Three alternative promoters of the rat gamma-glutamyl transferase gene are active in developing lung and are differentially regulated by oxygen after birth.

The rat gamma-glutamyl transferase mRNA transcripts I, II, and III are derived from three alternative promoters, P(I), P(II), and P(III). In the adult only mRNA III is expressed in the lung. We show that mRNA III gene expression is developmentally regulated in the fetal lung; it is first expressed in gestation. In contrast to the adult lung, the fetal lung expresses mRNA I, II, and III. The switch from the fetal to the adult pattern of gammaGT mRNA expression begins within the first 24 h of birth and is complete by 10 d of age. gammaGT mRNA II disappears within 24 h, mRNA I disappears by 10 d leaving mRNA III as the sole transcript. Alveolar epithelial type 2 cells (AT2) isolated from the adult lung express only mRNA III. When cultured in 21% O2 mRNA III is maintained, but when cultured in 3% O2 the fetal pattern of mRNA I, II and III expression is induced. When AT2 cells in hypoxia are exposed to carbon monoxide, mRNA II is suppressed suggesting that a heme-binding protein (responsive to oxygen) may suppress mRNA II expression and may be responsible for the decrease in lung mRNA II seen after birth. A reporter gene under the control of DNA sequences from the gammaGT P(III) promoter is activated in transient transfection studies in response to hyperoxia, while a deletion construct retaining an antioxidant responsive element is not. Oxygen appears to regulate each of the alternative promoters of the gammaGT gene, such that P(II) is rapidly repressed by a heme-dependent mechanism, P(I), is more gradually repressed by a nonheme mechanism and P(III) is activated by a putative oxygen response element. We hypothesize that similar oxygen-dependent mechanisms regulate other genes in the developing lung at birth.

Identification and characterization of the pulmonary alveolar type II cell Maclura pomifera agglutinin-binding membrane glycoprotein.

The lectin Maclura pomifera agglutinin (MPA) binds to the apical surface of pulmonary alveolar type II but not type I cells. We show that MPA binds to a single membrane glycoprotein in type II cells with a molecular mass of 230 kDa in the rabbit and 200 kDa in the rat. The glycoprotein has an abundance of terminal N-acetylgalactosamine residues. It is a hydrophilic integral membrane protein suggesting that it has an extensive extramembrane domain or is an ion channel. The glycoprotein is similar in rat and rabbit, with the exception that the rat glycoprotein is partially sialylated and is trypsin sensitive. The MPA-binding glycoprotein represents a new integral membrane marker of the apical domain of the pulmonary alveolar type II cell.

Atrial natriuretic peptide modulates alveolar type 2 cell adenylyl and guanylyl cyclases and inhibits surfactant secretion.

Alveolar epithelial type 2 (T2) cells isolated from the lungs of adult rats responded to exogenous atrial natriuretic peptide (ANP) by two signalling mechanisms. First, ANP induced a dose-dependent reduction of ligand-stimulated adenylyl cyclase activity and cAMP accumulation. This effect was inhibited by the addition of GDPbetaS or by pretreatment with pertussis toxin (PT), consistent with mediation by a Gi protein(s). PT-catalyzed [32P]ADP-ribosylation, immunoblots with specific antisera, and Northern blot analysis demonstrated that T2 cells contain the G-proteins Gi2 and Gi3 which could transduce this signal. ANP also promoted PT-insensitive, dose-dependent accumulation of cGMP, consistent with activation of a receptor guanylyl cyclase. Isoproterenol-stimulated phosphatidylcholine secretion was markedly attenuated by ANP, and this effect was inhibited by PT pretreatment, consistent with mediation by a Gi protein(s). These data indicate that in addition to the lung being a major clearance organ for circulating ANP, lung parenchymal cells are targets of ANP action.

Alveolar cell differentiation markers in human lungs.

We describe two apical surface integral membrane glycoproteins which appear to be differentiation markers of the human pulmonary alveolar type 2 cell which has as a major function the production of pulmonary surfactant. These membrane glycoproteins bind the lectin, Maclura pomifera agglutinin and can be found in detergent extract of whole lungs, lung membranes and isolated type 2 cells. One of the MPA binding glycoproteins (MPA-gp330) has an apparent molecular weight of 330 kD and is analogous to a similar membrane glycoprotein found in rat and rabbit type 2 cells. The other glycoprotein (MPA-gp350/390) is an antigen found on the surface of many human cancer cells. In studies of human fetal lung tissue we found that MPA-gp350/390 is expressed before known surfactant functions of the type 2 cell while MPA-gp330 appears later. Neither glycoprotein is influenced by glucocorticoids yet surfactant synthesis is hormone-dependent. These studies demonstrate that pulmonary type 2 cell differentiation is a more complex process than previously appreciated and that differentiation markers are expressed in a discoordinate fashion and regulated by different factors.

