Activating mutations in transient receptor potential vanilloid family member 4 (Trpv4) are known to cause a spectrum of skeletal dysplasias ranging from autosomal dominant brachyolmia to lethal metatropic dysplasia. To develop an animal model of these disorders, we created transgenic mice expressing either wild-type or mutant TRPV4. Mice transgenic for wild-type Trpv4 showed no morphological changes at embryonic day 16.5 but did have a delay in bone mineralization. Overexpression of a mutant TRPV4 caused a lethal skeletal dysplasia that phenocopied many abnormalities associated with metatropic dysplasia in humans, including dumbbell-shaped long bones, a small ribcage, abnormalities in the autopod, and abnormal ossification in the vertebrae. The difference in phenotype between embryos transgenic for wild-type or mutant Trpv4 demonstrates that an increased amount of wild-type protein can be tolerated and that an activating mutation of this protein is required to produce a skeletal dysplasia phenotype. © 2014 American Society for Bone and Mineral Research
Transient receptor potential vanilloid family member 4 (TRPV4) is a tetrameric, calcium-selective, gated ion channel that is selectively, but not exclusively, expressed in chondrocytes, where it plays a role in chondrocyte differentiation.[2, 3] Mutations in Trpv4, which encodes the TRPV4 calcium channel, result in a spectrum of dominantly inherited skeletal dysplasia phenotypes,[1, 4-7] as well as a group of peripheral neuropathies. At the mild end of the spectrum is familial digital arthropathy-brachydactyly (FDAB), a condition primarily characterized by osteoarthropathy in the hands. Autosomal dominant brachyolmia (BO) is a more generalized but relatively mild condition characterized by short stature, a short trunk, and scoliosis. Spondylometaphyseal dysplasia, Kozlowski type (SMDK) is a more severe disorder with pronounced short stature, kyphoscoliosis, and short limbs. Radiographically, overfaced pedicles of the vertebral bodies, delayed epiphyseal ossification (particularly of the carpal bones), and irregular metaphyses characterize this phenotype. TRPV4 mutations can also produce nonlethal metatropic dysplasia, in which there is severe short stature, progressive kyphoscoliosis, and marked shortening of the long bones, which frequently manifest a dumbbell shape.[5, 6] Finally, the perinatal lethal form of metatropic dysplasia has extremely severe skeletal abnormalities including a small chest, very short long bones, and small hands. Radiographically, there is a wafer-thin appearance to the vertebrae, a rounded pelvis, and long bones with short, narrow diaphyses and flared ends. With the exception of FDAB, which appears to involve reduced channel activity, the majority of mutations result in single amino acid substitutions that activate the channel. Activation of the channels has been established by expression of mutations in cultured cells,[1, 4, 5] demonstrating increased basal and agonist-stimulated channel activity as well as increased calcium concentrations within the cells. The same mutation in different individuals usually produces a similar phenotype; however, the substitutions found in each of the disorders are distributed across multiple domains of the TRPV4 molecule and the phenotype cannot be predicted from a previously unobserved genotype.
Because these conditions are mainly caused by a gain-of-function in TRPV4, a transgenic mouse approach was used to model the TRPV4 disorders. Wild-type or mutant Trpv4 was selectively expressed in cartilage using the Col2a1 promoter to test whether overexpression of wild-type Trpv4 was sufficient to generate a phenotype or if the expression of a mutant protein was necessary to produce the skeletal abnormalities that characterize the disorders in this spectrum.
Materials and Methods
The transgenic construct was created by PCR amplification of mouse Trpv4 from a plasmid generously provided by the Bernd Nilius Laboratory (Leuven, Belgium). The primers used for amplification incorporated a consensus Kozak sequence containing in-frame ATG translation initiation and TAG termination codons, flanked by PvuII endonuclease sites to facilitate cloning into the pCol2a1-Tyr transgenic vector. Mutant Trpv4 sequence was generated by means of site-directed mutagenesis, utilizing overlapping PCR primers containing the desired c.1781G > A mutation (M. musculus Trpv4 mRNA NM_022017). Constructs were fully sequenced after production to confirm orientation and eliminate clones containing PCR errors. The linearized transgenic construct (Fig. 1) was generated by PacI digestion and provided to the University of California, Irvine, transgenic facility for purification and microinjection into fertilized albino strain FVB oocytes. The microinjected zygotes were then implanted into pseudo-pregnant surrogate dams. Embryos were harvested 16 days after injection. Because of the presence of a tyrosinase reporter in the transgenic construct, successful incorporation and expression of the construct was initially determined by presence of black eye color and then confirmed by PCR analysis. All experiments were approved by the UCLA and UCI animal care and use committees, as appropriate.
