A Loss-of-Function Mutation in Natriuretic Peptide Receptor 2 (Npr2) Gene Is Responsible for Disproportionate Dwarfism in cn/cn Mouse

Abstract

The achondroplastic mouse is a spontaneous mutant characterized by disproportionate dwarfism with short limbs and tail due to disturbed chondrogenesis during endochondral ossification. These abnormal phenotypes are controlled by an autosomal recessive gene (cn). In this study, linkage analysis using 115 affected mice of F2 progeny mapped the cn locus on an ∼0.8-cM region of chromosome 4, and natriuretic peptide receptor 2 (Npr2) gene was identified as the most potent candidate for the cn mutant in this region. This gene encodes a receptor for C-type natriuretic peptide (CNP) that positively regulates longitudinal bone growth by producing cGMP in response to CNP binding to the extracellular domain. Sequence analyses of the Npr2 gene in cn/cn mice revealed a T to G transversion leading to the amino acid substitution of highly conserved Leu with Arg in the guanylyl cyclase domain. In cultured chondrocytes of cn/cn mice, stimulus with CNP did not significantly increase intracellular cGMP concentration, whereas it increased in +/+ mice. Transfection of the mutant Npr2 gene into COS-7 cells also showed similar results, indicating that the missense mutation of the Npr2 gene in cn/cn mice resulted in disruption of the guanylyl cyclase activity of the receptor. We therefore concluded that the dwarf phenotype of cn/cn mouse is caused by a loss-of-function mutation of the Npr2 gene, and cn/cn mouse will be a useful model to further study the molecular mechanism regulating endochondral ossification by CNP/natriuretic peptide receptor B signal. The achondroplastic mouse is a spontaneous mutant characterized by disproportionate dwarfism with short limbs and tail due to disturbed chondrogenesis during endochondral ossification. These abnormal phenotypes are controlled by an autosomal recessive gene (cn). In this study, linkage analysis using 115 affected mice of F2 progeny mapped the cn locus on an ∼0.8-cM region of chromosome 4, and natriuretic peptide receptor 2 (Npr2) gene was identified as the most potent candidate for the cn mutant in this region. This gene encodes a receptor for C-type natriuretic peptide (CNP) that positively regulates longitudinal bone growth by producing cGMP in response to CNP binding to the extracellular domain. Sequence analyses of the Npr2 gene in cn/cn mice revealed a T to G transversion leading to the amino acid substitution of highly conserved Leu with Arg in the guanylyl cyclase domain. In cultured chondrocytes of cn/cn mice, stimulus with CNP did not significantly increase intracellular cGMP concentration, whereas it increased in +/+ mice. Transfection of the mutant Npr2 gene into COS-7 cells also showed similar results, indicating that the missense mutation of the Npr2 gene in cn/cn mice resulted in disruption of the guanylyl cyclase activity of the receptor. We therefore concluded that the dwarf phenotype of cn/cn mouse is caused by a loss-of-function mutation of the Npr2 gene, and cn/cn mouse will be a useful model to further study the molecular mechanism regulating endochondral ossification by CNP/natriuretic peptide receptor B signal. The skeleton of vertebrates is formed by two different processes, namely intramembranous and endochondral ossifications. The latter process leads to the development of long bones that comprise the appendicular skeleton and vertebrae. During endochondral ossification, mesenchymal cells initially differentiate into chondrocytes, and progress through proliferating, maturating, and hypertrophic stages with strict columnar alignment. Distal hypertrophic chondrocytes then undergo apoptosis and are replaced by trabecular bone. A large number of genes have been implicated in the mechanisms regulating these processes (1.Erlebacher A. Filvaroff E.H. Gitelman S.E. Derynck R. Cell. 1995; 80: 371-378Abstract Full Text PDF PubMed Scopus (617) Google Scholar), and mutations of these genes often cause skeletal dysplasias with shortened extremities (2.Newman B. Wallis G.A. Clin. Genet. 