Ontogeny of pulmonary alveolar epithelial markers of differentiation.

We studied differentiation of the pulmonary epithelium in the periphery of fetal rat lung in vivo and in vitro by comparing the ontogeny of cell-surface glycoconjugates with that of surfactant phospholipids. Apical surface binding of the lectin Maclura pomifera agglutinin (MPA) and expression of a 200-kDa MPA-binding glycoprotein (MPA-gp200) was evident at 20 days gestation in type 2 cells, but did not correlate with ultrastructural features of type 2 cell differentiation. Epithelial cells isolated from peripheral lung of 18-day gestation fetal rats displayed hormone-sensitive surfactant synthesis prior to the hormone-insensitive expression of MPA-gp200. Expression of MPA-gp200 occurred in association with the appearance of many new apical surface proteins suggesting a hormone-independent process of polar membrane differentiation. Thus membrane and secretory differentiation are discordant and can be dissociated. In vivo binding of Ricinus communis 1 agglutinin (RCA1), an apical marker of the differentiated alveolar type 1 cell occurred in undifferentiated peripheral lung epithelial cells as early as 18 days gestation, disappeared from differentiating type 2 cells and appeared in differentiated type 1 cells. Both undifferentiated fetal epithelial cells at 18 days gestation and fully differentiated type 1 cells express multiple glycoproteins with terminal beta-linked galactose residues which bind RCA1. Some of these RCA1-binding glycoproteins appear to be similar. These observations suggest that alveolar epithelial type 1 cells may derive directly from undifferentiated peripheral lung epithelial cells as well as from fully differentiated type 2 cells. In addition, terminal differentiation of fetal lung peripheral epithelium into type 1 and type 2 cells may involve repression as well as induction of differentiation-related genes.

gamma -glutamyltransferase and its isoform mediate an endoplasmic reticulum stress response.

Although use of multiple alternative first exons generates unique noncoding 5'-ends for gamma-glutamyltransferase (GGT) cDNAs in several species, we show here that alternative splicing events also alter coding exons in mouse GGT to produce at least four protein isoforms. GGTDelta1 introduces CAG four bases upstream of the primary ATG codon and encodes an active GGT heterodimeric ectoenzyme identical to constitutive GGT cDNA but translational efficiency is reduced 2-fold. GGTDelta2-5 deletes the last eight nucleotides of exon 2 through most of exon 5 in-frame, selectively eliminating residues 96-231 from the amphipathic N-terminal subunit, including four N-glycan consensus sites, while leaving the C-terminal hydrophilic subunit intact. GGTDelta7 introduces 22 bases from intron 7 causing a frameshift and a premature stop codon so a truncated polypeptide is encoded terminating with 14 novel residues but retaining the first 339 residues of the native GGT protein. GGTDelta8-9 deletes the terminal four nucleotides of exon 8 plus all of exon 9 and inserts 24 bases from intron 9 in-frame so the C-terminal subunit of the encoded polypeptide loses residues 401-444 but gains eight internal hydrophobic residues. In contrast to the product of GGTDelta1, those derived from GGTDelta2-5, Delta7, Delta8-9 all lack transferase activity and persist as single-chain glycoproteins retained largely in the endoplasmic reticulum as determined by immunofluorescence microscopy and constitutive endoglycosidase H sensitivity in metabolically labeled cells. The developmental-stage plus tissue-specific regulation of the alternative splicing events at GGTDelta7 and GGTDelta8-9 implies unique roles for these GGT protein isoforms. The ability of the GGTDelta1 and GGTDelta7 to mediate the induction of C/EBP homologous protein-10, CHOP-10, and immunoglobulin heavy chain binding protein, BiP, implicates a specific role for these two GGT protein isoforms in the endoplasmic reticulum stress response.

The Bax inhibitor-1 gene is differentially regulated in adult testis and developing lung by two alternative TATA-less promoters.