Cleared skeleton preparation
After harvest, skin and organs were removed from the embryos, which were fixed in 95% ethanol overnight. Cartilage was stained overnight using 0.03% alcian blue dye (Sigma, St. Louis, MO, USA) in 80% ethanol and 20% acetic acid. After destaining in 95% ethanol, mineralized bone was stained in 0.005% alizarin red in 1% KOH for 4 hours. All embryos were then rinsed and cleared in 1% KOH at 4°C overnight before being transferred to 0.5% KOH in 50% glycerol for additional clearing and then 0.2% KOH in 80% glycerol for storage. Before microscopy, remaining soft tissue was excised and embryos were placed on gelatin-coated plates for imaging on a Leica (Buffalo Grove, IL, USA) dissecting microscope. Femur length measurements were made using a pinwheel reticle (Swift Microscope World, Carlsbad, CA, USA).
After removal of skin and organs, embryos were dissected and fixed in formalin overnight at 4°C. After decalcification in formic acid solution (Immunocal; Immunotech, Vaudreuil-Dorion, Canada), samples were rinsed, dried, and dehydrated in graded ethanol solutions (50% to 100%). Samples were then cleared with xylene and embedded in paraffin in a vacuum oven. The 0.5-µm sections were obtained using a Leica microtome and placed on precoated slides (Fisher Scientific, Pittsburgh, PA, USA).
Immunostaining of paraffin-embedded sections
Slide-mounted sections were deparaffinized in xylene and rehydrated in graded ethanol solutions (100% to 50%). To improve antigen accessibility, sections were treated with 1 mg/mL hyaluronidase (Sigma) at 37°C for 45 minutes. After washing, samples were blocked using a commercial blocking reagent (PerkinElmer, Waltham, MA, USA) and treated overnight at 4°C with a primary antibody in blocking reagent. Sections were next treated with a secondary biotinylated anti-rabbit antibody (Invitrogen, diluted 1:250) for 30 minutes in blocking buffer. Fluorescent signal was generated by staining with Alexa 555-tagged streptavidin (Invitrogen, Carlsbad, CA, USA). Samples were then counterstained with DAPI and imaged on an Olympus (Tokyo, Japan) fluorescent microscope. Anti-type II collagen antibody was purchased from Abcam (Cambridge, MA, USA; cat. no. ab34712), and anti-type X collagen antibody was purchased from Calbiochem (Merck KGaA, Darmstadt, Germany; cat. no. 234196). Both antibodies were used at 1:500 dilutions and were generated in rabbit. Rabbit anti-SOX9 antibody was purchased from Novus Biologicals (Littleton, CO, USA; cat. no. 85551) and used at a dilution of 1:200. Rabbit anti-rat TRPV4 antibody was generously provided by the Nilius Laboratory in Leuven, Belgium. Scale bars were determined using a pinwheel reticle (Swift Microscope World).
Hematoxylin and eosin staining
Sections were deparaffinized and rehydrated as described above. After washing with deionized water, samples were stained with hematoxylin QS (Vector Laboratories, Burlingame, CA, USA) for 3 minutes, rinsed in water, and destained in acid ethanol before rinsing again. Samples were then stained with 0.1% eosin for 30 seconds before being destained in ethanol and imaged on an Olympus light microscope.
All results are shown as the mean ± SE. Differences in length were analyzed with a Student's t test (Microsoft Excel, Microsoft Corporation, Redmond, WA, USA).
We used transgenic mice to determine whether overproduction of wild-type Trpv4 is sufficient to produce skeletal abnormalities or if expression of a mutant protein is required to generate a phenotype. Mice were generated that expressed either wild-type or mutant Trpv4 (R594H) under the control of the cartilage-selective Col2a1 promoter. The Col2a1 promoter was selected because it is highly expressed in cartilage and has been used in many studies to direct expression in chondrocytes, permitting analysis of the skeletal phenotype exclusive of other tissues. The R594H mutation was selected because it produces the mild to moderately severe, but nonlethal, SMDK phenotype in humans.[5, 15] The transgenic construct also incorporated a tyrosinase reporter gene (Fig. 1) to facilitate monitoring of gene expression in transgenic albino embryos.
No visible abnormalities were observed in the appearance of E16.5 embryos transgenic for wild-type Trpv4 (n = 7) compared with nontransgenic controls (n = 15). Morphologically, the whole embryos of both control and wild-type Trpv4 transgenic mice were of normal size (Fig. 2A, B). Additionally, the whole skeletons (Fig. 3A, B), vertebrae (Fig. 3E, F), long bones (Fig. 4A, B), and autopods (Fig. 5A, B) of both control and wild-type transgenic embryos were of normal size and appearance. Although the overall lengths of the femurs, like the patterning of the other skeletal elements, were nearly identical between these two groups of mice (Fig. 6A), both the absolute mineralized length (Fig. 6A) and the percent mineralized portion of the femurs (Fig. 6B) were statistically significantly lower in the wild-type transgenic embryos. Additionally, although not reaching statistical significance, trends toward delayed metacarpal and vertebral ossification could also be found (data not shown), further suggesting a delay in skeletal mineralization.
