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

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.

我们此前主要通过C型利钠肽（C-type natriuretic peptide，CNP）基因敲除突变小鼠模型，探究了C型利钠肽在软骨内骨生长中的生理作用，并证实CNP是小鼠生理性软骨内骨生长不可或缺的因子。然而，C型利钠肽基因敲除（knockout，KO）小鼠在出生后至10周龄时的存活率仅约70%，我们无法对其成年阶段的表型进行充分分析。为此，我们采用锌指核酸酶介导的基因组编辑技术构建了CNP KO大鼠模型。我们成功建立了两株完全缺失CNP的突变大鼠株系，其表型与CNP KO小鼠一致，即表现为软骨内骨生长严重受损的矮小体型。组织学分析显示，CNP KO大鼠的生长板宽度显著降低，尤其是肥大软骨细胞层的宽度；生长板软骨细胞的增殖活性也呈下降趋势。值得注意的是，CNP KO大鼠未出现咬合不正，且出生后存活时长可超过一年。在33周龄时，CNP KO大鼠的体型显著短于野生型（wild-type）大鼠，所有样本的股骨生长板均已闭合，而野生型大鼠未观察到该现象。组织学观察表明，在本次研究检测的所有机体组织中，CNP缺失仅对骨骼产生影响。综上，CNP KO大鼠可存活一年以上，且终生表现出软骨内骨生长缺陷与生长迟缓表型。