Signaling through fibroblast growth factor receptor 2b plays a key role in the development of the exocrine pancreas. (73/6597)

The development of the pancreas depends on epithelial-mesenchymal interactions. Fibroblast growth factors (FGFs) and their receptors (FGFRs 1-4) have been identified as mediators of epithelial-mesenchymal interactions in different organs. We show here that FGFR-2 IIIb and its ligands FGF-1, FGF-7, and FGF-10 are expressed throughout pancreatic development. We also show that in mesenchyme-free cultures of embryonic pancreatic epithelium FGF-1, FGF-7, and FGF-10 stimulate the growth, morphogenesis, and cytodifferentiation of the exocrine cells of the pancreas. The role of FGFs signaling through FGFR-2 IIIb was further investigated by inhibiting FGFR-2 IIIb signaling in organocultures of pancreatic explants (epithelium + mesenchyme) by using either antisense FGFR-2 IIIb oligonucleotides or a soluble recombinant FGFR-2 IIIb protein. Abrogation of FGFR-2 IIIb signaling resulted in a considerable reduction in the size of the explants and in a 2-fold reduction of the development of the exocrine cells. These results demonstrate that FGFs signaling through FGFR-2 IIIb play an important role in the development of the exocrine pancreas.  (+info)

Human sweat gland myoepithelial cells express a unique set of cytokeratins and reveal the potential for alternative epithelial and mesenchymal differentiation states in culture. (74/6597)

We have characterized precisely the cytokeratin expression pattern of sweat gland myoepithelial cells and have identified conditions for propagating this cell type and modulating its differentiation in culture. Rare, unstratified epithelioid colonies were identified in cultures initiated from several specimens of full-thickness human skin. These cells divided rapidly in medium containing serum, epidermal growth factor (EGF), and hydrocortisone, and maintained a closely packed, epithelioid morphology when co-cultured with 3T3 feeder cells. Immunocytochemical and immunoblot analysis disclosed that the cells differed from keratinocytes in that they were E-cadherin-negative, vimentin-positive, and expressed an unusual set of cytokeratins, K5, K7, K14, and K17. When subcultured without feeder cells, they converted reversibly to a spindle morphology and ceased K5 and K14 expression. Under these conditions, EGF deprivation induced flattening, growth arrest, and expression of alpha-smooth muscle actin ((&agr;)-sma). Coexpression of keratins and alpha-sma is a hallmark of myoepithelial cells, a constituent of secretory glands. Immunostaining of skin sections revealed that only sweat gland myoepithelial cells expressed the same pattern of keratins and alpha-sma and lack of E-cadherin as the cell type we had cultured. Interestingly, our immunocytochemical analysis of ndk, a skin-derived cell line of uncertain identity, suggests that this line is of myoepithelial origin. Earlier immunohistochemical studies by others had found myoepithelial cells to be K7-negative. We tested five K7-specific antibodies that can recognize this protein in western blots and in the assembled keratin filaments of mesothelial cells. Three of these antibodies did not recognize the K7 present in myoepithelial cell filaments or in HeLa cell filaments, indicating that some K7 epitopes are masked when K7 pairs with K17 instead of with its usual keratin filament partner, K19.  (+info)

Floor plate and netrin-1 are involved in the migration and survival of inferior olivary neurons. (75/6597)

