A syndrome of tricuspid atresia in mice with a targeted mutation of the gene encoding Fog-2.

Tricuspid atresia (TA) is a common form of congenital heart disease, accounting for 1-3% of congenital cardiac disorders. TA is characterized by the congenital agenesis of the tricuspid valve connecting the right atrium to the right ventricle and both an atrial septal defect (ASD) and a ventricular septal defect (VSD). Some patients also have pulmonic stenosis, persistence of a left-sided superior vena cava or transposition of the great arteries. Most cases of TA are sporadic, but familial occurrences with disease in multiple siblings have been reported. Gata4 is a zinc-finger transcription factor with a role in early cardiac development. Gata4-deficient mice fail to form a ventral heart tube and die of circulatory failure at embryonic day (E) 8.5 (refs 6,7). Zfpm2 (also known as Fog-2) is a multi-zinc-finger protein that is co-expressed with Gata4 in the developing heart beginning at E8.5 (refs 8-10). Zfpm2 interacts specifically with the N-terminal zinc finger of Gata4 and represses Gata4-dependent transcription. Here we use targeted mutagenesis to explore the role of Zfpm2 in normal cardiac development. Zfpm2-deficient mice died of congestive heart failure at E13 with a syndrome of tricuspid atresia that includes an absent tricuspid valve, a large ASD, a VSD, an elongated left ventricular outflow tract, rightward displacement of the aortic valve and pulmonic stenosis. These mice also display hypoplasia of the compact zone of the left ventricle. Our findings indicate the importance of Zfpm2 in the normal looping and septation of the heart and suggest a genetic basis for the syndrome of tricuspid atresia.

Androgen regulation of the largest subunit of RNA polymerase II in the rat ventral prostate.

One of the dramatic changes in the prostate during androgen manipulation is the alteration in cellular content of total RNA - the amount of total RNA in each cell. The abundance of cellular total RNA correlates with the RNA polymerase (RNAP) activity in the prostate. One possible mechanism of androgen regulation of RNAP activity involves the regulation of RNAP expression. Western blot analysis showed that the largest subunit of the RNAP II, an essential component of the transcriptional machinery for mRNA, is indeed regulated by androgens. Castration down-regulates the protein level of RNAP II, whereas androgen replacement up-regulates the protein. However, androgen manipulation does not have consistent effects on the phosphorylation of the C-terminal domain (CTD) of the RNAP II. Androgen regulation of the RNAP II protein expression was also observed in the seminal vesicles but not in the thymus and liver, indicating that androgen regulation of RNAP II protein expression appears to be limited to the male sex accessory organs. These observations suggest that RNAP II plays an essential role in androgen action in male sex accessory organs.

Adenoviral-mediated gene transfer of a constitutively active form of the retinoblastoma gene product attenuates neointimal thickening in experimental vein grafts.

PURPOSE: Inappropriate or excessive vascular smooth muscle cell proliferation leads to the development of occlusive lesions in up to 50% of vein grafts. The purpose of this study was to test the hypothesis that induced overexpression of a cytostatic nonphosphorylatable form of the retinoblastoma protein (DeltaRb) would attenuate neointimal thickening in experimental vein grafts. METHODS: A replication-deficient adenovirus vector that encoded a nonphosphorylatable, constitutively active form of DeltaRb was constructed (AdDeltaRb) and contained an NH2-terminal epitope tag from the influenza hemagglutinin molecule (HA). Forty-eight male New Zealand white rabbits underwent surgical exposure of the external jugular vein for transfection with either 3 x 10(10) plaque-forming units/mL AdDeltaRb (n = 16), 3 x 10(10) plaque-forming units/mL control adenovirus (AdBglII, n = 15), or vehicle (n = 17) for 10 minutes at 120 mm Hg. After vector exposure, the vein was excised and interposed end-to-end into the carotid circulation. After 5 days, 12 grafts (four from each group) were excised and assayed for genomic DeltaRb DNA with the polymerase chain reaction or for hemagglutinin molecule expression and localization with immunohistochemistry. The remainder of the grafts (n = 36) were perfusion-fixed after 4 weeks, and 5 microm sections prepared for digital planimetric analysis. RESULTS: Polymerase chain reaction results identified the DeltaRb gene only in the grafts that were transfected with AdDeltaRb. Immunohistochemical analysis results revealed transgene expression in most of the endothelial cells and in many of the smooth muscle cells. After 4 weeks, the grafts that were exposed to AdDeltaRb exhibited a 22% reduction in neointimal thickness (vehicle, 77 +/- 7 microm; AdBglII, 75 +/- 5 microm; AdDeltaRb, 60 +/- 5 microm; P =.05), and medial thickness, luminal diameter, and other parameters were unchanged (medial thickness: vehicle, 72 +/- 10 microm; AdBglII, 85 +/- 7 microm; AdDeltaRb, 69 +/- 9 microm; P = NS; luminal diameter: vehicle, 4.5 +/- 0.2 mm; AdBglII, 4.4 +/- 0.2 mm; AdDeltaRb, 4.7 +/- 0.1 mm; P = NS). CONCLUSION: With this delivery system, adenoviral-mediated gene transfer is highly efficient and induced overexpression of DeltaRb leads to a reduction in vein graft neointimal thickening.

