Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional Adrenomedullin gene

Abstract

Adrenomedullin, a recently identified potent vasodilator, is expressed widely and has been suggested to have functions ranging from reproduction to blood pressure regulation. To elucidate these functions and define more precisely sites of Adm expression, we replaced the coding region of the Adm gene in mice with a sequence encoding enhanced green fluorescent protein while leaving the Adm promoter intact. We find that Adm(-/-) embryos die at midgestation with extreme hydrops fetalis and cardiovascular abnormalities, including overdeveloped ventricular trabeculae and underdeveloped arterial walls. These data suggest that genetically determined absence of Adm may be one cause of nonimmune hydrops fetalis in humans.

Figure 1

Generation of Adm^−/− animals by homologous recombination. (a) Strategy to disrupt the Adm gene. (Top) Endogenous wild-type (WT) allele. (Middle) Linearized targeting vector. (Bottom) Targeted allele after homologous recombination. Open boxes represent exons 1–4; the checkered region in exon 4 codes for the mature Adm peptide; the initiator methionine of the propeptide is indicated (Met). The solid black box depicts a bovine growth hormone poly(A) addition sequence downstream of the EGFP cDNA. The locations of primer sequences for PCR (p1, p2, and p3) are shown by small arrows. The sequence used as a probe for the Southern-based detection strategy is indicated by a labeled line (PROBE). Restriction sites: A, AvrII; B, BamHI; Sc, SacI; Sp, SpeI; X, XhoI. Parentheses denote sites destroyed during cloning. (b) Detection of the targeted allele by Southern blot analysis. Genomic DNA from E9.5 embryos was digested with AvrII and probed with the SacI/AvrII fragment (PROBE) depicted in the bottom line of a. (c) Detection of the targeted allele by PCR. Genomic DNA from embryos was amplified with the primer sequences depicted in a. (d) Measurement of Adm RNA in total RNA extracts from E9.5 embryos by real-time quantitative reverse transcription–PCR. The relative quantity of Adm RNA in Adm^+/− and Adm^−/− embryos is expressed as a percentage of total Adm RNA in Adm^+/+ embryos. Error bars represent SEM.

Figure 2

Sites of Adm expression determined by EGFP fluorescence. (a) Frozen section of adrenal gland from an adult Adm^+/−. Note intense fluorescence in adrenal medulla (m) and the absence of fluorescence in adrenal cortex (c). (b) Confocal microscopy of an E9.5 Adm^+/− embryo. Moderate expression is seen in the heart (arrowhead h), and strong expression in the developing vasculature (arrowhead v) and forebrain (arrowhead b); a speck of nonspecific fluorescence has been covered with a black box. (c) Frozen section of an E13.5 Adm^+/− embryo, showing expression in the developing left ventricle. Arrowheads indicate the pericardial surface of the left ventricle. (d) Frozen section of an E16.5 Adm^+/− embryo, showing expression in the aorta (a), esophagus (oe), and thoracic duct (td).

Figure 3

Adm^−/− embryos have massive generalized edema. (a) Appearance of E14.5 Adm^−/− (Left) and Adm^+/+ (Right) embryos in their yolk sacs shortly after dissection from the uterus. Note that the yolk sac (arrowhead ys) of the Adm^−/− embryo is distended with fluid, and its blood vessels are thinner than those of its wild-type littermate. (b) The thoracic cavity of E13.5 Adm^−/− embryos is considerably enlarged (Left) compared with a wild-type littermate (Right). The black lines indicate the width of the thoracic cavity just above the diaphragm. (c) Severe hydrops fetalis is apparent in the E14.5 Adm^−/− embryos (Left), readily visible after their yolk sacs are removed. The arrowheads (s) indicate the outer skin layer of the embryos. (d) The extreme hydrops of the Adm^−/− embryos, dissected away from their yolk sacs, is completely general, as indicated by the uniformly swollen back and head. The arrowheads (s) again indicate the outer skin layer. (e and f) H&E stain of transverse sections through E14.5 Adm^+/+ (e) and Adm^−/− (f) embryos. The Adm^−/− embryo has a fluid-filled thoracic cavity, and the tissues external to the rib cage are markedly swollen (e and f, ×1).

Figure 4

Adm^−/− embryos have developmental heart defects. Transverse sections through the hearts of Adm^+/+ (Upper) and Adm^−/− (Lower) embryos were stained with H&E. (a) E13.5 Adm^+/+ littermate. (d) E13.5 Adm^−/− embryo. The heart of the Adm^−/− embryo is smaller than the heart of its wild-type littermate. (b) E14.5 Adm^+/+ littermate. (e) E14.5 Adm^−/− embryo. The heart of the Adm^−/− embryo is still smaller, and the left ventricle is significantly occluded with increased trabeculae compared with its Adm^+/+ littermate. (c) E14.5 Adm^+/+ littermate. (f) E14.5 Adm^−/− embryo. Higher-power magnification of the left ventricle at E14.5 shows an increase in the number of trabeculae, a thinner, convoluted compact zone (cz), and a generally delaminated appearance of the Adm^−/− heart compared with wild type. c, endocardial cushion; s, septum; t, tricuspid valve; m, mitral valve; rv, right ventricle; lv, left ventricle; ch, chamber; cz, compact zone (a, b, d, and e, ×2; c and f, ×10).

Figure 5

Adm^−/− embryos have thin arterial walls. Transverse sections through vessels of Adm^+/+ (Upper) and Adm^−/− (Lower) embryos at E13.5 were stained with H&E. (a and c) aorta; (b and d) carotid artery. Note the thin vascular walls (approximately three cells thick) of the Adm^−/− vessels compared with wild-type vessels (approximately five cells thick). a, aorta; c, carotid artery (×10).