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First published online September 9, 2005
doi: 10.1242/10.1242/dev.02011

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A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia

Ann M. Flenniken^1,*, Lucy R. Osborne^1,2,3,*, Nicole Anderson⁴, Nadia Ciliberti⁵, Craig Fleming¹, Joanne E. I. Gittens⁶, Xiang-Qun Gong⁶, Lois B. Kelsey¹, Crystal Lounsbury⁷, Luisa Moreno⁸, Brian J. Nieman^9,10, Katie Peterson¹, Dawei Qu⁸, Wendi Roscoe⁷, Qing Shao⁷, Dan Tong⁶, Gregory I. L. Veitch^6,7, Irina Voronina¹, Igor Vukobradovic¹, Geoffrey A. Wood¹, Yonghong Zhu¹¹, Ralph A. Zirngibl³, Jane E. Aubin^1,3, Donglin Bai⁶, Benoit G. Bruneau^3,11,12, Marc Grynpas^1,13, Janet E. Henderson¹⁴, R. Mark Henkelman^9,10, Colin McKerlie^1,13,15, John G. Sled^9,10, William L. Stanford^1,4,5, Dale W. Laird^6,7, Gerald M. Kidder⁶, S. Lee Adamson^1,12,16 and Janet Rossant^1,3,

¹ Centre For Modeling Human Disease, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada
² Department of Medicine, Medical Sciences Building, 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada
³ Department of Molecular and Medical Genetics, Medical Sciences Building, 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada
⁴ Institute of Medical Science, University of Toronto, Toronto, Ontario M5S 1A8, Canada
⁵ Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5G 1X8, Canada
⁶ Department of Physiology and Pharmacology, University of Western Ontario, Dental Science Building, London, Ontario N6A 5C1, Canada
⁷ Department of Anatomy and Cell Biology, University of Western Ontario, Dental Science Building, London, Ontario N6A 5C1, Canada
⁸ Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X8, Canada
⁹ Mouse Imaging Centre, The Hospital for Sick Children, 555 University Avenue Toronto, Ontario M5G 1X8, Canada
¹⁰ Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
¹¹ Cardiovascular Research, The Hospital for Sick Children, Toronto, Ontario M5S 1A8, Canada
¹² Heart and Stroke/Richard Lewar Centre of Excellence, University of Toronto, Toronto, Ontario M5S 1A8, Canada
¹³ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada
¹⁴ Department of Medicine and Centre for Bone and Periodontal Research, McGill University, 740 Avenue Dr Penfield, Montreal, Quebec H3A 1A4, Canada
¹⁵ Integrative Biology Research Program, The Hospital for Sick Children, Toronto, Ontario M5S 1A8, Canada
¹⁶ Department of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

View larger version (63K): [in a new window]   Fig. 1. Morphological characteristics of mice heterozygous for the Gja1Jrt mutation. (A,C) External plantar and x-ray images taken at 11 weeks of age show that Gja1Jrt/+ mice have variable soft tissue fusion of digits 2, 3 and 4 on the forelimb and hindlimb. (B,D) Faxitron analysis shows the digit fusion in Gja1Jrt/+ mice does not involve the bone. Gja1Jrt/+ are missing the middle phalange of the last digit on both the forelimb and hindlimb (arrows) and exhibit abnormal bone growth of digit 1 (pollex) on the forelimb (arrowhead). (E) Upper incisors are small and both upper and lower incisors are white in the Gja1Jrt/+ mice, instead of yellow as in wild-type (+/+) mice at 20 weeks of age. (F) Back-scatter scanning electron microscopy shows the enamel layer on Gja1Jrt/+ upper incisors is very thin compared with wild-type littermates (+/+), and is nearly absent in places. de, dentine; en, enamel. Scale bar: 1 mm. White boxes indicate the area of higher magnification as seen in the insets.

