Novel mechanisms in murine nitrofen-induced pulmonary hypoplasia: FGF-10 rescue in culture.

We evaluated the role of the key pulmonary morphogenetic gene fibroblast growth factor-10 (Fgf10) in murine nitrofen-induced primary lung hypoplasia, which is evident before the time of diaphragm closure. In situ hybridization and competitive RT-PCR revealed a profound disturbance in the temporospatial pattern as well as a 10-fold decrease in mRNA expression level of Fgf10 but not of the inducible inhibitor murine Sprouty2 (mSpry2) after nitrofen treatment. Exogenous FGF-10 increased branching not only of control lungs [13% (right) and 27% (left); P < 0.01] but also of nitrofen-exposed lungs [23% (right) and 77% (left); P < 0.01]. Expression of mSpry2 increased 10-fold with FGF-10 in both nitrofen-treated and control lungs, indicating intact downstream FGF signaling mechanisms after nitrofen treatment. We conclude that nitrofen inhibits Fgf10 expression, which is essential for lung growth and branching. Exogenous FGF-10 not only stimulates FGF signaling, marked by increased mSpry2 expression, in both nitrofen-treated and control lungs but also substantially rescues nitrofen-induced lung hypoplasia in culture.

Smad7 is a TGF-beta-inducible attenuator of Smad2/3-mediated inhibition of embryonic lung morphogenesis.

Smad7 was recently shown to antagonize TGF-beta-induced activation of signal-transducing Smad2 and Smad3 proteins. However, the biological function of Smad7 in the process of lung organogenesis is not known. Since Smad2/3-mediated TGF-beta signaling is known to inhibit embryonic lung branching morphogenesis, we tested the hypothesis that Smad7 regulates early lung development by modulating TGF-beta signal transduction. An antisense oligodeoxynucleotide (ODN) was designed to specifically block endogenous Smad7 gene expression at both transcriptional and translational levels in embryonic mouse lungs in culture. TGF-beta-mediated inhibition of lung branching morphogenesis was significantly potentiated in cultured embryonic lungs in the absence of Smad7 gene expression: abrogation of Smad7 potentiated TGF-beta-mediated inhibition of lung branching morphogenesis from 76 to 52% of the basal level in lungs cultured in the presence of 5 ng/ml TGF-beta1 ligand. Likewise, TGF-beta1 EC(50) (concentration of TGF-beta1 that induced half maximal branching inhibition) was reduced from 5 to 1 ng/ml when Smad7 gene expression was abrogated in lung culture, indicating an enhanced level of TGF-beta signaling in lung tissue with abolished Smad7 gene expression. By immunocytochemistry, Smad7 protein was co-localized with both Smad2 and Smad3 in distal bronchial epithelial cells, supporting the concept that Smad7 inhibits TGF-beta signaling by competing locally with Smad2 and Smad3 for TGF-beta receptor complex binding during lung morphogenesis. Furthermore, antisense Smad7 ODN increased the negative effect of TGF-beta1 on epithelial cell growth in developing lungs in culture. We also demonstrated that Smad7 mRNA levels were rapidly and potently induced upon TGF-beta1 stimulation of lungs in culture, suggesting that Smad7 regulates TGF-beta responses in a negative feedback loop. These studies define a novel function for Smad7 as an intracellular antagonist of TGF-beta-induced, Smad2/3-mediated inhibition of murine embryonic lung growth and branching morphogenesis in culture. The optimization of TGF-beta signaling during early lung development therefore requires a finely-regulated competitive balance between both permissive and inhibitory members of the Smad family.

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.

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.

Commitment and differentiation of lung cell lineages.

