of embryonic day (E)16.5 (A–H), E18.5 (I, J), newborn (K, L), wild-type (Wt) and Ror2-/- mice. Hematoxylin and eosin–stained sections of E16.5 (M–R) and E13.5 (S, T) Wt and Ror2-/- mice. A,B: Ventral view, showing truncated Meckel's cartilage and broadening of the skull in the mutant. The bar indicates the length of the hyoid in the mutant. C,D: Side view of Meckel's cartilage with thickened tip, pointing downward (arrow). E,F: Truncated nasal capsule in the mutant, thickened on the left side. Note that ossification of the maxilla in the wt is more advanced than in the mutant. G,H: Dorsal view showing an ectopic cartilage on the right-hand side extending out from the anterolateral process of the ala temporalis (arrow). Note shortening of the trabecular basal plate. I,J: Control and mutant distal tip of the mandible with disrupted and partially unfused rostral process. K,L: Control and mutant middle ear showing disruption of the malleus (arrow). M,N: Frontal section of a E16.5 Wt and mutant embryo with a cleft palate in the mutant. O,P: Upper molar of Wt and Ror2-/- mice indicating regular tooth formation. Q,R: Frontal section showing a normal pituitary gland with remnants of Rathke's pouch in the Wt and mutant. S,T: Genital tubercle of E13.5 Wt and mutant, significantly shorter in the Ror2-/- mouse. DP, dental pulpa; EK, enamel knot; FNP, frontonasal process; IN, Incus; MA, malleus; MC, Meckel's cartilage; OC, oral cavity; OP, os petrosum; PA, palate; PI, pituitary gland; SB, sphenoid bone; TBP, trabecular basal plate; TG, tongue; TR, tympanic ring.

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As Ror2 is expressed in the pituitary gland and developing tooth germs, we analyzed the pituitary gland and teeth. We found a normal anterior pituitary gland with remnants of Rathke's pouch in the mutant at E16.5 (Fig. 2Q,R). Furthermore, analysis of teeth revealed that the shape, size, and number of teeth was normal at E16.5 (Fig. 2O,P).

External Genital

Analysis of the genital tubercle of E13.5 Ror2-/- mice showed that the tip of the genital tubercle has reached only half the size of the control embryo (Fig. 2S,T). Because the external genitals of both sexes do not differ until E14.5, determination of the gender of the mice was not performed. We also demonstrated that Ror2 expression is present in the genital tubercle (Fig. 1P). These findings suggest that Ror2 is involved in genital outgrowth, consistent with the finding of micropenis and hypoplasia of clitoris/labia minora in RS patients.

Somitogenesis

Ror2-/- mice exhibited spondylocostal defects characterized by severe vertebral malformations of the cervical and thoracic spine consisting of fused and split vertebrae and an irregular rib pattern (Fig. 3A,B). The tail of the Ror2-/- mutant was markedly shortened, and sacral vertebrae were significantly smaller, tilted, and partially fused with compressed or occasionally missing intervertebral discs (Fig. 3C–F). The histologic analysis showed that somites of Ror2-/- mice were reduced in the anteroposterior dimension and had an irregular cone-like shape at E13.5 (Fig. 3G,H). Cells forming the epithelium and the basolateral region had an irregular size and shape, whereas cells in the center of the somite appeared normal.

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Figure 3. Vertebral malformations in Ror2-/- mutant. A–F: Skeletal preparations of the vertebral spine of newborn wild-type (Wt) and Ror2-/- mice stained with alizarin red/Alcian blue. The size markers refer to the length of the mutant vertebra. A,B: Ventral view of Wt and Ror2 thoracic spine, showing fused and split vertebrae and an irregular rib pattern. C–F: Tail of Wt and Ror2-/- mutant, showing proximal (C,D) and middle (E,F) sacral vertebrae. Note that vertebrae are smaller, tilted, and partially fused, and intervertebral discs are compressed and occasionally missing. G,H: Hematoxylin and eosin staining of somites of Wt and Ror2-/- mutant. The somites of the mutant are smaller, as indicated by size markers, referring to the length of the mutant somite. The epithelial cells exhibit an irregular size and shape in the mutant. ID, intervertebral disc; V, vertebra; SM, somite.