2003; 63: 241-251Crossref PubMed Scopus (34) Google Scholar, 3.Cohen Jr., M.M. Am. J. Med. Genet. 2002; 112: 304-313Crossref PubMed Scopus (65) Google Scholar). The natriuretic peptide (NP) 1The abbreviations used are: NP, natriuretic peptide; ANP, atrial NP; BNP, brain NP; NPRA, NP receptor A; NPRB, NP receptor B; NPRC, NP receptor C; CNP, C-type NP; AMDM, acromesomelic dysplasia Maroteaux type. family comprises atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) (4.Levin E.R. Gardner D.G. Samson W.K. N. Engl. J. Med. 1998; 339: 321-328Crossref PubMed Scopus (2066) Google Scholar), and three receptors for NPs, including natriuretic peptide receptor A (NPRA), natriuretic peptide receptor B (NPRB), and natriuretic peptide receptor C (NPRC), have been identified in mammals (5.Chinkers M. Garbers D.L. Chang M.S. Lowe D.G. Chin H.M. Goeddel D.V. Schulz S. Nature. 1989; 338: 78-83Crossref PubMed Scopus (890) Google Scholar, 6.Schulz S. Singh S. Bellet R.A. Singh G. Tubb D.J. Chin H. Garbers D.L. Cell. 1989; 58: 1155-1162Abstract Full Text PDF PubMed Scopus (491) Google Scholar, 7.Maack T. Suzuki M. Almeida F.A. Nussenzveig D. Scarborough R.M. McEnroe G.A. Lewicki J.A. Science. 1987; 238: 675-678Crossref PubMed Scopus (853) Google Scholar). NPRA and NPRB consist of extracellular ligand binding, transmembrane, protein kinase homology, and guanylyl cyclase catalytic domains. These receptors mediate ligand signals by producing an intracellular second messenger, cyclic GMP (cGMP) (2.Newman B. Wallis G.A. Clin. Genet. 2003; 63: 241-251Crossref PubMed Scopus (34) Google Scholar). Both ANP and BNP bind to NPRA with high affinity, and CNP prefers binding to NPRB (8.Suga S. Nakao K. Hosoda K. Mukoyama M. Ogawa Y. Shirakami G. Arai H. Saito Y. Kambayashi Y. Inouye K. Imura H. Endocrinology. 1992; 130: 229-239Crossref PubMed Google Scholar). NPRC has a ligand-binding domain and a short cytoplasmic domain that lacks guanylyl cyclase activity and is considered to be involved in NP clearance (7.Maack T. Suzuki M. Almeida F.A. Nussenzveig D. Scarborough R.M. McEnroe G.A. Lewicki J.A. Science. 1987; 238: 675-678Crossref PubMed Scopus (853) Google Scholar). ANP and BNP mainly reside in the atrium and ventricle, respectively, and act as cardiac hormones that regulate central fluid volume, blood pressure, and the development of cardiovascular tissues (9.Cameron V.A. Ellmers L.J. Endocrinology. 2003; 144: 2191-2194Crossref PubMed Scopus (144) Google Scholar). On the other hand, CNP gene (Nppc) and NPRB gene (Npr2) expressions have been detected in mice tibial growth plates (10.Yamashita Y. Takeshige K. Inoue A. Hirose S. Takamori A. Hagiwara H. J. Biochem. (Tokyo). 2000; 127: 177-179Crossref PubMed Scopus (23) Google Scholar, 11.Chusho H. Tamura N. Ogawa Y. Yasoda A. Suda M. Miyazawa T. Nakamura K. Nakao K. Kurihara T. Komatsu Y. Itoh H. Tanaka K. Saito Y. Katsuki M. Nakao K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4016-4021Crossref PubMed Scopus (369) Google Scholar) and in the chondrogenic cell line ATDC5 (12.Suda M. Tanaka K. Yasoda A. Komatsu Y. Chusho H. Miura M. Tamura N. Ogawa Y. Nakao K. J. Bone Miner. Metab. 2002; 20: 136-141Crossref PubMed Scopus (24) Google Scholar). Since CNP stimulates the longitudinal growth of cultured fetal mouse tibias (13.Yasoda A. Ogawa Y. Suda M. Tamura N. Mori K. Sakuma Y. Chusho H. Shiota K. Tanaka K. Nakao K. J. Biol. Chem. 1998; 273: 11695-11700Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) and CNP-deficient mice develop dwarfism due to impaired endochondral ossification (11.Chusho H. Tamura N. Ogawa Y. Yasoda A. Suda M. Miyazawa T. Nakamura K. Nakao K. Kurihara T. Komatsu Y. Itoh H. Tanaka K. Saito Y. Katsuki M. Nakao K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4016-4021Crossref PubMed Scopus (369) Google Scholar), the CNP/NPRB pathway has been considered to be involved in the process of endochondral ossification. However, there is no direct evidence that NPRB effects longitudinal bone growth by regulating endochondral ossification. The achondroplastic mouse is a mutant strain with an autosomal recessive gene (cn) that arose spontaneously in the AKR/J mouse strain (14.Lane P.W. Dickie M.M. J. Hered. 