Vascular expression of Notch pathway receptors and ligands is restricted to arterial vessels.

Mice with targeted mutations in genes required for Notch signal transduction die during embryogenesis, displaying overt signs of hemorrhage due to defects in their vascular development. Surprisingly, directed expression of a constitutively active form of Notch4 within mouse endothelial cells produces a similar vascular embryonic lethality. Moreover, patients with mutations in Notch3 exhibit the cerebral vascular disorder, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). These findings underscore the importance of Notch signaling in vascular development; however, they do not identify the specific functional defect. Here, we report that Notch1, Notch3, Notch4, Delta4, Jagged1 and Jagged2 are all expressed in arteries, but are not expressed by veins. These findings identify an aspect of Notch signaling that could contribute to the mechanism by which this pathway modulates vascular morphogenesis.

Regulation of follistatin messenger ribonucleic acid in porcine granulosa cells by epidermal growth factor and the protein kinase-C pathway.

Follistatin is a 35-kilodalton monomer isolated from follicular fluid by virtue of its ability to suppress FSH secretion from cultured pituitary cells. Experiments were designed to test the hypothesis that the accumulation of follistatin RNA in the ovary is regulated by epidermal growth factor (EGF) and activation of the protein kinase-C (PKC) pathway. Follistatin mRNA was quantitated by slot blot hybridization of total RNA from primary cultures of porcine granulosa cells treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA), an activator of PKC. PMA (0.1, 1.0, 10, and 100 nM) induced a dose-dependent increase in follistatin mRNA accumulation after 2 h, with a maximal increase of 40-fold over that in untreated control cultures at a dose of 10 nM. PMA (10 nM) induced a time-dependent increase in follistatin mRNA levels, with a maximal response at 2 h. Follistatin gene expression was induced by a 2-h incubation with EGF (3 nM), but not by LH (100 ng/ml), GnRH (10 nM) or prostaglandin F2 alpha (80 micrograms/ml). EGF (0.01, 0.1, 1, and 10 nM) induced a dose-dependent induction of follistatin gene expression in granulosa cells after 2-h incubation, with maximal stimulation of 33-fold at a dose of 1 nM. The time course of induction of follistatin mRNA by EGF was very similar to that induced by PMA, with maximal stimulation occurring at 2 h and declining thereafter. Pretreatment of granulosa cells for 24 h with PMA abrogated the EGF-induced stimulation of follistatin mRNA accumulation. However, cotreatment of granulosa cells with EGF and PMA for 2 h resulted in additive stimulation of follistatin mRNA. These results demonstrate that 1) follistatin gene expression in cultured porcine granulosa cells is acutely stimulated by PMA and EGF in a time- and dose-dependent manner; 2) follistatin gene expression may be regulated by the PKC pathway; and 3) the stimulatory effect of EGF on follistatin gene expression may require PKC.

Superovulatory and endocrine responses in heifers treated with FSH-P at different stages of the estrous cycle.

Forty-two Holstein heifers were superovulated with FSH-P (total dose, 30 mg) and cloprostenol. Treatment was initiated on Day 3 (Group D3, n = 11), Day 6 (Group D6, n = 11), Day 9 (Group D9, n = 10) or Day 12 (Group D12, n = 10) of the estrous cycle. Heifers were bled daily for serum progesterone and estradiol-17beta determinations and every 6 h for a 48-h duration at the expected time of estrus for luteinizing hormone (LH) assay. Ova and embryos were flushed from the reproductive tracts and the number of corpora lutea (CL) were recorded after slaughter on Day 7 post-estrus. Mean (+/- SEM) numbers of observed CL were higher (P < 0.05) in Group D9 (33.3 +/- 4.8) than in Group D3 (15.3 +/- 3.8), with Group D6 (17.0 +/- 2.9) and Group D12 (23.9 +/- 7.3) being intermediate. Similarly, mean (+/- SEM) numbers of fertilized embryos were highest (P < 0.05) in Group D9 (13.3 +/- 2.2). There was also a nonsignificant trend for the number of transferable embryos to be greatest in Group D9. Neither serum progesterone concentrations 3 d after the LH peak nor peak serum estradiol 17beta concentrations differed among groups, but both were significantly correlated with numbers of observed CL and total ova and embryos.

A retrospective clinical study of osteochondrosis dissecans in 21 horses.