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We analyzed the integrity of the PSM and somitogenesis in Ror2-/- mice with whole-mount in situ hybridization of developmental marker genes. For this purpose, embryos of stages E9.5 and E10.5 (Theiler stage 15 to 17) were used, when thoracic somites that are most severely affected in the mutant have been formed. In the Ror2-/- embryos, the size of the tail bud was reduced to approximately half the size of wt, as seen by the reduced area of expression of T (Fig. 4A,B), responsible for the formation of posterior mesoderm and axial development (Herrmann et al., 1990). In the notochord, T expression was normal, indicating the integrity of the notochord. The expression domain of Lunatic Fringe (Lfng), Dll1, and Notch1 was reduced, reflecting the smaller size of the PSM (Fig. 4C–J). However, these genes were expressed with normal intensity, and Lfng showed its regular oscillation pattern in the PSM and the posterior nascent (0) somite (Fig. 4C–F).

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Figure 4. Analysis of PSM marker genes in Ror2-/- mutant. Whole-mount in situ hybridization of tail buds of embryonic day (E) 9.5 (C–F) and E10.5 (A,B,G–N) wild-type (Wt) and Ror2-/- embryos with size markers referring to the mutant PSM. A,B: Expression of T shows a smaller PSM in the Ror2 mutant and a regular formed notochord. C–F: Cyclic expression pattern of Lfng expression in the PSM (C,D) and in the posterior part of the forming somite of control and mutant (arrows in E,F). G–J:Dll1 (G,H) and Notch1 (I,J) are expressed in a smaller PSM with regular intensity, indicating regular signalling of the Notch-Delta pathway. K,L: Expression of Mesp2 in the presumptive somite (−2, arrows) is reduced in the mutant, indicating that formation of the anterior portion of the somite is perturbed. M,N:Cer1 expression in the nascent and newly formed somite (arrows) is reduced in the mutant indicating that fewer cells in the PSM proliferate, and formation of the anterior somite portion is disturbed. NC, notochord; PSM, presomitic mesoderm; S 0, nascent somite; S + 1, newly formed somite; S −2, recently formed presumptive somite.

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To analyze the segmentation process of presumptive (−2, −1), nascent (0), and newly formed (+1) somites, we analyzed Mesp2 and Cerberus (Cer1) expression. Mesp2 is involved in the Notch signaling pathway and is needed for the initiation of somitogenesis and the specification of the anterior half of the somite (Burgess et al., 1996). The expression domain of Mesp2 in the recently formed presumptive somite (−2) was reduced in the mutant, indicating that fewer cells are available for the formation of somites (Fig. 4K,L). Consistently, Cer1, expressed in the nascent (0) and the anterior parts of the newly formed somite (+1; Biben et al., 1998), was reduced (Fig. 4M,N). Cer1 is not essential for murine development, as indicated by homozygous Cer1 knockout mice, which have no phenotype (Belo et al., 2000), but serves as a potent marker for somitogenesis.

To test whether the polarity of the somite was changed, we analyzed markers expressed in the anterior and posterior somite portion. Tbx18 expression in the anterior part of the somites (Kraus et al., 2001) was reduced in the mutant (Fig. 5I,J). Expression of Uncx 4.1 in the posterior halves of the somites (Mansouri et al., 1997) was also diminished but less than Tbx18 expression (Fig. 5K,L). These observations were most obvious in recently formed somites. Therefore, our findings indicate that fewer cells are available in the PSM of the Ror2-/- mutant, leading to a shortened tail and a reduced somite size, stronger affecting the anterior somitic compartment.

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Figure 5. Analysis of somitogenesis in Ror2-/- mutant. Whole-mount in situ hybridization and vibratome sections of prospective thoracic somites of embryonic day (E) 10.5 wild-type (Wt) and Ror2-/- embryos with size markers referring to the size of the somite in the mutant. A–F: Expression of Myf5 (A,B), Pax9 (C,D), and Paraxis (E,F) indicates formation of myotome, sclerotome, and dermomyotome in Wt and mutant with considerable epithelialization defects (arrow in B). The size markers in E and F show that the size of the somites is markedly reduced in the mutant. G,H:Fgf4 is expressed irregularly in the mutant, indicating improper positioning of epithelial somitic boundaries (arrow in H). I,J:Tbx18 expression in the anterior portion of the somite is reduced in the mutant. The size markers indicate smaller somites in the mutant. K,L:Uncx4.1 expression demarcates the posterior segments of somites. The posterior portion is reduced in the mutant, and these segments are tilted (arrow in L).