1968; 59: 300-308Crossref PubMed Scopus (72) Google Scholar). The homozygous (cn/cn) mouse exhibits disproportionate dwarfism with short limbs, short tail, and a domed skull. These phenotypes are distinguishable from normal littermates by 7 days after birth and thereafter gradually become more prominent (15.Silberberg R. Lesker P. Growth. 1975; 39: 17-33PubMed Google Scholar, 16.Johnson D.R. J. Anat. 1978; 125: 267-275PubMed Google Scholar). Histological analyses have revealed that the tibial epiphyseal growth plates of cn/cn mice are thinner than normal mice, and the hypertrophic chondrocyte zone is considerably narrowed (17.del Marco A. Reprod. Nutr. Dev. 1981; 21: 1025-1031Crossref PubMed Scopus (5) Google Scholar, 18.Wikstrom B. Hjerpe A. Reinholt F.P. Engfeldt B. Coll. Relat. Res. 1986; 6: 279-293Crossref PubMed Scopus (3) Google Scholar). These findings suggested that the dwarf phenotype of cn/cn mouse was caused by retarded longitudinal bone growth due to disturbed endochondral ossification. Thus, the cn/cn mouse has been believed to be a useful model for hereditary human skeletal dysplasias. However, the gene responsible for the skeletal abnormalities of the cn/cn mouse has not yet been identified. In this study, we determined the chromosomal localization of the cn locus and identified the causative mutation in the Npr2 gene. Mice—Heterozygotes (cn/+) of mutant mice were obtained from the Jackson Laboratory, and the strain was maintained by sib mating of heterozygotes. JF1/Ms mice were obtained from the National Institute of Genetics, Mishima, Japan. Linkage Analysis—F1 mice were generated by mating heterozygous (cn/+) mice with JF1 (+/+) mice, and the F2 progeny was subsequently obtained from intercrossing heterozygous (cn/+)F1 mice. Genomic DNA was extracted from mouse livers by phenol/chloroform extraction. PCR reactions for microsatellite markers proceeded as follows: 35 cycles at 94 °C for 30 s, 55–60 °C for 30 s, and 72 °C for 45 s. The primer sequences of newly generated microsatellite markers, D4Mok1 (5′-GATTTGAGTTTCTGACACCTCCC-3′ and 5′-ACTTGCTTGTTTGCGTGTGGGC-3′) and D4Mok2 (5′-TCCTTGGCTGATGCCTAAGC-3′ and 5′-GGTCTAGGAAGCTAACTCAGTGG-3′), were obtained from genomic sequences located at 42.2 Mb (GenBank™ accession number NT_039260) and 43.0 Mb (GenBank™ accession number NT_039296) positions on mouse chromosome 4, respectively. The reaction mixture for PCR (10 μl) contained 1× PCR buffer, 0.2 mm dNTP, a 0.5 μm concentration of each primer, and 0.25 unit of TaqDNA polymerase (Amersham Biosciences). The PCR products were fractionated on 3.0% agarose gels and stained with ethidium bromide. The data were analyzed using Map Manager computer software. Detection of Mutation in Npr2 Gene—Tibial RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The extracted RNA was then incubated with RNase-free DNase I (Takara, Ohtsu, Japan) for 1 h at 37 °C to remove contaminating genomic DNA. First strand cDNA was synthesized for reverse transcription-PCR using the Superscript preamplification system (Invitrogen) according to the manufacturer's instructions. Npr2 cDNA was amplified with specific primer (5′-TGTATCCGGGCCGACTAAGCTTGC-3 and 5′-GGAAACACAGTGACCATTGCCCATCC-3′) under the following conditions: 94 °C for 2 min followed by 35 cycles of 94 °C for 30 s, 64 °C for 20 s, and 72 °C for 4 min. The amplified fragments were sequenced using an ABI PRIZM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). To detect the mutation in the genomic DNA of mice by restriction enzyme digestion, DNA fragments, including exon 18 of Npr2 