In contrast to mice overexpressing wild-type Trpv4, mice transgenic for the SMDK-causing Trpv4R594H allele exhibited perinatal lethal skeletal abnormalities. Because the dam often consumed mutant pups, subsequent analyses were carried out on E16.5 embryos. The embryos expressing mutant Trpv4 (n = 8) exhibited a range of skeletal abnormalities, with the most severely affected embryos (n = 3) appearing hydropic at the time of collection (Fig. 2C, D). The phenotype included several features commonly found in the human TRPV4 skeletal dysplasias, such as small size with pronounced midface hypoplasia and short limbs. Mutant animals also had shortened tails consistent with axial abnormalities. Although there was a spectrum of severity for Trpv4R594H transgenic mice, overall the phenotype was much more severe than would be expected for a mutation that causes SMDK in humans. The cleared and stained skeletons of Trpv4R594H transgenic mice were much smaller (Fig. 3C, D) than those of the control animals, with abnormalities in the craniofacial, axial, and appendicular skeletal elements. The most severely affected mice showed a nearly complete lack of mineralization of all endochondral bones, although membranous bones appeared to ossify normally (Fig. 3D).
Compared with controls, the spines of most mice expressing Trpv4R594H had an abnormal pattern of mineralization with merging of the ossified regions of the central core and lateral processes (Fig. 3G). In the most severely affected Trpv4R594H transgenic animals (Fig. 3H), there was a complete lack of appropriate ossification of the vertebral bodies but a small amount of ectopic ossification in a portion of the lumbar spine.
The long bones of Trpv4R594H transgenic embryos were uniformly shorter than controls with narrow diaphyses and proportionally wider epiphyses (Fig. 4C, D). The more severely affected embryos also showed complete absence of mineralization of the long bones (Fig. 4D). There were marked irregularities in the shapes of the metacarpals and phalanges of both moderately (Fig. 5C) and severely (Fig. 5D) affected Trpv4R594H transgenic mice compared with controls. Additionally, the boundaries of the carpals in moderately affected Trpv4R594H transgenic mice appeared poorly defined, with the severely affected embryos exhibiting carpal fusions. Although the human TRPV4 disorders are characterized by delayed carpal ossification, mineralization does not occur in the mouse autopod until after E16.5 (see the control in Fig. 5A), so this aspect of phenotype could not be assessed in the mice.
To examine the organization of the growth plates, we performed histochemistry on samples of developing bone stained with hematoxylin and eosin. Consistent with the properly formed skeleton, both the nontransgenic (Fig. 6C) and wild-type transgenic (Fig. 6D) embryos had well-organized growth plates with clearly defined reserve, proliferative, and hypertrophic zones and clear boundaries between each zone. The moderately affected Trpv4R594H transgenic embryos (Fig. 6E) had a much narrower proliferative zone with a widened reserve zone and less well-defined boundaries between the different zones. In the severely affected Trpv4R594H transgenic embryos (Fig. 6F), the growth plate was highly disorganized with much more pronounced widening of the reserve zone, lack of an organized proliferative zone, and a narrow hypertrophic zone, with very poorly defined margins between each zone. These findings are similar to the growth plates studied from human fetuses with lethal metatropic dysplasia, which show fewer hypertrophic cells, overgrowth of the poorly organized reserve zone cartilage, and significantly decreased mineralization.
To define the pattern of expression of Trpv4 in the transgenic mice, growth plates from the distal femur were stained with anti-TRPV4 antibody. Although expression of native TRPV4 was too low to detect in nontransgenic mice (Fig. 7A), TRPV4 expression was detected in the reserve, proliferating, and hypertrophic zones of the growth plates of mice transgenic for wild-type Trpv4, and a low level of expression was apparent in perichondrium (Fig. 7B). A similar pattern of expression of mutant TRPV4 was observed in moderately affected (Fig. 7C) and severely affected (Fig. 7D) embryos, although in the severely affected mutant the zones were difficult to distinguish.
All embryos tested had normal patterns of type II collagen expression within the growth plate (Fig. 7E–H). The overall pattern of type X collagen expression was also normal (Fig. 7I–L), but severely affected Trpv4R594H transgenic embryos (Fig. 7L) had less type X collagen, consistent with the reduced number of hypertrophic chondrocytes in these mice.[16, 17] The expression pattern for SOX9 showed the highest level of expression across all genotypes in late proliferating chondrocytes (Supplemental Fig. S1). Because of the highly disrupted growth plate architecture, there was less expression in the most severely affected mutant embryos, consistent with a reduction in proliferating chondrocytes.