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Next, we analyzed markers involved in the specification of the myotome (Myf5, Fig. 5A,B), the sclerotome (Pax9, Fig. 5C,D), and the dermomyotome/sclerotome (Paraxis, Fig. 5E,F). The expression of these marker genes indicate that the cells of the somitic mesoderm differentiate into myotomal, sclerotomal, and dermomyotomal subpopulations. However, the expression patterns of Myf5, Pax9, and Paraxis were altered, as the regular expression stripes were perturbed, tilted, and sometimes fused, indicating an epithelialization defect in the mutant. The analysis of the epithelial marker Fgf4 (Fig. 4G,H), required for proper positioning and epithelialization of somitic boundaries (Dubrulle and Pourquie, 2002), confirmed that somite epithelialization is perturbed in the mutant.

Limb Development

Ror2-/- animals exhibited shortening of the long bones of the appendicular skeleton. The zeugopode was affected strongest in fore- and hindlimbs and was shortened by approximately 50%, whereas stylopode and autopode were only slightly reduced, leading to mesomelic shortening of the limb. Consistently, the zeugopode exhibited considerable delay of ossification, as seen by the loss of alizarin red staining, whereas the stylopode and autopode showed only a discrete delay of ossification. Approximately 20% of Ror2-/- animals showed a duplicated first digit of the right hindlimb, reminiscent of bifid thumbs of RS patients (data not shown).

To analyze the limb reduction defect in more detail, we compared histologic sections of humerus, radius, and ulna of E15.5 mutant embryos with wt (Fig. 6). During endochondral bone formation, mesenchymal cells condense and form chondrocytes that subsequently proliferate, hypertrophy, and are finally replaced by bone (Mundlos, 2000). We compared the different zones of the growth plate (Fig. 6; resting chondrocytes are referred to as immature due to their residual proliferation during embryogenesis) for their morphology and rate of proliferation. The mutant immature chondrocytes, located at the more distal part of the humerus and ulna, appeared normal, with a regular size and shape as well as normal proliferation. When chondrocytes differentiate further, they adopt a flat shape and arrange themselves in columns, referred to here as columnar (syn.: proliferating/prehypertrophic) chondrocytes. In the humerus of the mutant, however, few columns were observed. Instead, the chondrocytes had an oval shape, and the columnar zone was significantly reduced (Fig. 6A). In the ulna of the mutant, this was even more pronounced. Chondrocytes had a round shape, remained immature, and no columns were established at E15.5 (Fig. 6B). Next, chondrocytes undergo hypertrophy in the wt, whereas in the humerus of the mutant fewer and smaller hypertrophic chondrocytes were formed, resulting in a reduced hypertrophic zone (Fig. 6A). Corresponding to the more pronounced affection of the zeugopode, no hypertrophic chondrocytes were observed in the ulna of the mutant at E15.5 (Fig. 6B).

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Figure 6. Histologic analysis of growth plate in Ror2-/- mutant. A: Growth plate of the humerus of wild-type (Wt) and Ror2-/- mutant at embryonic day (E) 15.5 with hematoxylin and eosin staining (lateral) and bromodeoxyuridine (BrdU) proliferation analysis (center) shows a reduced columnar and hypertrophic zone. The arrangement of the columnar chondrocytes is perturbed, and the hypertrophic chondrocytes are smaller. Note regular proliferation as seen by brown BrdU staining in immature and columnar chondrocytes. B: Growth plate of the ulna of Wt and Ror2-/- as in A with loss of the columnar and hypertrophic zone in the mutant. The Ror2-/- chondrocytes show features comparable to immature chondrocytes in the Wt. IC, immature chondrocytes; CC, columnar chondrocytes; HC, hypertrophic chondrocytes.

