Conserved function of mSpry-2, a murine homolog of Drosophila sprouty, which negatively modulates respiratory organogenesis.

In Drosophila embryos, the loss of sprouty gene function enhances branching of the respiratory system. Three human sprouty homologues (h-Spry1-3) have been cloned recently, but their function is as yet unknown [1]. Here, we show that a murine sprouty gene (mSpry-2), the product of which shares 97% homology with the respective human protein, is expressed in the embryonic murine lung. We used an antisense oligonucleotide strategy to reduce expression of mSpry-2 by 96%, as measured by competitive reverse transcriptase PCR, in E11. 5 murine embryonic lungs cultured for 4 days [2]. Morphologically, the decrease in mSpry-2 expression resulted in a 72% increase in embryonic murine lung branching morphogenesis as well as a significant increase in expression of the lung epithelial marker genes SP-C, SP-B and SP-A. These results support a striking conservation of function between the Drosophila and mammalian sprouty gene families to negatively modulate respiratory organogenesis.

Abrogation of betaglycan attenuates TGF-beta-mediated inhibition of embryonic murine lung branching morphogenesis in culture.

Although betaglycan (TGF-beta type III receptor) is known to enhance TGF-beta ligand binding to its type II receptor in murine lung epithelial cell lines, the biological significance of this phenomenon in the process of lung organogenesis is not understood. Betaglycan gene expression was detected in embryonic murine lungs undergoing branching morphogenesis in ex vivo culture. Antisense betaglycan oligodeoxynucleotides (ODN) resulted in up to 56% stimulation of lung branching morphogenesis in culture, while betaglycan mRNA and protein expression levels were suppressed by 90 and 82%, respectively. Following abrogation of betaglycan expression with antisense oligodeoxynucleotide, embryonic lungs were relatively insensitive to TGF-beta: TGF-beta2 (0.5 ng/ml) and TGF-beta1 (20 ng/ml), respectively, down-regulated lung morphogenesis by 38 and 34% in control cultures, whereas TGF-beta-induced inhibition was attenuated to 13 and 26% respectively, in the presence of betaglycan antisense oligodeoxynucleotides. TGF-beta neutralizing antibodies also prevented TGF-beta-mediated inhibition of lung branching in culture, supporting the speculation that autocrine/paracrine TGF-beta signaling is minimal in the absence of betaglycan. Betaglycan was immunolocalized mainly to the epithelial cells in developing airways, a spatial distribution which overlaps with that of TGF-beta type II receptor. Furthermore, abrogation of endogenous betaglycan gene expression prevented the characteristic down-regulation of cyclin A and surfactant protein C (SP-C) mRNAs by exogenous TGF-beta ligands. These results show that betaglycan expression is essential for optimal TGF-beta signaling during embryonic lung development. We therefore conclude that the abrogation of endogenous betaglycan attenuates endogenous autocrine and/or paracrine TGF-beta-mediated negative regulation of lung organogenesis.

Spatial-specific TGF-beta1 adenoviral expression determines morphogenetic phenotypes in embryonic mouse lung.

The precise spatial-temporal role that expression and activation of transforming growth factor (TGF)-beta plays in mammalian organ morphogenesis remains incompletely understood. Using replication deficient adenoviral vectors containing engineered TGF-beta1 cDNAs, we studied the spatial effects of locally over-expressing either latent or mutated, constitutively active TGF-beta1 protein during embryonic mouse lung branching morphogenesis in culture. Transfer of exogenous genes into lung epithelium was achieved by intra-tracheal micro-injection of recombinant adenovirus, while submerging lungs in virus resulted in gene transfer into the pleura and subjacent mesenchymal cells, as revealed by cytochemical staining for beta-galactosidase. Only lungs transfected with active, but not latent TGF-beta1 gene, showed elevated levels of active TGF-beta. Epithelial over-expression of active, but not latent TGF-beta1, via intra-tracheal micro-injection inhibited lung branching morphogenesis by 36 %. In contrast, lungs submerged with either active or latent TGF-beta1 recombinant virus did not demonstrate an inhibitory effect upon branching. Pulmonary gene regulation was assayed by competitive polymerase chain reaction coupled with reverse transcription. Direct respiratory tract micro-injection of adenovirus over-expressing active TGF-beta1 resulted in a dose-dependent inhibition of epithelial surfactant protein (SP)-C and SP-B mRNA levels by up to 76 % and 70 %, respectively, while in contrast, fibronectin and matrix Gla protein (MGP) mRNA levels remained stable. However, lungs that had been submerged in adenovirus expressing active TGF-beta1 demonstrated a concentration-dependent induction of both fibronectin and MGP mRNA levels up to 4.3- and 4.7-fold respectively in the presence of 1 x 10(11) pfu/ml active TGF-beta1 virus. On the other hand, lungs treated with adenovirus expressing latent TGF-beta1 either by micro-injection or submerging failed to demonstrate any regulatory effect either upon epithelial or mesenchymal gene expression. We conclude that adenovector-mediated over-expression of activated TGF-beta1 in specific spatial compartments results respectively in either inhibition of branching morphogenesis and epithelium-specific gene expression, or in induction of matrix gene expression without affecting morphogenesis or epithelium-specific gene expression, depending on the route of administration. Also, the lack of effect of latent TGF-beta1 over-expression strongly suggests that TGF-beta activation per se provides an important locus of fine regulation of the spatial effects of TGF-beta signaling during embryonic lung branching morphogenesis.