During their circumferential migration, the nuclei of inferior olivary neurons translocate within their axons until they reach the floor plate where they stop, although their axons have already crossed the midline to project to the contralateral cerebellum. Signals released by the floor plate, including netrin-1, have been implicated in promoting axonal growth and chemoattraction during axonal pathfinding in different midline crossing systems. In the present study, we report experiments that strongly suggest that the floor plate could also be involved in the migration of inferior olivary neurons. First, we show that the pattern of expression of netrin receptors DCC (for deleted in colorectal cancer), neogenin (a DCC-related protein), and members of the Unc5 family in wild-type mice is consistent with a possible role of netrins in directing the migration of precerebellar neurons from the rhombic lips. Second, we have studied mice deficient in netrin-1 production. In these mice, the number of inferior olivary neurons is remarkably decreased. Some of them are located ectopically along the migration stream, whereas the others are located medioventrally and form an atrophic inferior olivary complex: most subnuclei are missing. However, axons of the remaining olivary cell bodies located in the vicinity of the floor plate still succeed in entering their target, the cerebellum, but they establish an ipsilateral projection instead of the normal contralateral projection. In vitro experiments involving ablations of the midline show a fusion of the two olivary masses normally located on either side of the ventral midline, suggesting that the floor plate may function as a specific stop signal for inferior olivary neurons. These results establish a requirement for netrin-1 in the migration of inferior olivary neurons and suggest that it may function as a specific guidance cue for the initial steps of the migration from the rhombic lips and then later in the development of the normal crossed projection of the inferior olivary neurons. They also establish a requirement for netrin-1, either directly or indirectly, for the survival of inferior olivary neurons.  (+info)

Glutamate acting at NMDA receptors stimulates embryonic cortical neuronal migration. (76/6597)

During cortical development, embryonic neurons migrate from germinal zones near the ventricle into the cortical plate, where they organize into layers. Mechanisms that direct neuronal migration may include molecules that act as chemoattractants. In rats, GABA, which localizes near the target destination for migrating cortical neurons, stimulates embryonic neuronal migration in vitro. In mice, glutamate is highly localized near the target destinations for migrating cortical neurons. Glutamate-induced migration of murine embryonic cortical cells was evaluated in cell dissociates and cortical slice cultures. In dissociates, the chemotropic effects of glutamate were 10-fold greater than the effects of GABA, demonstrating that for murine cortical cells, glutamate is a more potent chemoattractant than GABA. Thus, cortical chemoattractants appear to differ between species. Micromolar glutamate stimulated neuronal chemotaxis that was mimicked by microM NMDA but not by other ionotropic glutamate receptor agonists (AMPA, kainate, quisqualate). Responding cells were primarily derived from immature cortical regions [ventricular zone (vz)/subventricular zone (svz)]. Bromodeoxyuridine (BrdU) pulse labeling of cortical slices cultured in NMDA antagonists (microM MK801 or APV) revealed that antagonist exposure blocked the migration of BrdU-positive cells from the vz/svz into the cortical plate. PCR confirmed the presence of NMDA receptor expression in vz/svz cells, whereas electrophysiology and Ca2+ imaging demonstrated that vz/svz cells exhibited physiological responses to NMDA. These studies indicate that, in mice, glutamate may serve as a chemoattractant for neurons in the developing cortex, signaling cells to migrate into the cortical plate via NMDA receptor activation.  (+info)

Direct exposure of postimplantation mouse embryos to 5-bromodeoxyuridine in vitro and its effect on subsequent chondrogenesis in the limbs. (77/6597)

As maternally administered 5-bromodeoxyuridine (BudR) is very quickly degraded by the liver, a combination of whole embryo culture and organ culture techniques was adopted to expose postimplantation mouse embryos to the analog and to study the effects of long-term embryos were exposed to increasing concentrations of BudR for 12 or 24 h. Forelimbs of the treated embryos were then organ-cultured in drug-free medium and the extent of cartilage development in the explants examined. Exposure of embryos to 50-150 mug/ml of BudR for 24 h resulted in significant inhibition of chondrogenesis in the subsequent limb cultures and the effect was related to dose. After treatment with 150 mug/ml of the drug, the forelimbs of the early 11-day embryos (somite stage 26-29) showed an almost complete lack of cartilage, while the limbs of mid-11 th-day embryos (somite stage 32-34) were not nearly as sensitive and exhibited about 50% reduction in the amount of cartilage development. We conclude that if embryos in which the limb development is at a very early stage of development are exposed to BudR, the future course of limb differentiation is permanently and irreversibly damaged, resulting in a partial or even complete suppression of chondrogenesis in the organ. As both the dose and perhaps also the duration of treatment were critical, we suggest that the rather low frequency of reported limb malformations after in vivo injection of teratological doses of BudR may be due to only a small amount of the chemical reaching the embryos.  (+info)