We identified Bax inhibitor-1, BI-1, as a developmentally regulated gene product in perinatal lung using suppressive subtractive hybridization. BI-1 is a novel suppressor of apoptosis that was previously cloned as testis-enhanced gene transcript (TEGT). However, sequence analysis of lung BI-1 revealed unique nucleotides starting 29 bases upstream of the ATG initiation codon and extending to the 5' end of lung-derived BI-1 cDNA compared to the original transcript from the testis. Cloning and sequencing of the upstream region of the BI-1 gene revealed that these unique sequences originated from two alternative first exons, located in tandem and separated by approximately 600 bases. Neither was preceded by a TATA box in the usual position, and S1 nuclease mapping at each exon 1 revealed multiple transcription start points with a major site being overlapped by a consensus initiator element. Promoter activity from each region was documented by transient transfection analysis in vitro using DNA sequences ligated to a reporter gene. The proximal promoter, P1, may exhibit cell type-specific differences in fibroblasts versus epithelia, whereas the distal promoter, P2, may exhibit species-specific differences in rat versus human cells. RT-PCR analysis for expression in adult tissues using exon 1-specific 5' primers and common 3' primers revealed that P1 is tissue-specific; P2 is ubiquitously active. The developmental regulation of BI-1 in the late fetal and early postnatal lung is specific for P2, indicating that these two TATA-less promoters are differentially regulated in adult testis and developing lung. Since Bax inhibitor-1 functions as a suppressor of apoptosis, its expression could provide a survival advantage for select cell populations during the peak period of apoptosis that occurs at birth.

Human pulmonary alveolar type 2 cells contain an apical membrane glycoprotein common to malignant cells.

A monoclonal antibody, Ca1, raised against a detergent extract of Hep 2 cells derived from a human laryngeal carcinoma, was shown in these studies to bind to the apical surface of normal alveolar type 2 cells but not type 1 cells in the human lung. In lung specimens from patients with alveolitis, the antibody also bound to hyperplastic type 2 cells and to transition cells which were in the process of becoming alveolar type 1 cells. Ca1 binds to the apical plasma membrane and to internal membranes of cytoplasmic vesicles thought to be involved in the packaging of pulmonary surfactant. A surfactant-enriched fraction of human lung lavage did not bind the Ca1 antibody suggesting that the antigen was not an integral component of secreted surfactant. In normal human lung parenchyma, Ca1 binds only to type 2 cells, however it also binds to the apical surface of Clara cells in areas of cellular hyperplasia. Solubilized homogenates of whole lung, of a cell membrane fraction and of Hep 2 cells, immunoprecipitated with Ca2, separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with iodinated lectins, revealed that terminal glycosylation of the type 2 cell antigen differed from that of Hep 2 cells. Ca1 and a 330 kilodalton type 2 cell glycoprotein bind the lectin Maclura pomifera agglutinin. These two glycoproteins represent the first defined membrane markers of the apical surface of the human type 2 cell.

A rat alveolar type II cell line developed by adenovirus 12SE1A gene transfer.

The regulation of pulmonary alveolar type II cell proliferation and differentiation is poorly understood and has been difficult to study, in part due to lack of proliferation, cellular heterogeneity, and phenotypic instability of type II cells in primary culture. To develop a stable population of homogeneous cells capable of proliferation, we transfected type II cells isolated from the lungs of neonatal rats with an immortalizing oncogene, adenovirus 12SE1A, using a retroviral vector. Individual clones were isolated, screened for cytokeratin expression, and further characterized. One of the 12SE1A expressing clones, E1A-T2, has epithelial features such as cytokeratin expression and tight junctions, and coexpresses vimentin. E1A-T2 rapidly proliferate when grown in 10% fetal bovine serum, and slow their growth at confluence. A labeling index of greater than 90% during a 24-h pulse of [3H]thymidine reflects a uniform population of proliferating cells. E1A-T2 can be grown and passed in 0.4% fetal bovine serum, suggesting the production of an autocrine growth factor(s). The type II cell Maclura pomifera agglutinin (MPA)-binding glycoprotein, MPA-gp200, appears to be expressed in an incompletely glycosylated form, whereas other features of differentiated type II cells, such as lamellar bodies, surfactant protein A, and a high percentage of saturated phosphatidylcholine, are absent. Homogeneous, clonally derived type II cell lines, such as E1A-T2 may retain sufficient type II cell features of interest to test new hypotheses relating to cell proliferation and differentiation otherwise not feasible using primary cultures of type II cells.