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We compared the chondrocyte proliferation rate in different zones of wt and mutant. The proliferation of chondrocytes of the ulna of Ror2-/- mutants corresponds to the proliferation rate of the wt immature chondrocytes of the ulna (25.8 vs. 24.6%). In the columnar zone of the humerus, the proliferation was similar in mutant and wt (27.3 vs. 28.5%). These findings indicate that the skeletal phenotype in the Ror2-/- mutant is not caused by altered proliferation characteristics of the chondrocytes.

To closer analyze the disturbed chondrocyte differentiation, we tested marker genes for chondrogenesis. Collagen 2 (Col2a1) expression was normal in limbs of Ror2-/- mice, indicating regular formation of cartilage (Fig. 7A,B,G,H,M,N). Indian hedgehog (Ihh) is expressed in columnar chondrocytes and determines the number of chondrocytes undergoing differentiation to hypertrophic chondrocytes. Ihh was markedly reduced in the growth plate of the humerus and absent in ulna/radius of E15.5-old Ror2-/- mice (Fig. 7C,D,I,J), indicating a delay in chondrocyte differentiation. Collagen10 (Col10), which is normally expressed in the hypertrophic cartilage, was severely reduced/absent in humerus and radius/ulna, respectively, indicating defects in formation of hypertrophic chondrocytes (Fig. 7E,F,K,L). Expression of Pthlh, known to coordinate chondrocyte maturation by means of a Ihh/Pthlh feedback loop, was only detectable in low amounts at E15.5 in both wt and mutant (not shown).

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Figure 7. Analysis of chondrocyte differentiation in forearms in Ror2-/- mutant. 33P-labeled in situ hybridization analysis with chondrogenic marker genes of wild-type (Wt) and Ror2-/- humerus, ulna/radius at embryonic day (E) 15.5, and digits at E13.5. A,B:Col2 expression in the mutant humerus indicates regular formation of chondrocytes with delay of hypertrophy. C,D:Ihh expression is diminished in the mutant humerus, reflecting a delay of chondrocyte differentiation (arrow). E,F:Col10 expression is present in hypertrophic chondrocytes of humeri of Wt but not in Ror2-/- mice (arrow in F). G,H:Col2 expression in Wt and mutant ulna/radius. I–L:Ihh (I,J) and Col10 (K,L) expression is seen in the columnar/prehypertrophic and hypertrophic chondrocytes of the ulna/radius in Wt but not in Ror2-/- mice (arrows in J,L). M,N:Col2 expression in Wt and mutant hands. O,P:Ihh expression in hands of Wt and Ror2-/- mice is reduced in the digits of mutant mice (arrow in P).

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As missing middle phalanges have been reported in mice generated in another Ror2-/- loss of function mouse model (DeChiara et al., 2000), we analyzed the digits of Ror2-/- mice. We could not confirm the finding of missing middle phalanges in the present mice, however, metacarpals and digits had a shortened and thickened appearance. In the digits Col2 expression was normal, indicating the presence of chondrogenic condensations (Fig. 7M,N). Ihh expression was reduced, suggesting a delayed entry of chondrocytes into prehypertrophy as in the more proximal elements (Fig. 7O,P). Ihh target genes, Ptc, Gli1, Gli3, Pthlh, and Pthr1 did not exhibit any significant expression differences in the digits of the mutant (not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Genetic disorders of the skeleton comprise a large group of disorders with a wide spectrum of manifestations (Kornak and Mundlos, 2003). Their developmental origin illustrates the importance of multiple genes during skeletal development. Loss-of- function mutations in the RTK Ror2 are responsible for recessive Robinow syndrome, a multisystem disorder characterized by short stature; skeletal malformations of the face, spine, and limbs; as well as heart defects, developmental delay, and microgenitals. In the present study, we analyzed the developmental malformations of the Ror2-/- mouse as a model for human Robinow syndrome. In addition, the Ror2 expression pattern was evaluated for its functional relevance in mouse and human.