View larger version (27K): [in a new window]   Fig. 2. Micro-computed tomography of Gja1Jrt/+ skulls. Surface renderings of average skulls in orthographic projection were constructed from five Gja1Jrt/+ mice and five control mice (+/+) ranging in age from 54-60 weeks of age. There are differences seen in profiles along the dorsal surface of the skull. Average skull shapes were overlaid with the magnitude of the deformation needed to map the control skull (+/+) onto the average Gja1Jrt/+ skull. The false color range (indicative of deformation) is from 120 µm (black) to 720 µm (white). Colored regions were statistically significant (P<0.01) by a Hotelling T2 statistic comparing the two groups. There is a large deformation across the bridge of the nose depressing the nasal bone and eye sockets by 668±218 µm and 760±150 µm, respectively, as well as the outward displacement of the frontal bone and occipital bone of 680±265 µm and 460±269 µm, respectively.

View larger version (90K): [in a new window]   Fig. 3. Cardiac phenotype of Gja1Jrt/+ mutants. Histopathology revealed very few, tiny `gap junctions' in the longitudinal muscle fibers of the myocardium of mutants following immunofluorescence for Cx43 (green) (arrow) compared with wild-type controls (+/+) in which intense Cx43 staining is seen in the gap junctions at the intercalated disks (arrows) (A,B). Histopathology also revealed patent foramen ovale in some mutants (arrows in C,D). The body weight (BW) of Gja1Jrt/+ mutants was markedly reduced relative to controls both when young (8-14 weeks) and when old (50-67 weeks) (E). The left ventricular inner chamber dimension in diastole (LV IDd) was large relative to the body weight0.33 and the ventricular wall thickness in diastole (WTd) was reduced relative to the LV IDd in Gja1Jrt/+ mutants (E). In older mutants, there was a prolongation of the LV pre-ejection time (PET) and ejection time (ET) when compared with controls (E). Old mutants evaluated by echocardiography exhibited reduced right ventricular (RV) fractional shortening (FS) and reduced RV WTd, suggesting the development of RV failure with aging (E). LV FS did not change (not shown). *P<0.05, **P0.005. Scale bars: 20 µm in A,B; 500 µm in C,D. la, left atrium; ra, right atrium; lvw, left ventricular wall; ivs, interventricular septum.

View larger version (34K): [in a new window]   Fig. 4. ECG analysis of Gja1Jrt/+ x FVB mutants by radio-telemetry. Gja1Jrt/+ mutant mice crossed with FVB wild-type mice resulted in mice large enough to carry radio-telemetry implants for awake ECG analysis (A). Ultrasound (conducted at 7 weeks) and histopathology (conducted 10-12 weeks) analyses revealed no difference in Gja1Jrt/+ x FVB mice relative to controls (not shown). However, conscious ambulatory ECGs (11-13 weeks) revealed a prolongation of the PQ interval indicative of mild first degree atrioventricular block (A). The PQ intervals were variable, occasionally increasing up to 43 mseconds in length. In addition, P wave width was increased and the heart rate (HR) in Gja1Jrt/+ x FVB mutants was lower than controls. (B) Several sporadic events were noted in Gja1Jrt/+ x FVB mutants: 5/9 had bradycardia (HR<300 minute-1, with the lowest HR at 134 minute-1), 4/9 had sinus arrest, 2/9 had widened QRS complex, 1/9 had AV block and 1/9 had AV dissociation and junctional escape. In the control group, only one mouse had notable events, namely bradycardia and 2nd degree AV block (not shown). *P<0.05, **P0.005.