To form a large diffusible interface capable of conducting respiratory gases to and from the circulation, the lung must undergo extensive cell proliferation, branching morphogenesis, and alveolar saccule formation, to generate sufficient surface area. In addition, the cells must differentiate into at least 40 distinct lung cell lineages. Specific transcriptional factors, peptide growth factor receptor-mediated signaling pathways, extracellular matrix components, and integrin-signaling pathways interact to direct lung morphogenesis and lung cell lineage differentiation. Branching mutants of the respiratory tracheae in Drosophila have identified several functionally conserved genes in the fibroblast growth factor signaling pathway that also regulate pulmonary organogenesis in mice and probably also in man. Key transcriptional factors including Nkx2.1, hepatocyte nuclear factor family forkhead homologues, GATA family zinc finger factors, pou and homeodomain proteins, as well as basic helix-loop-helix factors, serve as master genes to integrate the developmental genetic instruction of lung morphogenesis and cell lineage determination. Lung mesenchyme serves as a 'compleat' inducer of lung morphogenesis by secreting soluble peptide growth factors. In general, peptide growth factors signaling through cognate receptors with tyrosine kinase intracellular signaling domains such as epidermal growth factor receptor, fibroblast growth factor receptors, hepatocyte growth factor/scatter factor receptor, c-met, insulin-like growth factor receptor, and platelet-derived growth factor receptor, stimulate lung morphogenesis, while the cognate receptors with serine/threonine kinase intracellular signaling domains, such as the transforming growth factor-beta receptor family are inhibitory. The extracellular matrix also plays a key role in determining branching morphogenesis. Pulmonary neuroendocrine (PNE) cells differentiate earliest in gestation among lung epithelial cells. PNE cells are principally derived from endoderm and not neural crest. PNE cells have been proposed to function as airway chemoreceptors, while PNE cell secretory granules contain many bioactive substances such as GRP which may direct proliferation of adjacent epithelial cells. Mammalian achaete-schute homolog-1 null mutant mice do not develop PNE cells. Candidate molecular switches in the transition from a quiescent to a proliferative alveolar epithelial cell (AEC) phenotype and back again following acute hyperoxia, include autocrine peptide growth factor signaling pathways and cell cycle regulatory elements. AEC type 2 also appear capable of reversible transdifferentiation into AEC type 1 and intermediate phenotypes in response to cues from extracellular matrix and cell shape, as well as soluble factors. Evidence for expression of telomerase by alveolar epithelial stem cells, which correlates with self-renewal potential, is now beginning to emerge. Lung regeneration following lobectomy in juvenile rodents is associated with co-ordinated cell proliferation, re-expression of elastin and formation of alveoli. Retinoic acid has recently shown promise as a stimulator of alveolization in juvenile rats. Our future goal is to devise new rational and gene therapeutic strategies to stimulating lung growth and maturation, ameliorating lung injury, augmenting lung repair, and inducing lung regeneration. The ideal agent or agents would therefore mimic the instructive role of lung mesenchyme, correctly inducing the temporospatial pattern of lung cell lineages necessary to restore pulmonary gas diffusing capacity.

Problem-based learning at the University of Southern California School of Dentistry.

Responding to the recent Institute of Medicine report on dental education, the Center for Craniofacial Molecular Biology (CCMB) of the University of Southern California School of Dentistry has developed a parallel track program in dental education leading to the D.D.S. degree. This program was proposed in May of 1995, and the first class of twelve students was admitted in September of that year. Currently two classes are enrolled and plans to admit a further twelve students (Class of 2001) are in place. The educational strategy for this program is totally problem-based. Students work in groups of six with a faculty facilitator, not necessarily a content expert. Facilitators are largely drawn from the multidisciplinary pool of research faculty at the center. All learning is mediated through biomedical and biodental problem cases. No formal lectures or classes are scheduled. The learning of clinical dental skills is promoted through focussed dental patient simulations in which students review clinical charts, radiographs, medical reports and then explore identified, hands-on learning needs using patient simulators in a clinical context. Early patient exposure is obtained through dental office visits and other special patient clinics. Initial experience with this program suggests that the problem-based learning (PBL) students learn as well (if not better) than their traditional program peers and develop excellent group and cognitive analytical skills. The absence of a pool of dentally related biomedical cases suitable for a PBL program has necessitated the use of innovative approaches to their development and presentation. It is believed that this educational approach will produce dental clinicians equipped with the self-motivated, life-long learning skills required in the ever-changing world of bio-dental sciences in the twenty-first century.