Ror2 Is Necessary for Craniofacial and Ear Development

Absence of Ror2 in mice resulted in a shortened mandible, defects of derivatives of Meckel's cartilage, a cleft palate, thickening of the nasal capsule, and the presence of ectopic cartilages. These findings overlap with many of the phenotypic aspects seen in Robinow patients, exhibiting characteristic midline and orofacial features, including hypertelorism, midface hypoplasia, and dental abnormalities. Of interest, the Ror2 mutant exhibits some additional craniofacial malformations, including defective ear ossicles, a cleft palate, and an additional skull bone, that have not been reported in Robinow patients.

During development, the lower jaw is formed from the mandibular component of the first pharyngeal arch. The pharyngeal arch is a bilateral structure that consists of two processes that grow out from both sides of the future mouth cavity and fuse at their distal tips. Later on during development, Meckel's cartilage is formed and serves as a lead structure for the formation of the mandible, that forms around it by desmal ossification. Ror2 is strongly expressed around Meckel's cartilage, suggesting that Ror2 is necessary for mandibular outgrowth and determination of its distal tip. In Ror2-/- mice, Meckel's cartilage is significantly shortened, and the tip of Meckel's cartilage points downward in the mutant, compared with an upward orientation in the wild-type. This could result in the triangular mouth shape seen in some Robinow patients. In addition, the clefting of the mandible in Ror2-/- mice can be explained by a delayed outgrowth of the mandibular processes, that fail to meet and fuse at the appropriate time. The mutant phenotype thus correlates with the midline clefting of the lower lip occasionally seen in RS patients.

Ror2 is also expressed around the malleus and incus, which are derived from the most proximal part of Meckel's cartilage, and mutants show a disruption of the malleus. It is possible that the defective malleus is a result of the presence of an ectopic cartilage between the anterolateral process of the ala temporalis and the tegmen tympani, resulting in a physical barrier for malleus formation. The presence of ectopic cartilages could be linked with a role for Ror2 in regulating chondrocyte function. To our knowledge, hearing defects have not been reported to be associated with RS.

In addition, the broadening of the nasal capsule in the mutant is remarkable, as a broad nose appears to be a typical feature of patients with recessive RS, indicating a perturbed midline patterning. Another midline developmental defect present in the Ror2-/- mouse, but not in RS patients is a cleft palate. In Robinow patients, some milder features of orofacial midline affection have been described as lower lip, ankyloglossia, and a bifid tongue. For palate closure during embryonic development, the maxillary framework containing the lateral palatine processes and the nasal septum must be established. Ror2 is strongly expressed in the palatal shelves, the nasal septum, and capsule, indicating its importance during the fusion of the secondary palate.

Numerous genes are required for proper closure of the secondary palate. Mice with deficient Prx1/2 (ten Berge et al., 1998), Msx1 (Satokata and Maas, 1994), and Dlx5/6 (Robledo et al., 2002) exhibit craniofacial and ear defects. Recent findings indicate that Ror2 associates with Dlxin-1 and regulates its intracellular distribution and affects the transcriptional function of Msx2 (Matsuda et al., 2003). Dlxin-1 has been shown to also bind Dlx5 and regulate its transcriptional function. These findings indicate that Ror2 exerts a critical function for craniofacial development and that some of the craniofacial malformations in Ror2-/- mice may be transferred by genes of the Msx and Dlx families.

Ror2 Is Essential for Somite Formation

Consistent with vertebral malformations of the spine and rib fusions of patients with RS Ror2-/- mice exhibited spondylocostal malformations and a short tail. The analysis performed indicates that Ror2 is needed for the formation of the tail bud and the subsequent formation and epithelialization of somites. The histologic and molecular analysis showed that the PSM and the somites were markedly reduced in size in the mutant. The expression domains but not the intensity of expression of T, Dll 1, Notch 1, and Lfng was diminished, reflecting a smaller PSM with normal oscillations. Somites were formed in regular time intervals, but the presumptive, nascent, and newly formed somites of the mutant were smaller, as reflected by reduction of Mesp2 and Cer1. Consistent with the role of Mesp2 in initiation of somite segmentation and formation of the anterior half of the somite, Mesp2-null embryos show severe somite defects due to a lack of the rostral somitic compartment (Takahashi et al., 2000).