gene, were amplified from genomic DNA using specific primers (5′-TTCACAGCGCTGTCAGCTGAG-3′ and 5′-ACTTAGGGAGCGCTGACTGTGG-3′). PCR reaction was carried out as follows: 35 cycles of 94 °C for 30 s, 62 °C for 20 s, and 72 °C for 30 s. The PCR products were digested with AflIII restriction enzyme at 37 °C, and electrophoresed on 3% agarose gel. Culture of Mouse Chondrocytes and COS-7 Cells—Chondrocytes were prepared from the costal cartilage of cn/cn or +/+ mice (n = 5) at postnatal day 7 as described (19.Lefebvre V. Garofalo S. Zhou G. Metsaranta M. Vuorio E. De Crombrugghe B. Matrix Biol. 1994; 14: 329-335Crossref PubMed Scopus (135) Google Scholar). COS-7 cells were obtained from the RIKEN cell bank (Tsukuba, Japan). These cells were cultured in Dulbecco's fetal and at 37 °C under in The chondrocytes were in and cGMP were the cells The COS-7 cells were at a of 1 in plates and used for after for of Npr2 in Npr2 cDNA was amplified by reverse transcription-PCR with tibial RNA extracted from cn/cn or +/+ mice using primers or the and PCR was at 94 °C for 2 min followed by 35 cycles of 94 °C for 30 s, 64 °C for 20 s, and 72 °C for 4 min. fragments were digested with and restriction enzyme and then into The COS-7 cells were with the DNA using and the cells were to cGMP after for of cGMP in cultured cells were with and incubated in Dulbecco's fetal and 0.5 mm at 37 °C for min. of CNP were to the and the proceeded at 37 °C for a further min. The of cGMP was using a cGMP enzyme system (Amersham Biosciences). the cn by Linkage the chromosomal localization of the cn locus by linkage heterozygous (cn/+) mice were with JF1/Ms (+/+) mice. (cn/+) of mice were and F2 progeny were including 115 affected and normal mice. The of affected to normal F2 progeny did not significantly from the of recessive We initially the linkage the cn locus and microsatellite markers on mouse with using the 115 affected mice. linkage was detected the cn locus and markers on the region of chromosome 4, indicating the localization of the cn locus on this region. To the of the cn we further markers located at the region of chromosome in no was the cn locus and and at was with the other These data that the cn locus was mapped on the and and Detection of Mutation in Npr2 of the linkage with the mouse two Npr2 and are to the phenotypes of cn/cn mice, were in the region of the cn locus in human of Npr2 and genes have been in with acromesomelic dysplasia Maroteaux H. P. R.M. S. D. S. T. J. V. M. J. J. B. Y. Am. J. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar) and M. H. K. R. B. G. R. S. A. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), respectively. These human are characterized by skeletal dysplasias with disproportionate short Thus, we considered that these genes are the most candidate genes for the cn To the causative mutation in these we amplified and sequenced the region of these genes and the cn/cn and +/+ mice. The of the gene showed no causative and the gene was therefore from the However, analysis of the Npr2 cDNA revealed a T to G transversion leading to an amino acid substitution of Leu to Arg at in cn/cn mice We this mutation by exon 18 of Npr2 genomic DNA in cn/cn mice not To the T to G substitution is to the cn mutant PCR fragments including exon 18 of the Npr2 gene were amplified from of mice and digested by the AflIII restriction for a is generated by the T to G a amplified from the cn mutant was digested with whereas the fragments from +/+ and the strain of cn/cn as as other were not digested These that the substitution of T to G in the Npr2 gene is specific to the cn mutant of the Mutation on of Npr2 gene encodes a NPRB, CNP signals cGMP by intracellular guanylyl cyclase domain. Since Leu at on the guanylyl cyclase domain of NPRB is highly conserved this amino acid for the domain To the of the amino acid substitution of NPRB on guanylyl cyclase we the of cGMP by CNP in the chondrocytes obtained from the costal cartilage of