Serum accelerates the loss of type II cell differentiation in vitro.

The differentiated phenotype of the alveolar type II cell is rapidly altered in vitro. To evaluate factors that might influence this process, we isolated and plated rat type II cells in serum-supplemented media to promote adherence and then maintained the cells in a simple nutrient medium in the absence (S- cells) or presence (S+ cells) of serum for 5 to 7 d. The type II S- cells remained metabolically active. Despite protein synthesis that was 50% that of S+ cells, S- cells continued to synthesize a broad spectrum of proteins and to express several features of type II cell differentiation. They synthesized an apical integral membrane glycoprotein, Maclura pomifera agglutinin (MPA)-gp200, and a cytokeratin, No. 19, while S+ cells did not. When supplemented with linoleic acid, S- cells contained lamellar and multivesicular bodies, incorporated cell surface MPA into these structures, and secreted their phosphatidylcholine (PC) in response to mastoparan. Despite the relative synthesis of higher levels of total and saturated PC in S- cells supplemented with linoleic acid, phosphatidylglycerol remained diminished. A surfactant protein (SP-A) was present in S- cells, but synthesis was not detected. These studies demonstrate that serum accelerates the loss of type II cell differentiation in vitro and that the expression of type II cell markers of differentiation is not inherently linked.

Ontogeny of gamma-glutamyltransferase in the rat lung.

gamma-Glutamyltransferase (gamma-GT) is a key enzyme in the metabolism of glutathione and glutathione-substituted molecules. The gamma-GT gene is expressed in two epithelial cells of the adult lung, the bronchiolar Clara cell and the alveolar type II cell. Because pulmonary glutathione metabolism may be important in the perinatal period, we studied gamma-GT ontogeny in the developing rat lung. In the late fetal and early postnatal lung, gamma-GT mRNA was below detectable limits on Northern blots. Pulmonary gamma-GT protein and enzyme activity were present at low levels after fetal day 18. gamma-GT protein appeared as a high-molecular-mass band (>95 kDa), with small amounts of enzymatically active gamma-GT heterodimer. Between the 2nd and 3rd postnatal wk, pulmonary gamma-GT mRNA expression increased in association with an increase in gamma-GT protein and enzyme activity that reached adult lung levels. At this time, gamma-GT protein appeared predominantly in the heterodimeric form with small amounts of the >95-kDa protein. Immunocytochemistry revealed that, in the fetal and early postnatal lung, gamma-GT was expressed only in the alveolar type II cell, whereas the Clara cell became the major site of gamma-GT mRNA and protein expression by 2-3 wk and in the adult. Type II cells isolated from the fetal lung express gamma-GT mRNA and synthesize the >95-kDa form of gamma-GT in excess of the heterodimer. These studies demonstrate that the alveolar type II cell is the only cell producing gamma-GT in the newborn lung and that it synthesizes a form of gamma-GT that appears to differ from that produced at a later time point by the Clara cell.

Nitrogen dioxide exposure activates gamma-glutamyl transferase gene expression in rat lung.

Exposure to nitrogen dioxide (NO2) has been shown to activate glutathione metabolism in lung and lung lavage. Since GGT is a key enzyme in glutathione metabolism and we have previously characterized GGT expression in distal lung epithelium and in lung surfactant, we examined the NO2 exposed lung for induction of gamma-glutamyl transferase (GGT) mRNA, protein, and enzyme activity. We found that the GGT gene product is induced in lung by NO2. The GGT mRNA level in lung increases 2-fold within 6 hr and 3-fold after 24 hr of exposure to this oxidant gas, and this 3-fold elevation persists even after 14 days of exposure. The pattern of GGT mRNA expression switches from the single GGT mRNA III transcript in the normal lung to the dual expression of GGT mRNA I and mRNA III. Enzyme activity in whole lung increases 1.6- to 2.5-fold while extracellular surfactant-associated GGT activity accumulates 5.5-fold and GGT protein accumulates in lung surfactant. Induction of GGT mRNA and protein is evident in cells of the bronchioles by in situ hybridization and immunolocalization, respectively. In contrast, alveolar type 2 cells lack an in situ hybridization signal and exhibit a reduction in the intensity of immunostaining with prolonged exposure. Our studies show that NO2 induces GGT mRNA expression, including GGT mRNA1, in lung and GGT protein and enzyme activity in lung and lung lavage in response to the oxidative stress of NO2 inhalation.