Misexpression of Paraxis and Fgf4 in Ror2-/- mutants shows defects in the epithelial formation of the somite. Paraxis is needed for the formation of epithelial somites and in Paraxis-null mice no epithelia form in the paraxial mesoderm (Burgess et al., 1996). Epithelialization defects subsequently lead to a perturbed formation of the sclerotome in differentiating somites as seen by misexpression of Pax9, Tbx18, and Uncx4.1. Taken together, our findings suggest that the small PSM in the Ror2 mutant results from reduced number of cells. The subsequent defects in somite formation cause a disturbed segmentation process with emphasis on the anterior somite portion as well as defects in epithelialization and sclerotome formation.

Other mouse mutants with short tails and defective somitogenesis comprise the Wnt3a and Wnt5a knockout mouse (Takada et al., 1994; Yamaguchi et al., 1999). The zebrafish mutant ogon, encoding a zebrafish homologue of Secreted Frizzled (Yabe et al., 2003), and pipetail, a hypomorphic allele of Wnt5a (Rauch et al., 1997), also exhibit short tails. Consecutively, it has been shown, that Wnt/beta-catenin signaling is involved in the establishment of the segmentation clock and that Wnt3a controls the segmentation process in vertebrates (Aulehla et al., 2003). Of interest, Ror2-/- and Wnt5a-/- (Yamaguchi et al., 1999) mice exhibit similar phenotypes, and Ror2 recently has been associated with the Wnt/JNK signaling pathway (Oishi et al., 2003). The experiments performed show that Wnt5a binds to the cysteine-rich domain (CRD) of Ror2, and Ror2 associates by means of the CRD with rFz2, a putative receptor for Wnt5a.

Size reduction of the PSM appears to be a characteristic Ror2 phenotype. It is believed, that the size of the PSM depends on an equilibrium between cells being removed from the rostral end for the formation of a new somite and the supply of new paraxial mesoderm cells from primitive streak at the caudal end of the PSM (Tam, 1986). Taking this into account, the expression domain of Ror2-/- in the primitive streak, the PSM, and the somite epithelium indicates that Ror2 is required to transfer signals for proliferation and/or differentiation of cells in the primitive streak, the PSM, and the somites. The reduction of the PSM and the defects in somite formation and differentiation in the Ror2-/- mutant together with the association of Ror2 with the Wnt/JNK pathway supports this concept.

Ror2 Is Required for Growth of the Appendicular Skeleton

Correct cartilage morphogenesis requires integration of proliferation and hypertrophy and defines the longitudinal axis of an appendicular skeletal element. Ror2-/- animals exhibited severe shortening of radius and ulna, corresponding to limb defects in human autosomal recessive RS, characterized by mesomelic limb shortening.

The histologic and molecular analysis demonstrated a perturbed chondrocyte differentiation and delay of ossification in all long bones of the appendicular skeleton in the mutant. At the same time, we found a normal rate of proliferation. Columnar chondrocytes had an oval shape, showed a poor columnar organization, and failed to undergo regular hypertrophy. Consistently the columnar and hypertrophic zones were reduced or absent in the mutant, and marker genes expressed in these zone were diminished or lost. Ihh is expressed in columnar/prehypertrophic chondrocytes and regulates chondrocyte proliferation and chondrocyte hypertrophy (Karp et al., 2000; St-Jacques et al., 1999). In the growth plate of Ror2-/- mutants, Ihh expression was reduced, reflecting a delay of differentiation of columnar chondrocytes. This finding suggests that Ror2 is an upstream positive regulator of the hedgehog (Hh) signaling pathway. Ihh knockout mice exhibit strongly reduced chondrocyte proliferation, maturation of chondrocytes at inappropriate position, and a failure of osteoblast development in endochondral bones (St-Jacques et al., 1999). In humans, homozygous mutations in IHH cause acrocapitofemoral dysplasia (Hellemans et al., 2003), an autosomal recessive disorder with short stature and cone-shaped epiphyses in hands and hips, and heterozygous mutations in the signaling domain lead to brachydactyly type A1 (Gao et al., 2001). In addition, Col10 expression was absent in the humerus and radius/ulna of the mutant at E15.5, indicating the failure to form proper hypertrophic chondrocytes. These findings and the expression pattern of Ror2 in cartilage suggest that Ror2 is needed to confer signals needed for chondrocyte maturation and differentiation, but not for proliferation.