Osteochondrosis dissecans was diagnosed clinically and radiographically in 31 joints of 21 horses. The horses ranged in age from 8 months to 5 years at the time of presentation. The usual age of onset of clinical signs was 18 to 24 months. Presenting complaints included joint effusion and lameness of either gradual or sudden onset. In Thoroughbred horses, the stifle joint was the most common site of lesions and in Standardbred horses lesions occurred more commonly in the hock. In 16 of the 21 horses, the contralateral joint was radiographed and 9 of these horses had bilateral lesions. Thoroughbred horses were affected most commonly, followed by Standardbred horses. The prevalence was higher in males than females, the male: female ratio being 2.5:1.

Regulation of follistatin gene expression in the ovary and in primary cultures of porcine granulosa cells.

Experiments were designed to test the hypotheses that (1) follistatin gene expression in granulosa cells is regulated during follicular growth, and (2) that alteration of follistatin mRNA concentration can be hormonally induced in primary cultures of porcine granulosa cells. RNA isolated from granulosa cells from small (1-3 mm diameter), medium (3-5 mm) and large (> 5 mm) follicles of prepubertal and postpubertal sows was analysed by hybridization to a porcine follistatin cDNA probe. Amounts of follistatin mRNA increased with follicular diameter, but no differences in follicular follistatin mRNA were detected between prepubertal and postpubertal sows. Treatment of cultured porcine granulosa cells with FSH or LH for 20 h stimulated follistatin mRNA concentration by a factor of two (100 ng FSH ml-1) and a factor of 1.5 (10 ng LH ml-1), respectively, over untreated controls. Treatment of cultured granulosa cells with 200 ng FSH ml-1, 200 ng LH ml-1, 10 mumol dibutyryl cAMP l-1, 30 mumol forskolin l-1 and 100 ng cholera toxin ml-1 stimulated follistatin mRNA accumulation in granulosa cells by factors of 4.9, 3.7, 1.6, 13.7 and 3.5, respectively, compared with control cultures. Stimulation of follistatin mRNA accumulation in cultured granulosa cells by dibutyryl cAMP (30, 100 and 300 mumol l-1) and forskolin (3, 10 and 100 mumol l-1) was dose dependent. FSH and forskolin induced time-dependent increases in follistatin mRNA concentration in cultured granulosa cells, with maximal induction occurring 72 h after treatment (a factor of 4.5 for FSH and 15.5 for forskolin).(ABSTRACT TRUNCATED AT 250 WORDS)

Jagged2: a serrate-like gene expressed during rat embryogenesis.

DSL (Delta, Serrate, Lag-2) ligands activate Notch signaling and thereby regulate the differentiation of many different cell types during development. We have isolated a novel Serrate-like gene, Jagged2, whose amino acid sequence and expression pattern during rat embryogenesis suggest that it functions as a ligand for Notch. In contrast to previously described DSL ligands for Notch, Jagged2 is not widely expressed in the developing central nervous system. However, Jagged2 and Notch1 are coexpressed in the apical ectodermal ridge (AER), suggesting a role for this ligand-receptor pair in limb development. Furthermore, unlike Jagged1, Jagged2 is coexpressed with Notch in the developing thymus, where it may induce Notch signaling to direct T-cell fate. Our data are consistent with the idea that the diversity of cell types regulated by Notch signaling is a consequence of activation of unique Notch isoforms by different DSL ligands.

Follistatin has characteristics of a primary response gene in porcine granulosa cells.

To explore the regulation of follistatin gene expression, porcine granulosa cells were incubated with the translational inhibitor, cycloheximide (CHX), for periods from 6-24 h. This resulted in a 3 to 10-fold increase in follistatin mRNA accumulation compared to vehicle treated control cultures. At 20 h, CHX augmented the follicle stimulating hormone (FSH) induced stimulation of follistatin mRNA accumulation by a mean of more than sixfold. Over 6 h, CHX elevated follistatin mRNA abundance twofold, while epidermal growth factor (EGF) increased the message threefold. CHX in the presence of EGF produced an effect additive to the EGF response. Results in the longer term differed, as pretreatment of granulosa cells with CHX for 20 h suppressed the induction of follistatin gene expression by both EGF and phorbol 12-myristate-13-acetate. By blockade of transcription with Actinomyocin D, an estimate of the half-life of follistatin mRNA between 4 and 8 h was made. Half-life did not appear to be affected by the CHX suppression of protein translation. From the observations of the occurrence of follistatin gene expression independent of protein synthesis, superinduction in the presence of CHX and FSH, and the interactions between CHX and EGF, it is concluded that follistatin is a primary response gene in porcine granulosa cells.