Molecular cloning of FOG-2: a modulator of transcription factor GATA-4 in cardiomyocytes.

GATA transcription factors are important regulators of both hematopoiesis (GATA-1/2/3) and cardiogenesis (GATA-4) in mammals. The transcriptional activities of the GATA proteins are modulated by their interactions with other transcription factors and with transcriptional coactivators and repressors. Recently, two related zinc finger proteins, U-shaped (USH) and Friend of GATA-1 (FOG) have been reported to interact with the GATA proteins Pannier and GATA-1, respectively, and to modulate their transcriptional activities in vitro and in vivo. In this report, we describe the molecular cloning and characterization of a third FOG-related protein, FOG-2. FOG-2 is an 1,151 amino acid nuclear protein that contains eight zinc finger motifs that are structurally related to those of both FOG and USH. FOG-2 is first expressed in the mouse embryonic heart and septum transversum at embryonic day 8.5 and is subsequently expressed in the developing neuroepithelium and urogenital ridge. In the adult, FOG-2 is expressed predominately in the heart, brain, and testis. FOG-2 associates physically with the N-terminal zinc finger of GATA-4 both in vitro and in vivo. This interaction appears to modulate specifically the transcriptional activity of GATA-4 because overexpression of FOG-2 in both NIH 3T3 cells and primary rat cardiomyocytes represses GATA-4-dependent transcription from multiple cardiac-restricted promoters. Taken together, these results implicate FOG-2 as a novel modulator of GATA-4 function during cardiac development and suggest a paradigm in which tissue-specific interactions between different FOG and GATA proteins regulate the differentiation of distinct mesodermal cell lineages.

Evidence for non-synchronous rotation of Europa. Galileo Imaging Team.

Non-synchronous rotation of Europa was predicted on theoretical grounds, by considering the orbitally averaged torque exerted by Jupiter on the satellite's tidal bulges. If Europa's orbit were circular, or the satellite were comprised of a frictionless fluid without tidal dissipation, this torque would average to zero. However, Europa has a small forced eccentricity e approximately 0.01 , generated by its dynamical interaction with Io and Ganymede, which should cause the equilibrium spin rate of the satellite to be slightly faster than synchronous. Recent gravity data suggest that there may be a permanent asymmetry in Europa's interior mass distribution which is large enough to offset the tidal torque; hence, if non-synchronous rotation is observed, the surface is probably decoupled from the interior by a subsurface layer of liquid or ductile ice. Non-synchronous rotation was invoked to explain Europa's global system of lineaments and an equatorial region of rifting seen in Voyager images. Here we report an analysis of the orientation and distribution of these surface features, based on initial observations made by the Galileo spacecraft. We find evidence that Europa spins faster than the synchronous rate (or did so in the past), consistent with the possibility of a global subsurface ocean.

Episodic plate separation and fracture infill on the surface of Europa. Galileo Imaging Team.

Images obtained by the Voyager spacecraft revealed dark, wedge-shaped bands on Europa that were interpreted as evidence that surface plates, 50-100 km across, moved and rotated relative to each other. This implied that they may be mechanically decoupled from the interior by a layer of warm ice or liquid water. Here we report similar features seen in higher resolution images (420 metres per pixel) obtained by the Galileo spacecraft that reveal new details of wedge-band formation. In particular, the interior of one dark band shows bilateral symmetry of parallel lineaments and pit complexes which indicates that plate separation occurred in discrete episodes from a central axis. The images also show that this style of tectonic activity involved plates < 10 km across. Although this tectonic style superficially resembles aspects of similar activity on Earth, such as sea-floor spreading and the formation of ice leads in polar seas, there are significant differences in the underlying physical mechanisms: the wedge-shaped bands on Europa most probably formed when lower material (ice or water) rose to fill the fractures that widened in response to regional surface stresses.

Genes regulated by androgen in the rat ventral prostate.

Genes that are regulated by androgen in the prostate were studied in the rat. Four of the less than 10 genes that are down-regulated by androgen in the ventral prostate of a 7-day castrated rat were identified; their mRNAs decayed with identical kinetics. Twenty-five of the estimated 56 genes that are up-regulated by androgen in the castrated prostate have been isolated. The up-regulated genes fall into two kinetic types. Early genes are significantly up-regulated by 6.5 hr whereas the delayed genes respond mainly after 24 hr from the time of androgen replacement. These androgen-response genes are also regulated in the prostate by castration, indicating that these genes could play important roles in androgen-induced regrowth and/or castration-induced regression of the prostate during hormonal manipulation. A survey of the tissue specificity showed that the androgen-response gene expression program in the prostate is mainly prostate-specific. Total RNA Northern blot analysis detects the expression of about 16 up-regulated genes and 3 down-regulated genes in the prostate only. Four up-regulated genes and one down-regulated gene are regulated by androgen in both the prostate and seminal vesicles but not in other organs. The expression of the remaining androgen-response genes is not limited to the prostate but is only responsive to androgen in the prostate. This survey of the androgen-response gene expression program provides insights into the molecular and cellular mechanisms of androgen action in the prostate.