Ror2 Is Necessary for Genital Outgrowth

In Robinow syndrome, a micropenis is present in males and may cause concern regarding gender assignment at birth. In females, there is hypoplasia of the clitoris and of the labia minora. The mechanisms that regulate early development of the genital tubercle are not well understood. Several genes, including Shh (Haraguchi et al., 2001), Fgf (Haraguchi et al., 2000), and Hox genes (Mortlock and Innis, 1997), have been implicated in the control of genital outgrowth. Loss of Wnt5a leads also to impaired distal outgrowth of limbs and the genital tubercle (Yamaguchi et al., 1999). Of interest, the development of limbs and genitals show some similarities, as both result from apical growth and involve epithelial–mesenchymal interactions and use similar genes (Kondo et al., 1997). In the Ror2-/- mouse, the genital tubercle is shortened, and Ror2 is strongly expressed in the genital tubercle at E13.5. These findings indicate that Ror2 is needed for the outgrowth of the genital primordium.

Although androgens play an important role during external genital development, outgrowth and patterning of the genitalia take place before the onset of androgen production. Normal androgen receptor activity and levels of 5α reductase activity have been shown in genital skin fibroblasts of RS patients (Lee et al., 1980). Low basal testosterone and a low testosterone response to human chorionic gonadotropin stimulation together with an empty sella in the majority of RS patients indicate a low sensitivity to circulating testosterone concentrations (Soliman et al., 1998). Of interest, we could demonstrate Ror2 expression in the anterior lobe of the pituitary gland at E14.5 and E16.5. However, in the Ror2-/- mutant the morphology of the pituitary gland appeared normal. These findings suggest that Ror2 may be directly or indirectly involved in the regulation of androgen sensitivity or stimulation.

In conclusion, our results indicate that Ror2 is essential at multiple sites during organogenesis providing a developmental basis for craniofacial, somite, limb, and genital development of human Robinow syndrome. The wide-spread expression pattern of Ror2 suggests that Ror2 may interact with a large number of molecular partners in different organs. The phenotypic analysis of men and mice with loss of Ror2 function may indicate subtle differences between different species and in different organ systems or some residual Ror2 function in human individuals with Robinow syndrome.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Mice

Research on Ror2−/−mice was approved by the institutional review board. Ror2-/-mice were obtained from Yasuhiro Minami, Department of Genome Sciences, Kobe University, Japan. The mutants carry an inserted neomycin gene fused to a HSV-TK cassette that replaces the Ror2 exon 1 (Takeuchi et al., 2000). Analysis of the phenotype was performed on a C57Bl6 background by mating of heterozygous knockout (k.o.) animals. The mice were genotyped by using a multiplex polymerase chain reaction (PCR) approach with a forward primer located within the 5′ UTR of Ror2 (5′- ctt aac tgt tct agg tca agt atg -3′) together with reverse primers located in the neomycin gene for k.o. (5′- atc gcc ttc tat cgc ctt ctt gac gag -3′) and in the exon harboring the Ig-like domain for wt (5′-cct act ata gac tct gat cct tct gcc -3′). PCR products had a fragment length of approximately 200 bp for the k.o. allele, and 500 bp for the wt allele. The following reaction conditions were used 95°C for 4 min, followed by 35 cycles at 95°C for 40 sec, 61°C for 40 sec, 73°C for 40 sec, and a final extension at 73°C for10 min. PCR was performed in 25-μl reactions containing 1× PCR buffer, 1.5 mM MgCl2, 100 μM of each dNTP, 1 μM of each primer and five units of Taq polymerase. PCR-amplified DNA products were visualized on 1.2% ethidium bromide–stained agarose gels.

Phenotype and Developmental Analysis

Timed matings were produced, and DNA from extraembryonic membranes served for genotyping. For skeletal preparations, four animals were analyzed for each time point at E14.5, E16.6, E18.5, and newborns. Skeletal preparations were performed as described previously (Mundlos, 2000). For histologic analysis, embryos and tissues were embedded in paraffin and sectioned at 7 μm. Only forelimbs were used and stained with hematoxylin and eosin.