Notch1 is essential for postimplantation development in mice.

The Notch gene of Drosophila encodes a large transmembrane protein involved in cell fate determination during embryonic and larval development. This gene is evolutionarily conserved, and Notch homologs have been cloned from several vertebrate species. To examine the in vivo role of the Notch1 gene, a mouse homolog of Notch, a mutation was introduced by targeted disruption in embryonic stem cells, and these cells were used to generate mutant mice. Intercrosses of animals heterozygous for the Notch1 mutation yielded no live-born homozygous mutant offspring. Homozygous mutant embryos died before 11.5 days of gestation. Morphological and histological analysis of the homozygous mutant embryos indicated that pattern formation through the first nine days of gestation appeared largely normal. However, histological analysis of mutant embryos subsequent to this stage revealed widespread cell death. Death of mutant embryos did not appear to be attributable to defects in placentation or vascularization. Examination of the RNA expression pattern of the Notch2 gene, another Notch gene family member, indicated that it partially overlapped the Notch1 expression pattern. Genetic analysis of the Notch1 mutation also demonstrated that it was not allelic to a mouse mutation described previously, Danforth's short tail (Sd). These results demonstrate that the Notch1 gene plays a vital role during early postimplantation development in mice.

Expression patterns of Jagged, Delta1, Notch1, Notch2, and Notch3 genes identify ligand-receptor pairs that may function in neural development.

Notch genes encode receptors for a signaling pathway that regulates neurogenesis. The DSL (Delta/Serrate/lag-2) genes encode ligands that bind and activate Notch. In situ hybridization was used to determine the spatiotemporal expression of Notch1, Notch2, and Notch3, and the DSL ligands, Jagged and Delta 1, in an effort to identify potential ligand-receptor pairs that function during development of the rat nervous system. Here we describe both distinct and overlapping expression patterns for these genes in neural progenitors that form both the central and the peripheral nervous systems. The punctate expression patterns we detected for Jagged and Delta 1 are consistent with their role in mediating lateral inhibition, a process proposed to regulate neural determination. Furthermore, within the ventricular zone of the neural tube and retina, Jagged and Delta 1 were expressed in complementary regions, suggesting that different DSL-Notch combinations may direct the development of distinct neural subtypes.

Jagged: a mammalian ligand that activates Notch1.

Here we report the isolation of a rat cDNA clone, Jagged, which we show encodes a ligand for vertebrate Notch. Our conclusion is based on three observations. First, sequence analysis reveals substantial homology between Jagged and invertebrate ligands for the LIN-12/Notch proteins. Second, in situ hybridization of rat embryos identifies both distinct and overlapping patterns of gene expression for Jagged with those for Notch1, Notch2, and Notch3. Finally, the biological activity of Jagged was tested using a cell culture assay in which Jagged activates rat Notch1 expressed in myoblasts and prevents muscle cell differentiation. Our data support the hypothesis that Notch-ligand interactions function in maintaining mammalian cells in an undifferentiated state.

Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1.

The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism essential for embryonic development in mammals. Mutations in the human JAGGED1 ( JAG1 ) gene, which encodes a ligand for the Notch family of transmembrane receptors, cause the autosomal dominant disorder Alagille syndrome. We have examined the in vivo role of the mouse Jag1 gene by creating a null allele through gene targeting. Mice homozygous for the Jag1 mutation die from hemorrhage early during embryogenesis, exhibiting defects in remodeling of the embryonic and yolk sac vasculature. We mapped the Jag1 gene to mouse chromosome 2, in the vicinity of the Coloboma ( Cm ) deletion. Molecular and complementation analyses revealed that the Jag1 gene is functionally deleted in the Cm mutant allele. Mice heterozygous for the Jag1 null allele exhibit an eye dysmorphology similar to that of Cm /+ heterozygotes, but do not exhibit other phenotypes characteristic of Cm /+ mice or of humans with Alagille syndrome. These results establish the phenotype of Cm /+ mice as a contiguous gene deletion syndrome and demonstrate that Jag1 is essential for remodeling of the embryonic vasculature.