Three new allelic mouse mutations that cause skeletal overgrowth involve the natriuretic peptide receptor C gene (Npr3)

Abstract

In 1979, a BALB/cJ mouse was identified with an exceptionally long body. This phenotype was found to be caused by a recessive mutation, designated longjohn (lgj), that mapped to the proximal region of chromosome 15. Several years later, a mouse with a similarly elongated body was identified in an outbred stock after chemical mutagenesis with ethylnitrosourea. This phenotype also was caused by a recessive mutation, designated strigosus (stri). The two mutations were found to be allelic. A third allele was identified in a DBA/2J mouse and was designated longjohn-2J (lgj(2J)). Analysis of skeletal preparations of stri/stri mice indicated that the endochondral ossification process was slightly delayed, resulting in an extended proliferation zone. A recent study reported that mice overexpressing brain natriuretic peptide, one of the members of the natriuretic peptide family, exhibit a skeletal-overgrowth syndrome with endochondral ossification defects. The Npr3 gene coding for type C receptor for natriuretic peptides (NPR-C), which is mainly involved in the clearance of the natriuretic peptides, mapped in the vicinity of our mouse mutations and thus was a candidate gene. The present study reports that all three mutations involve the Npr3 gene and provides evidence in vivo that there is a natriuretic-related bone pathway, underscoring the importance of natriuretic peptide clearance by natriuretic peptide type C receptor.

Figure 1

Phenotype. Wild-type sib of homozygous lgj^2J mouse (Upper Left), lgj^2J/lgj^2J mutant (Upper Right), lgj/lgj mutant (Lower Left), and stri/stri mutant (Lower Right) mice at 3 months of age.

Figure 2

Alizarin red S/alcian blue staining of forepaw at various postpartum developmental stages. (A) Hand schematic representation with legends. (B) Forepaw of 0.5-day-old control (Upper) and stri/stri mutant (Lower) mice. (C) Forepaw of 2.5-day-old control (Upper) and stri/stri mutant (Lower) mice. (D) Forepaw of 6.5-day-old control (Upper) and stri/stri mutant (Lower) mice.

Figure 3

Skeletal phenotype at the trunk level. (A) Alizarin red S/alcian blue staining of the trunk in 10.5-day-old control (Upper) and stri/stri mutant (Lower) mice. Dorsal view from lumbar and sacral region. Arrows denote the transverse processes. (B) Hematoxylin and eosin-stained histological sections of cervical-thoracic vertebrae in 21-day-old control (Upper) and stri/stri mutant (Lower) mice. Magnification ×2. Arrows denote the nucleus pulposus localized in the intervertebral disc. (C) Higher magnification (×40) of hematoxylin and eosin-stained histological sections of cervical–thoracic vertebrae in 21-day-old control (Upper) and stri/stri mutant (Lower) mice. Arrows denote the nucleus pulposus. Arrowhead denotes aberrant proliferation.

Figure 4

Genetic map and sequence chromatograms. (A) Two distinct interspecific backcrosses were made, and 1,401 backcross mice were analyzed. Markers were ordered by minimizing double recombinants. Number of recombinant animals is given for each interval. Genetic distances and confidence intervals (in brackets) were calculated at risk α = 0.05. (B) Sequence chromatograms for control (2 chromatograms, at the top) and lgj/lgj mutant (Lower) mice. Deletion site is indicated in the mutant chromatogram, the two chromatograms above are part of control sequence viewed as flanking the deletion. (C) Sequence chromatograms for control (Upper) and lgj^2J/lgj^2J mutant (Lower) mice. Arrow denotes mutation site. (D) Sequence chromatograms for control (Upper) and stri/stri mutant (Lower) mice. Arrow denotes mutation site.

Figure 5

Partial predicted amino acid sequence encoded by Npr3 gene compared among species. Amino acid sequence is numbered according to mouse sequence. The three mutation sites are boxed as follows: shaded box, lgj allele; normal box, lgj^2J allele; striated box, stri allele. Bold underlined amino acids denote sequencing discrepancies with previous published cDNA sequence, which leads to three changes in the predicted amino acid sequence resulting in a protein that shares three more conserved residues among species.