cn/cn and +/+ mice. The of cGMP in the chondrocytes of +/+ mice increased in response to CNP whereas the cGMP did not significantly in cn/cn mice we the guanylyl cyclase activity in COS-7 cells with the the mutant or Npr2 gene. cGMP increased in COS-7 cells with the gene after to CNP, no was in COS-7 cells with a mutant gene of NPRB in cells not These that the missense mutation in the Npr2 gene impaired the guanylyl cyclase activity of In this study, we mapped the cn locus on chromosome 4 and identified a missense mutation in the Npr2 gene. This mutation leads to the substitution of highly conserved with the in the guanylyl cyclase domain of We further that cGMP was not in response to CNP stimulus in cells the mutant Npr2 gene. it is that the guanylyl cyclase activity of NPRB in cn/cn mouse was by amino acid substitution in the highly conserved region of the guanylyl cyclase domain. of evidence have suggested that the Npr2 gene a in regulating endochondral ossification. The Npr2 gene is in the and chondrocytes of the epiphyseal growth (10.Yamashita Y. Takeshige K. Inoue A. Hirose S. Takamori A. Hagiwara H. J. Biochem. (Tokyo). 2000; 127: 177-179Crossref PubMed Scopus (23) Google Scholar, 11.Chusho H. Tamura N. Ogawa Y. Yasoda A. Suda M. Miyazawa T. Nakamura K. Nakao K. Kurihara T. Komatsu Y. Itoh H. Tanaka K. Saito Y. Katsuki M. Nakao K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4016-4021Crossref PubMed Scopus (369) Google cyclic as as CNP, stimulates longitudinal bone growth V. J.A. De J. Res. 2000; PubMed Scopus Google Scholar), and mice with a disruption of CNP or protein kinase a of develop dwarfism due to impaired endochondral ossification (11.Chusho H. Tamura N. Ogawa Y. Yasoda A. Suda M. Miyazawa T. Nakamura K. Nakao K. Kurihara T. Komatsu Y. Itoh H. Tanaka K. Saito Y. Katsuki M. Nakao K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4016-4021Crossref PubMed Scopus (369) Google Scholar, T. Ogawa Y. Chusho H. Yasoda A. Tamura N. Komatsu Y. A. Nakao K. Endocrinology. 2002; PubMed Scopus Google Scholar, A. A. U. P. R. Science. PubMed Scopus Google mutations in the gene have been identified in with dysplasia Maroteaux a hereditary human characterized by disproportionate dwarfism H. P. R.M. S. D. S. T. J. V. M. J. J. B. Y. Am. J. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar). We therefore concluded that the retarded longitudinal bone growth of cn/cn mouse resulted from cGMP in the chondrocytes during endochondral ossification caused by a loss-of-function mutation in the Npr2 gene. The cn/cn mice disproportionate dwarfism with short limbs and tail, and the prominent during endochondral ossification of of and hypertrophic chondrocytes in a narrowed epiphyseal growth (17.del Marco A. Reprod. Nutr. Dev. 1981; 21: 1025-1031Crossref PubMed Scopus (5) Google Scholar, 18.Wikstrom B. Hjerpe A. Reinholt F.P. Engfeldt B. Coll. Relat. Res. 1986; 6: 279-293Crossref PubMed Scopus (3) Google Scholar, D.R. J. Anat. Google Scholar, D.R. J. Anat. Google Scholar). These skeletal phenotypes of cn/cn mice are and similar to of CNP-deficient mice (11.Chusho H. Tamura N. Ogawa Y. Yasoda A. Suda M. Miyazawa T. Nakamura K. Nakao K. Kurihara T. Komatsu Y. Itoh H. Tanaka K. Saito Y. Katsuki M. Nakao K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4016-4021Crossref PubMed Scopus (369) Google Scholar). In of cGMP are detected in the of CNP-deficient mice and in cells the Npr2 gene (11.Chusho H. Tamura N. Ogawa Y. Yasoda A. Suda M. Miyazawa T. Nakamura K. Nakao K. Kurihara T. Komatsu Y. Itoh H. Tanaka K. Saito Y. Katsuki M. Nakao K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4016-4021Crossref PubMed Scopus (369) Google Scholar). CNP not to NPRB with high also NPRA with (8.Suga S. Nakao K. Hosoda K. Mukoyama M. Ogawa Y. Shirakami G. Arai H. Saito Y. Kambayashi Y. Inouye K. Imura H. Endocrinology. 