Presence of calbindin D28K and GAD67 mRNAs in both orthotopic and ectopic Purkinje cells of staggerer mice suggests that staggerer acts after the onset of cytodifferentiation.

We used in situ hybridization to study the expression of GAD67 and calbindin D28K mRNAs in developing mouse cerebellar Purkinje cells. Both genes are expressed prenatally; calbindin D28K mRNAs can be detected in Purkinje cells of embryonic day (E) 15 mice, whereas GAD67 mRNAs first appear slightly later, in E16 mice. The stunted Purkinje cells of staggerer (sg/sg) mutant mice maintain calbindin D28K and GAD67 expression. Our data suggest that the sg/sg mutation does not interfere with the transcriptional activation of these two genes, and might therefore act after the induction of specific gene expression in developing Purkinje cells.

Embryonic mouse lung epithelial progenitor cells co-express immunohistochemical markers of diverse mature cell lineages.

Developmental expression of marker genes representative of different mature cell types can be used to study differentiation of cell lineages. We used immunohistochemistry to study expression in developing mouse lung of calcitonin gene-related peptide (CGRP), Clara cell 10-KD protein (CC10), and surfactant protein-A (SP-A), markers that are differentially expressed in neuroendocrine cells, Clara cells, and Type II alveolar cells. Two distinct developmental phases were revealed. The earlier phase (embryonic days 13-15; E13-E15) was characterized by CGRP, CC10, and SP-A immunostaining in all epithelial cells of the distal airways, with the three patterns being virtually identical in adjacent sections. The later phase (E16-E18) was characterized by emergence of staining of the differentiated cell types. These expression patterns were recapitulated in serumless organ culture, demonstrating that information necessary to generate both phases of gene expression is present within the lung analage by E11. We conclude that CGRP, CC10, and SP-A are co-expressed in most or all cells of the distal lung epithelium at E13-E15 and later become restricted to different cell lineages. This transient expression in progenitor cells of gene products characteristic of diverse differentiated cell types may reflect an underlying mechanism of gene regulation.

Molecular diversity of the SCG10/stathmin gene family in the mouse.

SCG10 is a neuronal growth-associated protein that shares an amino acid sequence similarity with an 18- to 19-kDa phosphoprotein named stathmin (also called p19, p18, Op18, pp17, prosolin, pp20, 19K, and leukemia-associated phosphoprotein, Lap18), which is more broadly expressed in a variety of cell types of the neural, immune, and reproductive systems. The sequence similarity has suggested that SCG10 and stathmin have been derived from structurally and evolutionarily related genes. To explore the structural and evolutionary relationships between these genes, we have isolated a series of cosmid and phage clones that covers the entire region of the mouse stathmin gene and most of the mouse SCG10 gene. The SCG10 transcription unit spans at least 30 kb, while the stathmin gene is 6 kb in length. Both genes consist of five exons, and many of the intron/exon boundaries fall into the homologous regions of conserved domains of these two proteins. However, the promoter-proximal regions are distinct in the two genes, suggesting that they have evolved by fusion of the duplicated coding exons to unique promoters. Southern blot analysis indicates that SCG10 mRNA is encoded by a single gene in the mouse genome, while stathmin cDNA probes detect multiple genes. Chromosome mapping experiments reveal that the SCG10 gene is localized at the proximal region of mouse chromosome 3 and is linked to Il-7, while the stathmin gene loci are distributed to three chromosomes; the authentic stathmin gene lies on chromosome 4, whereas the loci on chromosomes 9 and 17 are likely to be pseudogenes. These data are consistent with the idea that the neuron-specific SCG10 gene evolved by duplication and modification of the more broadly expressed stathmin/Lap18 gene.

Role of epidermal growth factor expression in early mouse embryo lung branching morphogenesis in culture: antisense oligodeoxynucleotide inhibitory strategy.

