Hydrops fetalis, cardiovascular defects, and embryonic lethality in mice lacking the calcitonin receptor-like receptor gene

Abstract

Adrenomedullin (AM) is a multifunctional peptide vasodilator that is essential for life. To date, numerous in vitro studies have suggested that AM can mediate its biological effects through at least three different receptors. To determine the in vivo importance of the most likely candidate receptor, calcitonin receptor-like receptor, a gene-targeted knockout model of the gene was generated. Mice heterozygous for the targeted Calcrl allele appear normal, survive to adulthood, and reproduce. However, heterozygote matings fail to produce viable Calcrl-/- pups, demonstrating that Calcrl is essential for survival. Timed matings confirmed that Calcrl-/- embryos die between embryonic day 13.5 (E13.5) and E14.5 of gestation. The Calcrl-/- embryos exhibit extreme hydrops fetalis and cardiovascular defects, including thin vascular smooth muscle walls and small, disorganized hearts remarkably similar to the previously characterized AM-/- phenotype. In vivo assays of cellular proliferation and apoptosis in the hearts and vasculature of Calcrl-/- and AM-/- embryos support the concept that AM signaling is a crucial mediator of cardiovascular development. The Calcrl gene targeted mice provide the first in vivo genetic evidence that CLR functions as an AM receptor during embryonic development.

FIG. 1.

Generation of Calcrl^−/− animals by homologous recombination. (a) Strategy to disrupt the Calcrl gene. (Top) Endogenous wild-type allele. (Middle) Targeting vector. (Bottom) Targeted allele following homologous recombination. Primer locations for PCR (p1, p2, p3, and p4) are shown by arrows. The location of the probe used for the Southern-based detection strategy is indicated by a labeled line (probe). The targeting vector plasmid sequence is indicated by a thin wavy line. Restriction sites: H, HindIII; X, XhoI. The initiator methionine and terminator codons are indicated as Met and STOP. (b) Detection of targeted ES cells by Southern blot analysis. Digestion of genomic DNA with HindIII results in a 9.0-kb fragment for the WT allele and a 7.4-kb fragment for the targeted allele when probed with the fragment depicted in panel a. (c) Primers depicted in panel a were used to amplify genomic DNA from embryos. (d) Measurement of Calcrl expression from total RNA extracts by real-time quantitative reverse transcription-PCR. The relative quantity of Caclrl RNA in Calcrl^+/− and Calcrl^−/− embryos is represented as a percentage of total Calcrl RNA in WT embryos. Error bars represent standard errors of the mean.

FIG. 2.

Calcrl^−/− embryos have massive generalized edema. (a) Generalized edema is observed throughout the entire body in Calcrl^−/− embryos at E12.5. (b) By E13.5, the generalized edema in Calcrl^−/− embryos has progressed to severe hydrops fetalis. (c and d) H&E stain of transverse sections through E13.5 WT (c) and Calcrl^−/− (d) embryos. The thoracic cavity and interstitial tissues are filled with fluid and distended (d, arrows). Magnification, ×1.

FIG. 3.

Calcrl^−/− embryos have thin arterial walls due to reduction in vascular smooth muscle cell proliferation. (a to f)Transverse sections through the descending aorta of wild-type and Calcrl^−/− embryos at E11.5, 12.5, and 13.5 were stained with H&E. The thickness of the vessel walls at E11.5 is similar in wild-type and Calcrl^−/− sections. However, by E12.5 and E13.5, the vascular walls are thinner in Calcrl^−/− mice (approximately three cells thick) compared to wild-type controls (approximately six cells thick). Asterisks indicate the accumulation of interstitial edema in Calcrl^−/− embryos. The percentage of proliferating, BrdU-positive cells in the aortic walls of Calcrl^−/− mice is significantly less than in those of wild-type controls (g). (h and j) Vascular smooth muscle cells in wild-type and Calcrl^−/− aortas stain positive for α-SMA. (i and k) Anti-PECAM staining shows that wild-type and Calcrl^−/− aortas have normal endothelial patterning. Magnification, ×10. Scale bar, 50 μm.

FIG. 4.

Calcrl^−/− embryos have developmental heart defects. Transverse sections through the hearts of wild-type and Calcrl^−/− embryos at E11.5, 12.5, and 13.5 were stained with H&E. (a and d) At E11.5, the overall heart size is similar in both wild-type and Calcrl^−/− embryos. (b and e) At E12.5, the overall heart size in the Calcrl^−/− embryo is smaller than that of its wild-type littermate. (c and f) At E13.5, the overall heart size in the Calcrl^−/− embryo is approximately two-thirds the size of that of its wild-type littermate. c, endocardial cushion; s, septum; t, tricuspid valve; m, mitral valve; rv, right ventricle; lv, left ventricle. Magnification, ×4. Scale bar, 200 μm.

FIG. 5.

Calcrl^−/− embryos show thin and disorganized compact zones of the heart. Transverse sections through the hearts of wild-type and Calcrl^−/− embryos at E11.5, 12.5, and 13.5 were stained with H&E. The compact zone in wild-type and Calcrl^−/− embryos at E11.5 (a and e) and E12.5 (b and f) is similar in thickness and cellular organization. At E13.5 (c and g), the compact zone is thinner in the Calcrl^−/− embryo. Higher magnification (d and h) reveals a discontinuous and convoluted organization of the compact zone in the Calcrl^−/− section. ch, chamber; cz, compact zone. Magnification, ×10 (a to c and e to g); ×20 (d and h). Scale bar, 100 μm (a to c and e to g); 50 μm (d and h).

FIG. 6.

Calcrl^−/− and AM^−/− embryos have defects in cardiac cell proliferation. The proliferation index of cardiac cells was determined as the number of BrdU-positive cells per total area in transverse heart sections. (a) No significant difference was found between wild-type and Calcrl^−/− embryos at E11.5. However, at E12.5 the proliferation index was significantly lower in Calcrl^−/− embryos than in wild-type controls. (b) The proliferation index of AM^−/− embryos at E13.5 is significantly lower than that of wild-type littermates.

FIG. 7.

Calcrl^−/− and AM^−/− embryos at E13.5 have increased levels of cardiac apoptosis. Typical pictures of TUNEL-stained transverse sections. (a) The number of TUNEL-positive cells is greater in Calcrl^−/− (b) and AM^−/− (d) hearts than in hearts of wild-type littermates (a and c).