Rats deficient C-type natriuretic peptide suffer from impaired skeletal growth without early death

<div><p>We have previously investigated the physiological role of C-type natriuretic peptide (CNP) on endochondral bone growth, mainly with mutant mouse models deficient in CNP, and reported that CNP is indispensable for physiological endochondral bone growth in mice. However, the survival rate of CNP knockout (KO) mice fell to as low as about 70% until 10 weeks after birth, and we could not sufficiently analyze the phenotype at the adult stage. Herein, we generated CNP KO rats by using zinc-finger nuclease-mediated genome editing technology. We established two lines of mutant rats completely deficient in CNP (CNP KO rats) that exhibited a phenotype identical to that observed in mice deficient in CNP, namely, a short stature with severely impaired endochondral bone growth. Histological analysis revealed that the width of the growth plate, especially that of the hypertrophic chondrocyte layer, was markedly lower and the proliferation of growth plate chondrocytes tended to be reduced in CNP KO rats. Notably, CNP KO rats did not have malocclusions and survived for over one year after birth. At 33 weeks of age, CNP KO rats persisted significantly shorter than wild-type rats, with closed growth plates of the femur in all samples, which were not observed in wild-type rats. Histologically, CNP deficiency affected only bones among all body tissues studied. Thus, CNP KO rats survive over one year, and exhibit a deficit in endochondral bone growth and growth retardation throughout life.</p></div

Histological analysis of the growth plates of CNP KO rats.

<p>(A) Histological pictures of tibial growth plates of three-week-old male WT, homozygous Δ11, and homozygous Δ774 mutant rats. WT rats from Δ774 mutant line were used for this analysis. Upper panels show the results of Alcian blue-H&E staining, while the middle and lower panels show the results of immunohistochemical staining for type II and type X collagens, respectively. (B) and (C) Graphs of the widths of total growth plates (B) and their hypertrophic chondrocyte layers (C). n = 3 in each group and each value is expressed as a ratio (%) of the WT value. *, <i>P</i> < 0.01 vs WT. (D) Histological pictures showing BrdU staining of the tibial growth plates of three-week-old male WT, homozygous Δ11, and homozygous Δ774 mutant rats. (E) Graphs of the number of BrdU positive chondrocytes in the proliferative chondrocyte layers of the growth plates.</p

Sequences of the genotyping PCR primers for the detection of mutations in the CNP gene.

<p>Sequences of the genotyping PCR primers for the detection of mutations in the CNP gene.</p

Gross appearance of and growth curves for Δ11 mutant rats.

<p>(A) and (B) Gross appearance of male WT rat and rat with a homozygous Δ11 mutation at two weeks (A) and 14 weeks (B) of age. Black scale bar in each picture denotes 10 mm. (C) Growth curves for Δ11 mutant rats. Upper panels show naso-anal lengths of male (left) and female (right) rats and lower panels exhibit body weight. n = 6, 15, and 10 for male WT, heterozygous Δ11, and homozygous Δ11 rats, respectively; n = 8, 14, and 5 for female WT, heterozygous Δ11, and homozygous Δ11 rats, respectively. *, <i>P</i> < 0.01 vs WT, §, <i>P</i> < 0.01 vs WT, and ¶, <i>P</i> < 0.05 vs WT.</p

Typical examples of H&E-stained specimens of the heart of female WT (left) and CNP KO (homozygous Δ11 mutant, right) rats.

<p>Scale bar shows 1 mm.</p

Growth curves for Δ774 mutant rats.

<p>Upper panels show naso-anal lengths of male (left) and female (right) rats and lower panels exhibit body weight. n = 6, 6, and 7 for male WT, heterozygous Δ774, and homozygous Δ774 rats, respectively; n = 11, 7, and 4 for female WT, heterozygous Δ774, and homozygous Δ774 rats, respectively. *, <i>P</i> < 0.01 vs WT, §, <i>P</i> < 0.01 vs WT, and ¶, <i>P</i> < 0.05 vs WT.</p

Generation of targeted mutations in the CNP-encoding gene <i>Nppc</i> by ZFN.

<p>(A) Schematic representation of the CNP protein and the target site within it (arrow). The empty (white) portion represents the signal peptide (23 a.a.), the filled (black) portion represents the NT-proCNP (50 a.a.), and the gray portion indicates the mature CNP with either 53 or 22 a.a. (B) Schematic representation of the target site in the rat <i>Nppc</i> (NC_005108.4) locus. (C) Schematic representation of the locations of PCR primers for genotyping.</p

Body length, body weight, heart weight, and kidney weight of female CNP KO and WT rats at 33 weeks of age.

<p>Body length, body weight, heart weight, and kidney weight of female CNP KO and WT rats at 33 weeks of age.</p

Skeletal analyses of CNP KO rats.

<p>(A) Soft X-ray pictures of homozygous Δ11 mutant (left) and homozygous Δ774 mutant (right) rats compared with WT rats. (B) Lengths of each indicated bone in male WT, homozygous Δ11, or homozygous Δ774 mutant rats at 20 weeks of age, measured from soft X-ray pictures. WT rats from both Δ11 and Δ774 lines were used for this analysis. n = 3–4, each, *, <i>P</i> < 0.01 vs WT.</p

Gross appearance and femoral growth plates of CNP KO rats at 33 weeks of age.

<p>(A) Gross appearance of female WT (left) and homozygous Δ11 mutant (right) rats. (B) Typical examples of an H&E-stained specimen of the femoral bone (proximal and distal region) in WT and KO rats at 33 weeks of age. Scale bar shows 1 mm.</p