1992; 130: 229-239Crossref PubMed Google Scholar, Lowe D.G. N. K. H. Goeddel D.V. J. PubMed Scopus Google Scholar), NPRA is in mouse (13.Yasoda A. Ogawa Y. Suda M. Tamura N. Mori K. Sakuma Y. Chusho H. Shiota K. Tanaka K. Nakao K. J. Biol. Chem. 1998; 273: 11695-11700Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), and mice no skeletal S. V. J. Garbers D.L. A. Nature. 1995; PubMed Scopus Google Scholar). these findings that the pathway the CNP to longitudinal bone growth by regulating endochondral ossification. the phenotypes of cn/cn and CNP-deficient mice are similar in of and in the of tibial growth the of these mice are The of CNP-deficient mice is at days of (11.Chusho H. Tamura N. Ogawa Y. Yasoda A. Suda M. Miyazawa T. Nakamura K. Nakao K. Kurihara T. Komatsu Y. Itoh H. Tanaka K. Saito Y. Katsuki M. Nakao K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4016-4021Crossref PubMed Scopus (369) Google Scholar), that of the cn/cn mice used in this study was than not CNP not in endochondral ossification also in cardiovascular and T. T. T. Mori K. S. Y. M. K. Endocrinology. PubMed Scopus Google Scholar). In CNP has been detected in tissues as the and H. E. M. T. 2000; PubMed Scopus Google Scholar). Thus, the CNP by other including NPRA, a in these tissues other than skeletal for this in is and Dickie (14.Lane P.W. Dickie M.M. J. Hered. 1968; 59: 300-308Crossref PubMed Scopus (72) Google Scholar) described that most cn/cn mice used in study of The cn/cn mice used in study an AKR/J the cn/cn mice used in this study a of and (14.Lane P.W. Dickie M.M. J. Hered. 1968; 59: 300-308Crossref PubMed Scopus (72) Google Scholar, D.R. J. Anat. Google Scholar). Since it is cn/cn and the CNP-deficient mice from the further on the cause of of cn/cn and the CNP-deficient mice with an a of CNP NPRB in tissues other than in bone dysplasia Maroteaux is an autosomal recessive human characterized by disproportionate short and of the extremities P. B. E. Med. Google Scholar), of the of a model for this in the gene have been identified in affected and guanylyl cyclase of NPRB of mutant have also been H. P. R.M. S. D. S. T. J. V. M. J. J. B. Y. Am. J. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar). to the of AMDM, the short limbs become more during than during the postnatal and PubMed Scopus Google Scholar). The skeletal abnormalities of cn/cn mice also more with (15.Silberberg R. Lesker P. Growth. 1975; 39: 17-33PubMed Google Scholar, 16.Johnson D.R. J. Anat. 1978; 125: 267-275PubMed Google Scholar). cn/cn mouse is a useful model for the and of is that CNP and cGMP signals in the of endochondral ossification. However, the of the pathway through NPRB during endochondral ossification The cn/cn mouse is the model with a Npr2 gene and be used to the of In cn/cn mice, the of chondrocytes in the epiphyseal growth are significantly than in mice D.R. J. Anat. Google Scholar, D.R. J. Anat. Google Scholar). The of and hypertrophic chondrocytes are also in cn/cn mice (17.del Marco A. Reprod. Nutr. Dev. 1981; 21: 1025-1031Crossref PubMed Scopus (5) Google Scholar, 18.Wikstrom B. Hjerpe A. Reinholt F.P. Engfeldt B. Coll. Relat. Res. 1986; 6: 279-293Crossref PubMed Scopus (3) Google Scholar). The pathway through NPRB therefore to positively chondrocyte and However, further are to this pathway regulates chondrocyte and growth is a of the most that regulate the and of chondrocytes 1998; 125: Google Scholar). similar abnormalities in the epiphyseal growth plates to of cn/cn mice 1998; 125: Google Scholar). CNP mice the achondroplastic phenotype of mutant mice by the pathway through and the of CNP/NPRB have been suggested for the of human A. Komatsu Y. Chusho H. Miyazawa T. A. Miura M. Kurihara T. T. Tanaka S. Suda M. Tamura N. Ogawa Y. Nakao K. Med. PubMed Scopus Google Scholar). CNP signals by NPRB regulate chondrocyte and during endochondral ossification through of the from using cn/cn mice, as as CNP-deficient mice, will to the of the pathway in chondrocytes during endochondral ossification and to develop for human skeletal dysplasias.