Retinoic acid induces changes in the pattern of airway branching and alters epithelial cell differentiation in the developing lung in vitro.

Retinoids have been shown to influence pattern formation during development and regeneration in numerous systems such as limbs, vertebrae, and neural tube although there is little information about the effects of retinoids on pattern formation in visceral organs. We investigated the effects of exogenous retinoic acid on the in vitro pattern of airway branching and on lung epithelial cell differentiation. Histology, [3H]thymidine autoradiographies and reverse transcriptase/polymerase chain reaction (RT/PCR) amplification were used to assess the effects of retinoids and the expression of lung epithelial markers of differentiation. We found that retinoic acid interferes, in a dose-dependent fashion, with the expression of epithelial genes that are found in distal segments of the fetal lung (surfactant-associated proteins SP-A, SP-B, and SP-C). At high concentrations, retinoic acid (RA) dramatically altered the developmental pattern of the lung, favoring growth of structures that resemble proximal airways and concomitantly suppressing distal epithelial buds. We hypothesize that this in vitro "proximalizing" effect on the developing lung may be related to alterations in the expression of pattern-related genes.

gamma-Glutamyl transferase (GGT) deficiency in the GGTenu1 mouse results from a single point mutation that leads to a stop codon in the first coding exon of GGT mRNA.

GGTenul, a recently described genetic murine model of gamma-glutamyl transferase (GGT) deficiency, was induced by the point mutagen N-ethyl-N-nitrosourea and is inherited as an autosomal recessive trait. The phenotype of systemic GGT deficiency suggested a mutation site within the cDNA coding region which is common in all GGT transcripts. To identify this site, total lung and kidney RNA was isolated from normal and mutant mice, amplified by RT-PCR using GGT-specific primers, cloned as two overlapping approximately 1 kb GGT cDNA fragments, sequenced and compared with that in the literature. A single base pair substitution was identified in the coding region at position 237, where thymidine became adenine, and this mutation replaced a leucine codon, TTG, with a termination codon, TAG. This mutation site was confirmed in mutant genomic DNA by PCR using primers that flanked the predicted site and spanned the intron between the common GGT non-coding exon and the first GGT coding exon. This PCR product was sequenced directly with the secondary 3' PCR primer, the mutation site identified and the protocol then utilized to genotype animals. In addition to this mutation, the steady-state level of GGT mRNA in mutant kidney is reduced 3-fold compared with the control. Heterodimeric GGT protein is not detectable by western blot in either whole kidney homogenate or a microsomal membrane fraction. The steady-state mRNA level of gamma-glutatmyl cysteinyl synthetase was unchanged in mutant mice compared with normal, but that of heme oxygenase-1 and Cu,Zn-SOD was induced 4- and 3-fold, respectively. Hence, the GGTenul mouse model of GGT deficiency results from a single point mutation in the first coding exon of GGT mRNA and the resulting impairment in glutathione turnover induces oxidative stress in the kidney.

Synthesis and release of amphipathic gamma-glutamyl transferase by the pulmonary alveolar type 2 cell. Its redistribution throughout the gas exchange portion of the lung indicates a new role for surfactant.

gamma-Glutamyl transferase (gamma-GT) catalyzes a transpeptidation reaction which is involved in the metabolism of glutathione. Glutathione is abundant within the epithelial lining fluid of the lung. However, little is known about gamma-GT expression in the epithelial cells of the lung alveolus. Herein we show that the pulmonary alveolar epithelial type 2 cell expresses the gene for gamma-GT. We were unable to detect expression in the pulmonary alveolar epithelial type 1 cell or in the pulmonary alveolar macrophage. gamma-GT expression in the pulmonary alveolar epithelial type 2 cell is via mRNA III, a transcript that was initially cloned from the liver. This cell synthesizes gamma-GT protein and releases enzyme activity into a surfactant-associated pool within the lung alveolus. The specific activity of this surfactant-associated enzyme is almost 10-fold higher than that of whole lung. This activity results from amphipathic gamma-GT since it partitions with lung surfactant phospholipid and with the detergent phase of Triton X-114. Activity can be dissociated from each by papain proteolysis. These results demonstrate that gamma-GT is expressed in the differentiated pulmonary alveolar epithelial type 2 cell and that amphipathic gamma-GT protein is released by this cell along with lung surfactant. These results suggest that surfactant may serve an expanded role in lung cell biology as the vehicle for the redistribution of amphipathic signal anchored proteins throughout the gas exchange surface of the lung.