Regression of hypertrophied rat pulmonary arteries in organ culture is associated with suppression of proteolytic activity, inhibition of tenascin-C, and smooth muscle cell apoptosis. (78/6597)

Increased elastase activity and deposition of the matrix glycoprotein tenascin-C (TN), codistributing with proliferating smooth muscle cells (SMCs), are features of pulmonary vascular disease. In pulmonary artery (PA) SMC cultures, TN is regulated by matrix metalloproteinases (MMPs) and mechanical stress. On attached collagen gels, MMPs upregulate TN, leading to SMC proliferation, whereas on floating collagen, reduced MMPs suppress TN and induce SMC apoptosis. We now investigate the response of SMCs in the whole vessel by comparing attached and floating conditions using either normal PAs derived from juvenile pigs or normal or hypertrophied rat PAs that were embedded in collagen gels for 8 days. Normal porcine PAs in attached collagen gels were characterized by increasing activity of MMP-2 and MMP-9 assessed by zymography and TN deposition detected by Western immunoblotting and densitometric analysis of immunoreactivity. PAs on floating collagen showed reduced activity of both MMPs and deposition of TN. Tenascin-rich foci were associated with proliferating cell nuclear antigen immunoreactivity, and TN-poor areas with apoptosis, by terminal deoxynucleotidyl transferase-mediated nick end labeling assay, but no difference in wall thickness was observed. Although normal rat PAs were similar to piglet vessels, hypertrophied rat PAs showed an amplified response. Increased elastase, MMP-2, TN, and elastin deposition, as well as SMC proliferating cell nuclear antigen positivity, correlated with progressive medial thickening on attached collagen, whereas reduced MMP-2, elastase, TN, and induction of SMC apoptosis accompanied regression of the thickened media on floating collagen. In showing that hypertrophied SMCs in the intact vessel can be made to apoptose and that resorption of extracellular matrix can be achieved by inhibition of elastase and MMPs, our study suggests novel strategies to reverse vascular disease.  (+info)

Mesonephric cell migration induces testis cord formation and Sertoli cell differentiation in the mammalian gonad. (79/6597)

In mammals a single gene on the Y chromosome, Sry, controls testis formation. One of the earliest effects of Sry expression is the induction of somatic cell migration from the mesonephros into the XY gonad. Here we show that mesonephric cells are required for cord formation and male-specific gene expression in XY gonads in a stage-specific manner. Culturing XX gonads with an XY gonad at their surface, as a 'sandwich', resulted in cell migration into the XX tissue. Analysis of sandwich gonads revealed that in the presence of migrating cells, XX gonads organized cord structures and acquired male-specific gene expression patterns. From these results, we conclude that mesonephric cell migration plays a critical role in the formation of testis cords and the differentiation of XY versus XX cell types.  (+info)

Evaluation of the effect of interleukin-6 and human extracellullar matrix on embryonic development. (80/6597)

Extracellular matrices and their associated growth factors can modulate the in-vitro growth of cells. In this study, the effects of culture substrata and the cytokine interleukin-6 (IL-6) on embryonic development were investigated. In-vitro fertilized mouse oocytes were pooled and randomly distributed amongst treatment groups. The test treatments were: (i) IL-6, at either 500 or 1000 pg/ml; (ii) human extracellular matrix (HECM) applied to organ culture dishes at either 5.0 or 10.0 microg/ml; and (iii) HECM and IL-6 combined. A total of 1285 embryos was evaluated. The effect of IL-6 on embryos was dose dependent. Treated embryos exhibited higher blastulation and hatching rates than untreated control embryos. Culture of embryos on human matrix proteins versus standard culture surfaces significantly improved in-vitro hatching. The combination of both of these treatments was superior to the medium alone control, and the mean cell count per blastocyst was higher (131.7 +/- 29.7 versus 82. 5 +/- 14.3 in control embryos; P < 0.0001). In a pilot study with human triploid embryos, the HECM/IL-6 culture system appeared to support embryonic compaction, blastulation and hatching. This work suggests that extracellular matrix components in combination with growth factors/cytokines may be another avenue for formulating more physiological culture systems.  (+info)