The docking protein FRS2␣ is a major mediator of fibroblast growth factor (FGF) signaling. However, the physiological role of FRS2␣ in vivo remains unknown. In this report, we show that Frs2␣-null mouse embryos have a defect in anterior-posterior (A-P) axis formation and are developmentally retarded, resulting in embryonic lethality by embryonic day 8. We demonstrate that FRS2␣ is essential for the maintenance of self-renewing trophoblast stem (TS) cells in response to FGF4 in the extraembryonic ectoderm (ExE) that gives rise to tissues of the placenta. By analyzing chimeric embryos, we found that FRS2␣ also plays a role in cell movement through the primitive streak during gastrulation. In addition, experiments are presented demonstrating that Bmp4 expression in TS cells is controlled by mitogen-activated protein kinase-dependent FGF4 stimulation. Moreover, both the expression of Bmp4 in ExE and activation of Smad1/5 in epiblasts are reduced in Frs2␣-null embryos. These experiments underscore the critical role of FRS2␣ in mediating multiple processes during embryonic development and reveal a potential new link between FGF and Bmp4 signaling pathways in early embryogenesis.
Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in phosphoinositide turnover and is involved in a variety of physiological functions. We analyzed PLC␦1 knockout mice and found that PLC␦1 is required for the maintenance of skin homeostasis. However, there were no remarkable abnormalities except hair loss and runting in PLC␦1 knockout mice, even though PLC␦1 is broadly distributed. Here, we report that mice lacking both PLC␦1 and PLC␦3 died at embryonic day 11.5 (E11.5) to E13.5. PLC␦1/PLC␦3 doubleknockout mice exhibited severe disruption of the normal labyrinth architecture in the placenta and decreased placental vascularization, as well as abnormal proliferation and apoptosis of trophoblasts in the labyrinth area. Furthermore, PLC␦1/PLC␦3 double-knockout embryos supplied with a normal placenta by the tetraploid aggregation method survived beyond E14.5, clearly indicating that the embryonic lethality is caused by a defect in trophoblasts. On the basis of these results, we conclude that PLC␦1 and PLC␦3 are essential in trophoblasts for placental development.Phosphoinositide-specific phospholipase C (PLC) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol mediates the activation of protein kinase C, and inositol 1,4,5-trisphosphate releases calcium from intracellular stores (1,19).PLC can be categorized into six types, , ␥, ␦, ε, , and , on the basis of sequence homology and activation mechanism (5,13,20,21,23,24,34). Each isozyme is composed of subtypespecific and conserved domains. All PLC isozymes contain catalytic X and Y domains, as well as various regulatory domains, including the C2 domain, EF-hand motif, and pleckstrin homology domain. Subtype-specific domains contribute to the specific regulatory mechanisms. These domains include the src homology domain in PLC␥ (23) and the Ras-associating domain and Ras-GTPase exchange factor-like domain in PLCε (15,29).PLC␦ types are evolutionarily conserved from lower to higher eukaryotes, and these isozymes are thought to be the primary forms expressed in mammals. Therefore, PLC␦ is expected to have important and basic physiological functions. There are three PLC␦ isozymes, PLC␦1, -␦3, and -␦4 (10). It has been suggested that PLC␦1 is involved in Alzheimer's disease (27) and essential hypertension (14,35). Recently, we analyzed PLC␦ knockout (KO) mice and found that PLC␦1 has an important role in skin homeostasis (18) and that PLC␦4 is involved in the acrosome reaction of sperm (6, 7). These results provide some clarification of the physiological functions of PLC␦1 and PLC␦4; however, the function of PLC␦3 remains unknown.In the present study, to elucidate the physiological roles of PLC␦3, we analyzed PLC␦3 KO mice. Thus far, PLC␦3 KO mice have exhibited no obvious abnormalities. Because PLC␦3 is most similar to PLC␦1, we generated mice lacking both PLC␦1 and PLC␦3 genes (PLC␦1/PLC␦3 double-knockout [DKO] mice). We found that simultaneous disruption of PLC␦...
Intense expression of mRNA of endothelin-B receptor (ETBR) has been detected in the Bergmann glia of cerebellum by in situ hybridization, but the intracellular localization has not been reported because of the absence of a useful antibody for immunohistochemical investigations. We made polyclonal antibodies against the carboxyl terminus of human ETBR (420-442) and ETAR (403-427), and performed light- and electron-microscopic immunohistochemistry of the wild-type and ETBR-deficient (sl/sl) rat cerebella. Localization of ETBR during postnatal development was examined by double-staining immunofluorescence using antibodies against ETBR and S-100 beta. In the wild-type rats, ETBR immunoreactivity appeared from postnatal day 5 (P5) and was distributed diffusely in the processes and cell bodies of S-100 beta-positive glial cells. By P14, ETBR immunoreactivity was concentrated in the Golgi apparatus of Bergmann glial cell soma and the plasma membrane of its processes. The ETBR-positive astrocytes in the granular layer decreased in number during P7-14 and had disappeared by week 3. At 3 weeks, ETBR immunoreactivity was restricted to the Golgi apparatus of Bergmann glia. In the sl/sl rats, ETBR immunoreactivity was not observed at all. In contrast to ETBR, ETAR immunoreactivity appeared transiently in the cytoplasm of all astrocytes (Bergmann glia and astrocytes in the granular layer) in the 9- to 14-day-old wild rats and 7- to 14-day-old sl/sl rats, and disappeared within 3 weeks in both. Granule cells did not express immunoreactivity for ETBR and ETAR from the neonatal stage to adulthood. Changes in the intracellular localization of ETBR and transient expression of ETAR may be correlated with the changes of glial functions and proliferation during postnatal development of rat cerebellum.
Subepithelial fibroblasts form a cellular network with gap junctions under the epithelium of the gastrointestinal tract. Previously, we have reported their unique characteristics, such as reversible rapid cell-shape changes from a flat to a stellate configuration induced by dBcAMP and endothelins (ETs), and Ca2+ responses to, for example, ETs, ATP, and substance-P. We have now investigated the subtypes of ET receptors both in the rat small intestine and in primary cultured subepithelial fibroblasts isolated from rat duodenal villi. Their properties were compared between wild-type and endothelin-B-receptor-mutant sl/sl rats. Light- and electron-microscopic immunohistochemistry showed intense ETA immunoreactivity in the subepithelial fibroblasts from the small intestine and colon of both wild-type and sl/sl rats. In culture, immunocytochemistry, reverse transcription/polymerase chain reaction analysis, Ca2+ response measurements, and cell-shape change analysis indicated functional ETA and ETB receptors in the wild-type cells, but only ETA in the sl/sl cells. However, wild-type cells were more sensitive to ET-1 than to ET-3 by about one order of magnitude. ETA seemed to be dominant both in vivo and in vitro. The relationship between cell-shape change and gap junction permeability was examined by fluorescence recovery after photobleaching; the gap junctions were usually open but were blocked by carbenoxolone. Permeability did not change significantly with cell-shape change. This network of differentiated subepithelial fibroblasts may maintain intercellular communication via gap junctions to transduce signals evoked in the local network to the whole network. The cell-shape change of the cells through ETA activation may play an important role as a barrier and for intercellular signaling in the intestinal villi.
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