Responses of frog skin, Rana pipiens, calcium ion transport to parathyroid hormone, calcitonin, and vitamin D3.

The responses of cutaneous Ca2+ transport to parathyroid hormone, calcitonin, and vitamin D3 were evaluated in intact Rana pipiens and isolated skins from this species. Parathyroid hormone (12 pmol g-1) stimulated Ca2+ influx in intact frogs but not in isolated skin (10-100 pmol ml-1) unless the frogs had been pretreated with vitamin D3 injections (100-300 microgram day-1) for 3 days prior to isolating the skins for flux measurements. Pretreatment of intact frogs with vitamin D3 for 3 days (100 microgram day-1) also resulted in greater responses to PTH (6-12 pmol g-1). Vitamin D3 (most likely after conversion to 1,25(OH)2D3) had a direct stimulatory effect on Ca2+ transport in isolated skins after three daily doses of 500 microgram of D3. Calcitonin also stimulated Ca2+ influx in both intact frogs (24 pmol g-1) and isolated skins (25-100 pmol ml-1). We conclude that parathyroid hormone, facilitated by vitamin D, stimulates Ca2+ influx in the skin of R. pipiens. We also conclude that calcitonin is stimulatory for cutaneous Ca2+ influx in this species.

Nicotine stimulates branching and expression of SP-A and SP-C mRNAs in embryonic mouse lung culture.

Although the effects of maternal smoking on fetal growth and viability are overwhelmingly negative, there is a paradoxical enhancement of lung maturation as evidenced, in part, by a lower incidence of respiratory distress syndrome in infants of smoking mothers. Other epidemiologic and experimental evidence further support the view that a tobacco smoke constituent, possibly nicotine, affects the development of the lung in utero. We are studying the direct effects of nicotine on murine lung development using a serumless organ culture system. We have found that embryonic lungs explanted at 11 days gestation showed a 32% increase in branching after 4 days in culture in the presence of 1 microM nicotine and 7- to 15-fold increases in mRNAs encoding surfactant proteins A and C after 11 days. The effect of nicotine exposure on surfactant gene expression is apparently mediated by nicotinic acetylcholine receptors because it was blocked by D-tubocurarine. The nicotine-induced stimulation of surfactant gene expression could, in part, account for the effect of maternal smoking on the incidence of respiratory distress syndrome.

The Shc 66 and 46 kD isoforms are differentially downregulated at parturition in the fetal mouse lung.

Many of the signaling pathways regulating fetal lung mesenchymal cell proliferation are mediated by the Shc intracellular signaling proteins. Shc is expressed as three isoforms: 52 kD and 46 kD proteins (Shc 52 and Shc 46, respectively) translated from the same mRNA, and a 66 kD form (Shc 66) translated from a separate mRNA. Shc 52 is an activator of Ras and mitogen-activated protein kinase, whereas Shc 66 antagonizes Ras activation. The function of Shc 46 is unclear. We hypothesized that the Shc isoforms are differentially regulated during fetal mouse lung morphogenesis. Relative Shc 66 and Shc 46 protein expression are high until parturition (term = 18.5 d), when a dramatic decrease begins; by postconceptual d 20, relative Shc 66 and Shc 46 expression have fallen by 75 and 69%, respectively. A similar pattern of decreasing Shc 66 mRNA expression in the peripartum period was detected by reverse transcription and competitive polymerase chain reaction during the same period. By isoform-specific immunohistochemistry, Shc 66 is widely distributed in the embryonic lung but becomes restricted to the bronchial smooth muscle and overlying epithelia, periarterial smooth muscle, and the interlobar pleura late in gestation. After parturition, Shc 66 is virtually absent from the lung. All three Shc isoforms are phosphorylated by epidermal growth factor stimulation in fetal lung mesenchymal cells, indicating that Shc 66 is functional in these cells. These data indicate that Shc isoforms are differentially regulated during lung development.

Inhibition of vascular and epithelial differentiation in murine nitrofen-induced diaphragmatic hernia.

Neonates with congenital diaphragmatic hernia (DH) die of pulmonary hypoplasia and persistent pulmonary hypertension. We used immunohistochemical localization of alpha-smooth muscle actin (alpha-SMA), platelet endothelial cell adhesion molecule (PECAM)-1, thyroid transcription factor (TTF)-1, surfactant protein (SP) A, SP-C, and competitive RT-PCR quantitation of TTF-1, SP-A, SP-C, and alpha-SMA mRNA expression to characterize the epithelial and vascular phenotype of lungs from ICR fetal mice with a nitrofen-induced DH. Nitrofen (25 mg) was gavage fed to pregnant mice on day 8 of gestation. Fetal mice were delivered on day 17. The diaphragm was examined for a defect, and the lungs were either fixed, sectioned, and immunostained or processed for mRNA isolation. In comparison with control lungs, DH lungs showed increased expression of alpha-SMA mRNA, fewer and more muscular arterioles (alpha-SMA), less well-developed capillary networks (PECAM-1), delayed epithelial development marked by a persistence of TTF-1 in the periphery, and decreased SP-A mRNA and SP-A expression. These data suggest that in the murine nitrofen-induced DH, as in human congenital DH, pulmonary insufficiency is due to an inhibition of peripheral pulmonary development including terminal airway and vascular morphogenesis.