Mechanisms of mastoparan-stimulated surfactant secretion from isolated pulmonary alveolar type 2 cells.

Mastoparan, a tetradecapeptide component of wasp venom, is a potent activator of secretion in a variety of cell types, and has been shown to activate purified G-proteins reconstituted into phospholipid vesicles with a preferential activation of Gi over Gs (Higashijima, T., Uzu, S., Nakajima, T., and Ross, E. R. (1988) J. Biol. Chem. 263, 6491-6494). To identify the biochemical activities of mastoparan in a cellular system, we characterized the effects of mastoparan on signal transduction pathways in rat pulmonary alveolar type 2 epithelial cells, which synthesize and secrete pulmonary surfactant. Mastoparan inhibited adenylylcyclase activity in a manner that was dose-dependent (IC50 = 30 microM), but sensitive to neither guanine nucleotide nor pertussis toxin (PT). Mastoparan induced a PT-sensitive increase in cellular inositol trisphosphate and a rapid rise in cytosolic calcium released from intracellular stores; the time to onset of the calcium rise, but neither the rate nor the amplitude of the rise, were PT-sensitive. Mastoparan also caused a dose- (EC50 = 16 microM) and time-dependent activation of arachidonic acid release that was completely insensitive to pretreatment with PT. Secretion of pulmonary surfactant was increased by mastoparan approximately 8-fold over constitutive levels at 1 h with an EC50 = 20 microM, and mastoparan-stimulated secretion was partially sensitive to PT at late time points and to inhibitors of arachidonic acid metabolism, but not to the protein kinase C inhibitor H7. These findings are consistent with the activation of Gi proteins in type 2 cells by mastoparan, although the lack of predicted triphosphoguanine nucleotide and PT sensitivity for some activities indicates that mastoparan does not act in a manner strictly analogous to liganded receptors or that some activities are not mediated by activation of Gi. While mastoparan is a potent secretagogue in several cell types, its secretory activity appears to have only a limited dependence on the activation of Gi proteins in type 2 cells.

Barking up the wrong tree? Use of polymerase chain reaction to diagnose syphilitic aortitis.

The presentation of syphilitic aortitis is often atypical and available serological tests are non-specific. The diagnostic gold standard remains direct identification of microorganisms in tissue. We present a case of syphilitic aortitis that presented as a mediastinal mass and report the use of polymerase chain reaction for Treponema pallidum to diagnose syphilitic aortic disease.

Cloning, characterization, and development expression of a rat lung alveolar type I cell gene in embryonic endodermal and neural derivatives.

We report here the identification and characterization of a novel gene, T1 alpha, expressed in high abundance in adult rat lung, fetal lung, and early fetal brain. T1 alpha was identified by a monoclonal antibody previously shown to be specific for an antigen expressed by alveolar epithelial type I cells. The cDNA for T1 alpha is 1.85 kb and identifies a single mRNA species of the same size on Northern blots of adult rat lung. The longest open reading frame of the cDNA is 498 bases which would encode a protein of approximately 18 kDa. The protein has a putative membrane spanning domain near the C-terminus but lacks consensus sequences for N-glycosylation. Northern blots and RT-PCR show high expression of T1 alpha in adult lung, with marginally detectable expression in adult brain, intestine, and kidney. RT-PCR analysis shows expression of T1 alpha in freshly isolated type I cells (50-60% purity) but not in highly purified type II cells or other lung cells. We believe therefore that T1 alpha is primarily if not uniquely expressed in alveolar type I cells in the adult rat. Polyclonal antisera against a 16-amino-acid peptide identified in the deduced sequence reacts with the apical membranes of adult type I cells in lung tissue sections but does not label other cell types. The above antiserum as well as the original monoclonal antibody recognize a single approximately 18-kDa protein derived from bacterial expression of a construct containing the T1 alpha open reading frame. By RT-PCR T1 alpha is detected in rat lung from Day 13.5 onward, but is detected by in situ hybridization earlier in lung, brain and neural derivatives, and foregut. Expression is down-regulated in all but lung tissues as development proceeds.