Transcription of endogenous genes in preimplantation 1- and 2-cell mouse embryos was determined by monitoring the incorporation of BrUTP by plasma membrane-permeabilized embryos. Incorporation is observed starting by mid-S phase in the 1-cell embryo and increases progressively; the amount of incorporation by the 1-cell embryo in G2 is about 20% that of the 2-cell embryo in G2. Incorporation by the male pronucleus is always about four to five times greater than that of the female pronucleus. Nevertheless, the amount of incorporation by the female pronucleus present in parthogenetically activated eggs is similar to the total amount of incorporation in inseminated eggs, i.e., the transcriptional capacity of the female pronucleus is not inherently less than that of the male pronucleus. Inhibiting the first round of DNA replication does not prevent the initiation of transcription in the 1-cell embryo, but does inhibit the extent of BrUTP incorporation by 35%. The transcriptional machinery of the 1-cell embryo appears to be rate-limiting, since the total amount of BrUTP incorporation by parthenogenetically activated and dispermic eggs is similar to that in monospermic eggs; trispermic eggs incorporate BrUTP to only about 60% the level of monospermic eggs. A transcriptionally repressive state may start to develop in the 2-cell embryo, since inhibiting the second round of DNA replication results in an 50% increase in BrUTP incorporation. Trapoxin treatment, which induces histone hyperacetylation, enhances incorporation by 2-cell embryos 1.8-fold and suggests that histone hyperacetylation can relieve this repression.

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Abstract-Gene targeting in the mouse has become a standard approach, yielding important new insights into the genetic factors underlying cardiovascular development and disease. However, we still have very limited understanding of how mutations affect developing cardiovascular function, and few studies have been performed to measure altered physiological parameters in mouse mutant embryos. Indeed, although in utero lethality due to embryonic heart failure is one of the most common results of gene targeting experiments in the mouse, the underlying physiological mechanisms responsible for embryonic demise remain elusive. Using in utero ultrasound biomicroscopy (UBM), we studied embryonic day (E) 10.5 to 14.5 NFATc1 Ϫ/Ϫ embryos and control littermates. NFATc1 Ϫ/Ϫ mice, which lack outflow valves, die at mid-late gestation from presumed defects in forward blood flow with resultant heart failure. UBM showed increasing abnormal regurgitant flow in the aorta and extending into the embryonal-placental circulation, which was evident after E12.5 when outflow valves normally first develop. Reduced NFATc1 Ϫ/Ϫ net volume flow and diastolic dysfunction contributed to heart failure, but contractile function remained unexpectedly normal. Among 107 NFATc1 Ϫ/Ϫ embryos imaged, only 2 were observed to be in acute decline with progressive bradyarrhythmia, indicating that heart failure occurs rapidly in individual NFATc1 Ϫ/Ϫ embryos. This study is among the first linking a specific physiological phenotype with a defined genotype, and demonstrates that NFATc1 Ϫ/Ϫ embryonic heart failure is a complex phenomenon not simply attributable to contractile dysfunction.