Based on normal TUNEL staining, there did not appear to be increased apoptosis (data not shown). Staining with PCNA and phosphohistone H3 antibodies also gave a normal pattern (data not shown), suggesting that abnormal proliferation of growth plate chondrocytes did not explain the reduced number of cells.
To better understand the role of TRPV4 in skeletal development, we created transgenic mice overexpressing either wild-type or mutant Trpv4 in chondrocytes. Embryos overexpressing wild-type Trpv4 had normally patterned bones with well-organized growth plates but a mild delay in mineralization. These data are consistent with previous studies showing that overexpressing wild-type Trpv4 in cells increased intracellular calcium levels[4, 5] and indicate that there is a phenotypic consequence to overexpression of wild-type Trpv4. Thus, in contrast with the normal expression levels of TRPV4 with an activating mutation that cause a range of skeletal dysplasias,[1, 4-6, 10] overexpression of normal TRPV4 in vivo produces only subtle changes in skeletal mineralization, at least at the level of TRPV4 overexpression in the animals studied. There is evidence in zebrafish that more severe skeletal phenotypes can result from generalized overexpression of wild-type TRPV4, presumed to correlate with the degree of overexpression. Currently, we do not know how this mild defect would affect skeletal development beyond E16.5 or at higher levels of overexpression.
Embryos transgenic for Trpv4R594H exhibited severe skeletal abnormalities that recapitulate what is found in humans with metatropic dysplasia and much more severe than individuals with SMDK caused by the R594H mutation. These embryos exhibited multiple skeletal abnormalities including dumbbell-shaped long bones, midface hypoplasia, platyspondyly, and a small ribcage, reminiscent of what is found in humans with metatropic dysplasia. The findings demonstrate that the overexpression of Trpv4R594H is sufficient to cause a skeletal dysplasia. In humans, the SMDK phenotype produced by the R594H mutation is moderately severe, but many of the transgenic mouse embryos presented a phenotype more similar to lethal metatropic dysplasia.
The more severe phenotype is likely the result of several factors. Use of the Col2a1 promoter resulted in strong, chondrocyte-specific expression of Trpv4, resulting in a level of TRPV4 in excess of normal endogenous expression. Additionally, TRPV4 is a tetramer, and the overexpression of mutant TRPV4 would be predicted to result in the overwhelming majority of functional tetramers containing only mutant subunits, another possible explanation for the unexpected phenotypic severity. We also cannot rule out the possibility that mice may be more susceptible to the effects of either this particular mutation or to Trpv4 mutations in general.
Histological analysis showed impaired differentiation of hypertrophic chondrocytes in the growth plates of moderately affected Trpv4R594H transgenic embryos with a near absence of hypertrophic chondrocytes in the growth plates of severely affected Trpv4R594H transgenic embryos. In extreme cases, no distinct hypertrophic zone could be detected by immunostaining for type X collagen. This defect in the hypertrophic zone accounts for the decreased or complete lack of mineralization in severely affected embryos transgenic for mutant TRPV4.
This study has demonstrated two important principles. First, increased levels of wild-type TRPV4 can have phenotypic consequences, but these appear to be mild. Second, expression of mutant Trpv4 is sufficient to cause a skeletal dysplasia reminiscent of Trpv4-based skeletal dysplasias in humans. The ability to model Trpv4-based skeletal dysplasias in mice will now allow a deeper exploration of the in vivo molecular mechanisms that produce these disorders, which in turn should open the door to new levels of understanding of the TRPV4 diseases.
All authors state that they have no conflicts of interest.
This study was supported in part by grant HD22657 from NICHD/NIH and funds from the Orthopaedic Hospital Research Center at UCLA. MMW was supported by an NIH training grant (5 T32 AR 59033-2). We thank Bernd Nilius and colleagues for providing a construct containing the mouse Trpv4 cDNA and for TRPV4 antibodies and Faith Hall-Glenn for her assistance with the skeletal preparations. We also thank Karen Lyons and Deborah Krakow for their help in reviewing this manuscript. The study was also supported by the BCM Intellectual and Developmental Disabilities Research Center (HD024064) from the Eunice Kennedy Shriver National Institute of Child Health & Human Development, and the Rolanette and Berdon Lawrence Bone Disease Program of Texas.
Authors' roles: Drafting manuscript: MMW and DHC. Revision of manuscript: MMW, SWT, BL, DHC. Performing experiments: MMW, SWT, YC. Approving final version of manuscript: MMW, SWT, YC, BL, DHC.