View larger version (90K): [in a new window]   Fig. 5. Bone characteristics of Gja1Jrt/+ mice. (A) Dual energy x-ray absorptiometry (PIXImus) to measure bone mineral content (BMC), bone area and bone mineral density (BMD) of femurs (males; 22 weeks) showed that BMC and BMD were significantly lower in Gja1Jrt/+ x FVB mice compared with wild-type littermates (+/+). (B) The distal metaphysis of the left femurs were scanned by micro-CT. Two-dimensional images were used to generate 3D reconstructions that clearly showed reduced trabeculae and thin cortices in Gja1Jrt/+ mice compared with wild-type littermates (+/+) at 12 weeks and 6 weeks (data not shown). Morphometric parameters, including percent bone, trabecular thickness distribution, trabecular connectivity, structure model index and cortical thickness, calculated with 3D Creator software supplied with the instrument confirmed the osteopenia in Gja1Jrt/+ animals (not shown). (C) Hematoxylin and Eosin-stained paraffin sections of distal femurs in control (+/+) and in Gja1Jrt/+ mice. A reduction in bone trabeculae was seen as early as 8 weeks of age in the Gja1Jrt/+ mice versus the control mice (i), and progressive bone marrow atrophy was observed in Gja1Jrt/+ mice at 17-18 weeks (ii) and 25 weeks (iii). With aging, the bone marrow space was almost completely atrophied in 51 week Gja1Jrt/+ mice versus the 62 week control (+/+) (iv).

View larger version (27K): [in a new window]   Fig. 6. Flow cytometric analysis of affected bone marrow populations in Gja1Jrt/+ mice. (A) TER119+ erythroblast population was dramatically diminished in affected Gja1Jrt/+ mice compared with control littermates (+/+). (B,C) Gating of the side population (SP) of Hoechst dye effluxing cells from viable whole bone marrow, which are highly enriched in hematopoietic stem cells and primitive progenitors. (B) Young, 15-week-old and (C) 57-62 week old Gja1Jrt/+ mice exhibit an amplified population of SP cells (indicated by box) compared with control littermates (+/+), suggesting increased stem and/or progenitor cells in the affected mice.

View larger version (78K): [in a new window]   Fig. 7. Immunostaining and intercellular coupling via gap junctions in primary granulosa cells. (A) Immunostaining for Cx43 (green) in granulosa cells in vivo and (B) in vitro showed only a few scattered gap junction-like plaques in Gja1Jrt/+ x FVB granulosa cells. O, oocyte. Scale bars: 20 µm. (C,D) Lucifer dye injection (asterisks mark injected cells) revealed strong dye coupling among wild-type granulosa cells (+/+), whereas dye coupling among granulosa cells from cultured Gja1Jrt/+ x FVB mutant follicles was severely restricted. O, oocyte. Scale bar: 50 µm. (E) Graphical representation of the mean number of neighboring cells receiving dye after injection where the number of cells tested is shown in parentheses above each bar. (F) The mean conductance of cells that were electrically coupled, as indicated by capacitative current transients, showed that coupling was severely reduced in Gja1Jrt/+ x FVB granulosa cells. The number of cells tested is shown in parentheses above each bar. (G) Representative current transients from wild type (+/+), Gja1-null (Gja1-/Gja1-) and Gja1Jrt/+ x FVB granulosa cells show that Gja1Jrt/+ x FVB granulosa cells exhibited either very weak coupling or a complete lack of coupling (12/17 weakly coupled; 5/17 not coupled). In vivo and in vitro experiments were performed on primary granulosa cells isolated from ovaries on both genetic backgrounds with similar results. (H) Western blots reveal that the level of total Cx43 and especially the slower migrating phosphorylated species, was greatly reduced in heart and ovary from Gja1Jrt/+ versus wild-type (+/+) mice (11 weeks). GAPDH was used as a gel loading control.

View larger version (60K): [in a new window]   Fig. 8. Illustration of Cx43 protein structure showing known human ODDD mutations. Known human mutations resulting in ODDD (Kjaer et al., 2004; Paznekas et al., 2003; Richardson et al., 2004; van Steensel et al., 2005; Vitiello et al., 2005) are highlighted in red with the Gja1Jrt mutation (G60S) highlighted in black. Missense mutations are denoted by the correct amino acid followed by the number and the substituted amino acid. The duplication (dup) and frameshift (FS) mutations are indicated by amino acid number followed by dup and FS, respectively.

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