Disruption of ECE-1 and ECE-2 reveals a role for endothelin-converting enzyme-2 in murine cardiac development
Hiromi Yanagisawa, Robert E. Hammer, James A. Richardson, Noriaki Emoto, S. Clay Williams, Shin-ichi Takeda, David E. Clouthier, Masashi Yanagisawa
Hiromi Yanagisawa, Robert E. Hammer, James A. Richardson, Noriaki Emoto, S. Clay Williams, Shin-ichi Takeda, David E. Clouthier, Masashi Yanagisawa
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Disruption of ECE-1 and ECE-2 reveals a role for endothelin-converting enzyme-2 in murine cardiac development

Abstract

Endothelin-converting enzyme-1 and -2 (ECE-1 and -2) are membrane-bound metalloproteases that can cleave biologically the inactive endothelin-1 (ET-1) precursor to form active ET-1 in vitro. We previously reported developmental defects in specific subsets of neural crest–derived tissues, including branchial arch–derived craniofacial structures, aortic arch arteries, and the cardiac outflow tract in ECE-1 knockout mice. To examine the role of ECE-2 in cardiovascular development, we have now generated a null mutation in ECE-2 by homologous recombination. ECE-2 null mice develop normally, are healthy into adulthood, are fertile in both sexes, and live a normal life span. However, when they are bred into an ECE-1–null background, defects in cardiac outflow structures become more severe than those in ECE-1 single knockout embryos. In addition, ECE-1–/–; ECE-2–/– double null embryos exhibited abnormal atrioventricular valve formation, a phenotype never seen in ECE-1 single knockout embryos. In the developing mouse heart, ECE-2 mRNA is expressed in the endocardial cushion mesenchyme from embyronic day (E) 12.5, in contrast to the endocardial expression of ECE-1. Levels of mature ET-1 and ET-2 in whole ECE-1–/–; ECE-2–/– embryos at E12.5 do not differ appreciably from those of ECE-1–/– embryos. The significant residual ET-1/ET-2 in the ECE-1–/–; ECE-2–/– embryos indicates that proteases distinct from ECE-1 and ECE-2 can carry out ET-1 activation in vivo.

Authors

Hiromi Yanagisawa, Robert E. Hammer, James A. Richardson, Noriaki Emoto, S. Clay Williams, Shin-ichi Takeda, David E. Clouthier, Masashi Yanagisawa

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Generation of ECE-2–/– mice by homologous recombination. (a) Targeting s...
Generation of ECE-2–/– mice by homologous recombination. (a) Targeting strategy. The exon encoding the zinc-binding domain of ECE-2 is replaced with a neor cassette driven by the RNA polymerase II promoter. Two tandem repeats of thymidine kinase (TK) are used for the negative selection with FIAU (see Methods). PCR primers in the neor gene and 3′ region external to the short arm of targeting vector are shown by arrows. AExon numbers are indicated according to the equivalent exons of the human ECE-1 gene (32); other ECE-2 exons are not shown. (b) Southern blot analysis of tail DNA from the offspring of ECE-2+/– intercrosses. DNA was digested with BamHI and hybridized with a 3′ probe (indicated by the gray box in panel a). k.o., targeted allele; w.t., wild-type allele. (c) RT-PCR of the ECE-2 transcript using total RNA extracted from the brain of ECE-2+/+, ECE-2+/–, and ECE-2–/– mice. Primers amplify the exon encoding the essential zinc-binding domain of ECE-2. The upper panel shows that a 160-bp transcript fragment is detected in wild-type and ECE-2+/– mice, but is absent in ECE-2–/– mice. The middle panel shows that no transcripts are detected without RT incubation. The lower panel shows detection of β-actin transcripts, indicating intact RNA preparations in all lanes.