Epidermal growth factor (EGF) expression and branching morphogenesis were inhibited using a 5' 15-mer antisense oligodeoxynucleotide (ODN) directed against EGF precursor mRNA in embryonic mouse lung in culture under chemically defined, serumless conditions. Antisense EGF ODN resulted in > 90% inhibition of EGF immunoreactive peptide synthesis, 75% reduction in branching morphogenesis, 73% decrease in DNA content, 64% decrease in RNA content, 73% decrease in protein synthesis, and 65% decrease in [3H]thymidine incorporation into DNA compared to Embryonic Day 11 controls in culture for 4 days. Sense ODN results were similar to control. Supplementing antisense ODN with EGF partially reversed antisense effects. The results further support a role for EGF in pulmonary organogenesis.

Mammalian achaete-scute homolog 1 is transiently expressed by spatially restricted subsets of early neuroepithelial and neural crest cells.

Using monoclonal antibodies, we have examined the expression pattern of MASH1, a basic helix-loop-helix protein that is a mammalian homolog of the Drosophila achaete-scute proteins. In Drosophila, achaete-scute genes are required for the determination of a subset of neurons. In the rat embryo, MASH1 expression is confined to subpopulations of neural precursor cells. The induction of MASH1 precedes, but is extinguished upon, overt neuronal differentiation. MASH1 is expressed in the forebrain by spatially restricted domains of neuroepithelium and in the peripheral nervous system exclusively by precursors of sympathetic and enteric neurons. The features of early and transient expression, in spatially restricted subpopulations of neural precursors, are similar to those observed for achaete-scute. Thus, the amino acid sequence conservation between MASH1 and achaete-scute is reflected in a parallel conservation of cell type specificity of expression, similar to the case of mammalian MyoD and Drosophila nautilus. These data support the idea that helix-loop-helix proteins may represent an evolutionarily conserved family of cell-type determination genes, of which MASH1 is the first neural-specific member identified in vertebrates.

Lurcher Purkinje cells express glutamic acid decarboxylase and calbindin mRNAs.

Purkinje neurons in immature Lurcher (Lc/+) mice are destined to die as a result of a defect intrinsic to the dying cells. We have used in situ hybridization to determine whether the Lc allele interferes with the normal program of gene expression in the doomed Purkinje cells. In P21 mice, degeneration of Purkinje cells is well underway, but the surviving Purkinje cells continue to express the mRNAs for both glutamate decarboxylase and calbindin D28K, two proteins whose expression is characteristic of normal Purkinje neurons. We conclude that the Lc allele probably does not interfere with the developmental program but acts to cause cell death in already differentiated Purkinje neurons.

Analysis of SCG10 gene expression in transgenic mice reveals that neural specificity is achieved through selective derepression.

SCG10 is a neural-specific, growth-associated protein that is broadly expressed in the embryonic central and peripheral nervous systems. Transgenic mice harboring a chimeric gene containing 4 kb of SCG10 5' flanking DNA fused to the bacterial CAT gene exhibit expression in brain but not in nonneuronal tissues. A low level of expression is detected in adrenal gland as well, consistent with the behavior of endogenous SCG10. Such a transgene is also activated at the same relative stage of embryonic development as its endogenous counterpart. Deletion of the 5'-most 3.7 kb of SCG10 sequence yields deregulated expression of the transgene in numerous nonneuronal tissues, although expression remains highest in brain. In contrast to other tissue-specific genes, therefore, the specificity of SCG10 expression appears to be achieved predominantly through selective repression in nonneuronal tissues.

Chromatin structure as a molecular marker of cell lineage and developmental potential in neural crest-derived chromaffin cells.

Adrenal medullary chromaffin cells have the capacity to transdifferentiate into sympathetic neurons. We show here that SCG10, a neural-specific gene that is induced during this transdifferentiation, is maintained in mature chromaffin cells in a potentially active chromatin conformation marked by two DNAase I hypersensitive sites (HSS). A low level of transcription is associated with this conformation. The HSS are also present in neurons expressing high levels of SCG10, but not in nonneuronal cells. Experiments using transgenic mice suggest that these HSS can in principle form in any cell type expressing the gene, but that a cis-repression mechanism normally prevents their assembly in nonneuronal cells. We suggest that the SCG10 HSS may represent a molecular marker of the lineage and phenotypic plasticity of chromaffin cells.