In Situ Hybridization

Whole-mount in situ hybridization was performed as described previously (Albrecht et al., 2002) by using embryos of stages E10.5 obtained by timed matings from the Ror2 k.o. cross. Analysis and photography of embryos was performed by using a binocular microscope and camera (Leica, Bensheim, Germany). Results were confirmed testing three mutant embryos for each stage and probe and compared with wt. Sections of whole-mount stained embryos were performed as described previously (Nacke et al., 2000).

For section in situ hybridization analysis, embryos and tissues were prepared as described for histologic procedure. Section in situ hybridization was performed on paraffin-embedded tissue by using 33P-labeled antisense RNA-probes as described previously (Vortkamp et al., 1996). Pretreatment with proteinase K was 3 min, 10 μg/ml at room temperature and hybridization was performed overnight at 70°C. Slides were washed and dipped in photoemulsion (Kodak, Rochester, NY), dried and exposed for 2 to 8 days. For counterstaining 0.1% toluidine blue-O was used. Slides were mounted, analyzed using darkfield microscopy, and documented using a digital camera (Leica, Bensheim, Germany).

Antisense specific riboprobes for whole-mount and section in situ hybridization were specific for mouse, except Col2 that derived from rat cDNA. Probes were as follows: rat Col2 (Kohno et al., 1984), Col10 (Jacenko et al., 1993), Ihh (Bitgood and McMahon, 1995), Fgf4 (Niswander et al., 1994), Gli1 (Hui et al., 1994), Mesp2 (Saga et al., 1997), Myf5 (Montarras et al., 1991), Notch1 (Conlon et al., 1995), Paraxis (Burgess et al., 1995), Uncx4.1 (Mansouri et al., 1997), and Tbx18 (Kraus et al., 2001).

Riboprobes for Ror2, Dll1, Pax9, and T-Gen were produced by using PCR amplifications of gene-specific exon. The amplifying primers were as follows: Ror2, 5′-GGT ACT CCA ACC AGG ACG TG-3′ and 5′-CCC AGG AGT TCA GTC TCA GG-3′; Dll1, 5′-GAT CAT TGA AGC CCT CCA TAC-3′ and 5′-GGG TAT CGG ATG CAC TCA TC-3′; Pax9, 5′-GTA CTG CTC AGA GCA ATG GA-3′ and 5′-CAG AAT GTC CGT GAC GGA GT-3′; T, 5′-TGT TCT ACA GCC TCT TGT TTG-3′ and 5′-TTT CTG CAG ATT GTC TTT GGC-3′. Amplified exons were cloned using TOPO TA Cloning kit (Invitrogen, Carlsbad, CA) containing RNA polymerase SP6 and T7 promoters.

Bromodeoxyuridine Proliferation Analysis

Chondrocyte proliferation was analyzed by using bromodeoxyuridine (BrdU) labeling of pregnant mice with intraperitoneal injection with 0.3 mg BrdU/g body weight dissolved in phosphate buffered saline (PBS, 10 mg/ml). We collected E15.5 embryos after 8 hr. Forelimbs were dissected, fixed in 4% paraformaldehyde, and embedded in paraffin. Sections were pretreated with proteinase K (Roche, Mannheim, Germany; 10 μg/ml for 10 min at 37°C) and with hyaluronidase (Sigma-Aldrich, Taufkirchen, Germany; 10 mg/ml in PBS for 1 hr at 37°C) and 2 N HCl (10 min at 37°C). Detection of labeled DNA was performed as described by the manufacturer (Roche, Mannheim, Germany). The percentage of positive cells within the humerus and ulna of E15.5 limbs was counted in immature and columnar chondrocytes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Achim Gossler, Andreas Kispert, Gail Martin, and Peter Gruss for providing some of the used in situ probes. The expert technical assistance of Britta Hoffman is appreciated, along with the observations on the craniofacial phenotype made by Michael DePew. S.M. was funded by a grant from the Deutsche Forschungsgemeinschaft to S.M.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES