QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 19 March 2008
doi: 10.1242/dev.015149

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Raz, R. Articles by Economides, A. N. PubMed PubMed Citation Articles by Raz, R. Articles by Economides, A. N.

The mutation ROR2^W749X, linked to human BDB, is a recessive mutation in the mouse, causing brachydactyly, mediating patterning of joints and modeling recessive Robinow syndrome

¹ Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.
² Max-Planck Institute for Molecular Genetics Berlin, and Institute for Medical Genetics, University Medicine Berlin, Charité, Berlin, Germany.
³ Saint Francis Hospital and Medical Center, Hartford, CT, USA.
⁴ The University of Connecticut School of Medicine, Farmington, CT, USA.

View larger version (39K): [in this window] [in a new window]   Fig. 1. Targeted truncation of ROR2 at W749. (A) Structure of the ROR2 receptor showing the immunoglobulin-like (Ig), frizzled-like (Frz), kringle (Kr), transmembrane (TM), tyrosine-kinase (TK), serine/threonine-rich (ST1, ST2) and proline-rich (PR) domains, and the site of the BDB mutation (black line). SP, signal peptide. (B) Targeting strategy in the region spanning the last exon of Ror2, encoding the TK and ST1/PR/ST2 domains. The targeting vector carries a replacement of the region encoding W749-D930 with a FLAG-encoding sequence fused in frame, followed by a selectable marker (NEO, neomycin resistance gene). Target sites for primers are indicated by white (cDNA genotyping) and black (genomic genotyping) arrowheads. (C) PCR analysis of yolk sac DNA from wild-type and mutant embryos showing the products from the wild type (WT, 419 bp) and targeted (T, 174 bp) alleles. (D) RT-PCR analysis confirming the loss of wild type Ror2 mRNA (455 bp product, WT) in Ror2W749FLAG/W749FLAG E15.5 embryos (KI), and the presence of mRNA for the truncated ROR2 (321 bp product, T). (E) Immunoblot analysis of ROR2 receptor from wild-type (WT) and Ror2W749FLAG/W749FLAG (KI) tissues. Antibodies used for immunoprecipitation (IP) and western (W), and sizes (kDa) and migration of molecular standards, are indicated. (F,G) Confocal microscopy of full-length and truncated ROR2 receptors overexpressed in Cos1 cells.

View larger version (57K): [in this window] [in a new window]   Fig. 2. Reduced body mass, altered body composition and skeletal defects of Ror2W749FLAG/W749FLAG mutants. (A,B) Comparison of 5-month-old control (A) and Ror2W749FLAG/W749FLAG littermate (B) mice; the mutants display a smaller body and a shorter, kinked tail. (C-F) Radiographic analysis of skeletons from wild-type (C) and Ror2W749FLAG/W749FLAG (D-F) adult mice. Ror2W749FLAG/W749FLAG animals display reduced length of the axial skeleton due to shorter vertebrae (D) and have a missing phalange in digits II-IV (inset, arrowheads). (E) Distal tail radiography from two mutant mice showing fused vertebrae (arrowhead) and hemivertebrae with misaligned articular surfaces, causing tail kinks (arrows). (F) Right hindlimb radiography from a Ror2W749FLAG/W749FLAG mouse with preaxial polydactyly (arrow). Note a missing phalanx in all the other digits (arrowhead). (G) Weight curves of heterozygous (HET) and homozygous (KI) Ror2W749FLAG male mice from a single litter, n=3-4. (H) Measurements of lean and fat mass in Ror2W749FLAG/W749FLAG mice (KI) and litter-matched wild-type controls (WT). (I) Percentage fat mass (white stacked bar) and lean mass (dotted bar) in Ror2W749FLAG/W749FLAG mice (KI) and controls (WT). Eight Ror2W749FLAG/W749FLAG and five wild-type littermate male mice were evaluated at 21 weeks of age (H,I). Values are means±s.e.m. Significantly different from controls: **P<0.01; *P<0.05.

View larger version (39K): [in this window] [in a new window]   Fig. 3. The craniofacial defects in Ror2W749FLAG/W749FLAG mutants are due to changes in the nasal bones and orbital width. (A) Facial phenotype of Ror2W749FLAG/W749FLAG mice (right), showing shorter snout, increased intercanthal distance and entropion eyes. (B-D) Comparison of crania from heterozyogous control (left) and homozygous mutant (right) Ror2W749FLAG mice (9-month-old males), viewed from dorsal (B) and lateral (C,D) aspects. The double arrows indicate the craniometric linear measurements that showed significant differences: (1) nasal length, (2) intermaxillar width, (3) intraorbital width and (4) mandible anterior length. (E-H) Average lengths and widths (mm) of craniometric parameters that were significantly different between Ror2W749FLAG/W749FLAG mice and control littermates. Values represent the mean±s.e.m. of triplicate measurements from five heterozyogous (HET) and five homozyogous (KI) mice. Significantly different from controls: **P<0.01; *P<0.05.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Transient decrease in trabecular bone volume in Ror2W749FLAG/W749FLAG mutants. (A,B) Representative femur sections from a 3-week-old Ror2W749FLAG/W749FLAG mouse (B) and a wild-type littermate (A), stained with Toluidine Blue. (C) Static and dynamic histomorphometric parameters in Ror2W749FLAG/W749FLAG male mice (white bars) and control littermates (black bars) at 3, 8 and 24 weeks. Parameters analyzed were: bone volume/tissue volume (BV/TV, %); trabecular number (Tr.N,/mm); trabecular separation (Tr.Sp, µm); number of osteoblasts/tissue area (N.Ob/T.Ar,/mm2); osteoblast surface/bone surface (Ob.S/BS, %); mineralizing surface/bone surface (MS/BS, %); mineral apposition rate (MAR, µm/day); bone formation rate/bone surface (BFR/BS, µm3/µm2/day). For static histomorphometry, sections were stained with Toluidine Blue. For dynamic histomorphometry, unstained sections were analyzed by fluorescence microscopy. Values are means±s.e.m. (n=6-11). Significantly different from controls: **P<0.01; *P<0.05.

View larger version (58K): [in this window] [in a new window]   Fig. 5. Compromised fertility of Ror2W749FLAG/W749FLAG males. (A) Percentage of test females that became plugged or pregnant after controlled mating with Ror2+/W749FLAG (gray bars, HET) or Ror2W749FLAG/W749FLAG (black bars, KI) littermate mice. Values are mean±s.e.m. (n=60 matings, 10 matings per male). (B) Relative combined testicular weight, expressed as a per mille () of body weight (BW), in Ror2W749FLAG/W749FLAG (black bars, KI) and control (gray bars, HET) littermates. Values are mean±s.e.m. (n=6, 2- to 3-month-old males). Significantly different from controls: **P<0.01; *P<0.05. (C-F) Histology of heterozygous (C,E) and homozygous (D,F) Ror2W749FLAG testes from 16-day-old animals. The homozygous gonad exhibited decreased tubular density, with seminiferous tubules containing a single cell layer (asterisk). (G-I) Histology of a Ror2W749FLAG/W749FLAG testes (2-month-old) showing a focal area of tubular degeneration (G, arrow). (H) High magnification of a normal seminiferous tubule in the mutant testis. (I) High magnification of an abnormal seminiferous tubule, lined by clumps of Sertoli cells along the basal membrane and devoid of spermatogonia or spermatids.

View larger version (47K): [in this window] [in a new window]   Fig. 6. The defects in skeletogenesis in Ror2W749FLAG/W749FLAG mutants stem from abnormalities in early chodrogenic condensation and specification of the distal digital joint. (A-I) Whole-mount cartilage staining of Ror2W749FLAG/W749FLAG (center) and heterozygous litter-matched E14.5 embryos (left), compared with Ror2TMlacZ/TMlacZ embryos (right). (A-C) Lateral view showing the reduction of all skeletal elements in Ror2W749FLAG/W749FLAG and Ror2TMlacZ/TMlacZ embryos. (D-F) Dorsal view of the thoracic spine. Ror2W749FLAG/W749FLAG and Ror2TMlacZ/TMlacZ vertebrae are hypoplastic and irregularly stacked (arrows). (G-I) Dissected forelimbs (top) and hindlimbs (bottom), showing reduced cartilage condensations in the homozygous mutant embryos. Note the absence of joint formation in the phalanx anlagens of the mutant forelimbs (arrowheads) and the greater reduction of condensation size in the Ror2TMlacZ/TMlacZ limbs. (J-T) Cleared skeletal preparations from control Ror2+/W749FLAG (left), littermate Ror2W749FLAG/W749FLAG (center) and Ror2TMlacZ/TMlacZ (right) E18.5 embryos. (J-L) Side view of skulls showing shortened nasal bones in the homozygous mutant embryos. (M-O) Dorsal view of ribcages showing smaller and tilted vertebrae in Ror2W749FLAG/W749FLAG and Ror2TMlacZ/TMlacZ embryos (N,O, arrows). Fused ribs were present with full penetrance in Ror2TMlacZ/TMlacZ embryos (O). (P-R) View of the hindlimb showing delayed ossification of metacarpals and phalanges in the homozygous mutant embryos (Q,R, arrows) and a severely misshapen Ror2TMlacZ/TMlacZ stylopod (arrowhead). (S-U) Comparison of control Ror2+/W749FLAG and Ror2W749FLAG/W749FLAG forepaws (S,T) with a Ror2-/- forepaw (U). Note the normal number of phalanges in the Ror2-/- digits despite being as affected in length as the Ror2W749FLAG/W749FLAG digits.

View larger version (113K): [in this window] [in a new window]   Fig. 7. Anlagen growth and chondrocyte proliferation are unaffected in Ror2W749FLAG/W749FLAG embryos. (A,B) Forelimb sections from Ror2+/W749FLAG (HET, A) and Ror2W749FLAG/W749FLAG (KI, B) E13.5 embryos stained with HAB reveal decreased cell numbers in the cartilagenous condensations of the mutant limbs. Stylopod (s), zeugopod (z) and autopod (a) are indicated. (C-F) High magnification of E13.5 ulna sections stained with HAB (C,E) or HGF (D,F), showing normal cell density and intensity of matrix staining. (G,H) Forelimb sections from Ror2+/W749FLAG (G) and Ror2W749FLAG/W749FLAG (H) E15.5 embryos stained with HAB and HGF. No overt histological alterations are observed in cell density or matrix staining, but the Ror2W749FLAG/W749FLAG bones are shorter, and have a smaller region of hypertrophy and primary spongiosa. (I,J) Sections of E18.5 humeri from three individual Ror2+/W749FLAG (I) and Ror2W749FLAG/W749FLAG (J) embryos stained with an anti-BrdU antibody and counterstained with AB. Ror2W749FLAG/W749FLAG humeri have a reduced length and shorter epiphyses (epiphysis range is indicated with horizontal lines). No difference was observed in the density or distribution of BrdU-positive cells. (K-N) BrdU incorporation analysis in E12.5 digital rays (K,L) and proliferative zone of postnatal P5 femurs (M,N, zone delineated) from Ror2+/W749FLAG (HET) and Ror2W749FLAG/W749FLAG (KI) littermate mice. (K,L) Proliferative rates were assayed in E12.5 rays by measuring the number of BrdU-positive nuclei (red) relative to the total number of nuclei (blue) in the condensation. No significant difference was observed between the genotypes (12.2±0.04% versus 12.5±0.05% in the controls, P=0.069). (M,N) BrdU incorporation analysis in postnatal P5 femurs showed no significant difference between genotypes (383±41.9 versus 373±60.5 cells/mm2 in the controls, P=0.90). (O,P) High magnification of the growth plates from the sections shown in M and N, respectively, revealing a normal architecture of the columnar chondrocytes.

View larger version (111K): [in this window] [in a new window]   Fig. 8. Absence of Gdf5 expression in the distal phalangeal interzone of Ror2W749FLAG/W749FLAG mutants. Gdf5 expression was analyzed by in situ hybridization in limbs from wild-type (WT, A,C,E) and Ror2W749FLAG/W749FLAG (B,D,F) embryos. (A,B) Whole-mount in situ hybridization of forelimbs from E12.5 embryos, showing identical patterns of Gdf5 expression in the mutant. (C,D) Sections of E13.5 forelimbs, revealing reduced expression of Gdf5 in the proximal phalangeal interzone of Ror2W749FLAG/W749FLAG digits (D, 1/2). (E,F) Sections from E15.5 digits, showing normal Gdf5 expression levels in the proximal phalangeal interzone of the Ror2W749FLAG/W749FLAG digit (F, 1/2), but absence of signal in the presumptive distal interzone (2/3). MC, metacarpal; P1, phalanx 1; P2, phalanx 2.