The abnormal cerebellar organization of Weaver and reeler mice does not affect the cellular distribution of three neuronal mRNAs.

We used in situ hybridization of 35S-labeled antisense RNAs to study the cellular distribution of three neuronal mRNAs. We compared the expression of these RNAs in cerebellar Purkinje neurons in wild-type (C57Bl-6J) mice and in two mutants (Weaver and reeler) known to have abnormal cerebellar morphologies. In normal mice, GAD mRNA is present in four sets of neurons in the cerebellar cortex while calbindin mRNA is present only in Purkinje neurons. Proenkephalin mRNA is present in Golgi II neurons as well as in a set of neurons in the deep part of the molecular layer. Despite the dramatic differences in structural organization and inputs of Purkinje neurons in the cerebella of adult Weaver and reeler mice, the expression of these RNAs appears unchanged. These results support the hypothesis that Purkinje cell cytodifferentiation proceeds autonomously after its inception in early embryonic life.

Comparative distribution of mRNAs for glutamic acid decarboxylase, tyrosine hydroxylase, and tachykinins in the basal ganglia: an in situ hybridization study in the rodent brain.

Neurotransmitter-related messenger RNAs were detected by in situ hybridization in sections of rat and mouse brains by using 35S-radiolabelled RNA probes transcribed from cDNAs cloned in SP6 promoter-containing vectors. The distribution of messenger RNAs for glutamic acid decarboxylase, tachykinins (substance P and K), and tyrosine hydroxylase was examined in the striatum, pallidum, and substantia nigra. Dense clusters of silver grains were observed with the RNA probe complementary of the cellular messenger RNA for glutamic acid decarboxylase (antisense RNA) over most large neurons in the substantia nigra pars reticulata and medium-sized to large neurons in all pallidal subdivisions. A few very densely and numerous lightly labelled medium-sized neurons were present in the striatum. Among the areas examined, only the striatum contained neurons labelled with the antisense tachykinin RNA. Most of these neurons were of medium size, and a few were large. With the antisense tyrosine hydroxylase RNA, silver grains were found over neurons of the substantia nigra pars compacta and adjacent A10 and A8 dopaminergic cell groups. No signal was observed with RNAs identical to the cellular messenger RNA for glutamic acid decarboxylase or tachykinin (sense RNA). These results show a good correlation with immunohistochemical studies, suggesting that documented differences in the distribution and the level of glutamic acid decarboxylase, tyrosine hydroxylase, and substance P immunoreactivities in neurons of the basal ganglia are related to differences in the level of expression of the corresponding genes rather than to translation accessibility, stability, or transport of the gene products.

In situ hybridization to localize mRNA encoding the neurotransmitter synthetic enzyme glutamate decarboxylase in mouse cerebellum.

Glutamate decarboxylase (GAD; EC 4.1.1.15) is responsible for the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). We have used a cDNA sequence encoding GAD to produce a single-stranded RNA hybridization probe for GAD mRNA. This probe detects GAD mRNA in individual cells in sections of mouse cerebellum. The specificity of in situ hybridization with this probe rests on four criteria: the distribution of labeled cells matched the results we and others obtain with GAD immunohistochemistry (Purkinje, Golgi II, stellate, and basket neurons were labeled, whereas granule cells and glia were not); a negative control probe having a sequence identical to GAD mRNA did not specifically label any cerebellar cells; prior treatment of the sections with RNase abolished specific labeling; the labeling showed the melting behavior typical of nucleic acid hybrids. Translation of GAD mRNA is apparently restricted to neuronal cell bodies since GAD mRNA was detectable in neuronal perikarya but not in terminals. Also, the choice of GABA as a neurotransmitter appears to be made at the level of transcription since granule neurons did not contain detectable GAD mRNA. The level of GAD mRNA varied among the classes of neurons as well as from cell to cell within each neuron type.