©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Gene Structure and Expression of Human Thyroid Transcription Factor-1 in Respiratory Epithelial Cells (*)

(Received for publication, December 14, 1994; and in revised form, January 19, 1995)

Kazushige Ikeda Jean C. Clark Jessica R. Shaw-White Mildred T. Stahlman (1) Christopher J. Boutell Jeffrey A. Whitsett (§)

From the Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039 and the Division of Neonatology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2370

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The human gene encoding thyroid transcription factor-1 (TTF-1), a homeodomain-containing nuclear transcription protein of the Nkx2 gene family, was isolated and characterized. Human TTF-1 was encoded by a single gene locus spanning approximately 3.3 kilobases and consisted of two exons and a single intron. The TTF-1 cDNA and polypeptide of 371 amino acids have been highly conserved, sharing 98% identity with the rat TTF-1 polypeptide. Human TTF-1 mRNA and polypeptide were selectively expressed in human and mouse pulmonary adenocarcinoma cell lines. In addition to its presence in thyroid gland epithelium, the human TTF-1 protein was detected by immunohistochemistry in human fetal lung as early as 11 weeks of gestation, being localized in the nuclei of epithelial cells of the developing airways. After birth, TTF-1 was selectively expressed in Type II epithelial cells in the alveoli and in subsets of bronchiolar epithelial cells in the conducting regions of the lung. The 5`-flanking region of the human TTF-1 gene directed transcription of luciferase cDNA in a lung epithelial cell-selective manner. The conservation and distribution of TTF-1 in the human respiratory tract support its role in the regulation of lung development and surfactant homeostasis.


INTRODUCTION

Thyroid transcription factor-1 (TTF-1) (^1)is a 38-kDa nuclear protein initially identified as a mediator of thyroid-specific gene transcription(1, 2) . TTF-1 contains a highly conserved homeobox domain and a 17-amino acid domain characterizing it as a member of the Nkx2 family of homeodomain transcription factors. TTF-1 activates thyroglobulin and thyroperoxidase gene transcription in thyroid adenocarcinoma cells and is expressed in epithelial cells of the developing and mature thyroid gland in the rat, consistent with its role in thyroid epithelial cell gene expression(2, 3, 4) . A potential role for TTF-1 in lung epithelial cell gene expression was recently demonstrated by the finding that TTF-1 activates the transcription of human surfactant protein B gene(5) . TTF-1 binds to two closely apposed TTF-1 DNA binding sites located -80 to -100 base pairs from the start of SP-B gene transcription. TTF-1 is also expressed in the embryonic forebrain and in the respiratory epithelium of the rat, appearing in fetal lung buds of the embryonic rat lung on postconceptional day 9 or 10(4) .

Like the thyroid gland, the lung develops as an outpouching of epithelial cells from the foregut endoderm. The fetal lung then undergoes branching morphogenesis and cellular differentiation to form the complex respiratory epithelium characteristic of the mature lung. Morphologic and biochemical criteria distinguish subsets of respiratory epithelial cells that line the proximal and distal respiratory tract in mammals. A number of proteins are selectively expressed by respiratory epithelial cells, including pulmonary surfactant proteins A, B, and C and the Clara cell secretory protein (CCSP) (for review, see (6) ). These cellular markers distinguish subsets of cells in the conducting and alveolar region of the lung in nonciliated respiratory epithelial and alveolar Type II epithelial cells. The surfactant proteins play important roles in surfactant homeostasis, enhancing the spreading of alveolar phospholipids and contributing to the regulation of the uptake and recycling of surfactant by Type II epithelial cells. The similarity of the temporal-spatial distribution of TTF-1 and surfactant protein B in the developing lung provides further support for the potential role of TTF-1 in the regulation of surfactant function of lung epithelial cell gene expression. In the work of Bohinski et al.(5) , the promoter elements for surfactant proteins B and C as well as the CCSP gene were transactivated by TTF-1 in vitro, suggesting that TTF-1 may also play a more general role in lung epithelial cell gene expression.

While the cDNAs encoding rat (1) and canine TTF-1 (^2)have been isolated and characterized, there is less information regarding the structure and function of the TTF-1 gene and its distribution in the human lung. Because of the potential importance of TTF-1 in the differentiation and function of the respiratory epithelium, we have assessed the immunohistochemical localization of this homeodomain protein in the human respiratory epithelium. The human TTF-1 gene was isolated, and the promoter elements that contribute to the regulation of expression of TTF-1 mRNA in lung cells were identified.


MATERIALS AND METHODS

Reagents, Bacterial Strains, and Plasmids

Restriction endonucleases and enzymes used in cloning reactions were purchased from Life Technologies, Inc. A random primer kit (Stratagene) was used to radiolabel cDNA fragments with [alpha-P]dCTP. Oligonucleotides were labeled with [-P]ATP by kinase reaction. Radioisotopes were purchased from DuPont NEN. Escherichia coli DH5alpha or DH5alphaF^1 was used as a host strain for pUC and pBluscript plasmids and M13 phage.

Identification of Genomic Clone

A human cosmid (pWE15, Stratagene) genomic library was kindly provided by Dr. A. Menon (University of Cincinnati College of Medicine) and screened using a 1.3-kb rat TTF-1 cDNA clone, a gift from Dr. R. Di Lauro (Stazione Zoologica ``Anton Dohrn,'' Naples, Italy). Hybridization was performed at 60 °C under conditions recommended for Hybond (Amersham Corp.). The final wash was in 0.2 times SSC (1 times SSC, pH 7.0: 150 mM NaCl, 15 mM sodium citrate) at 65 °C. Positive colonies were screened at lower density an additional three times to achieve colony purity. Filters were exposed to Kodak XAR film at -80 °C for 2 nights. Three genomic equivalents were screened in duplicate, and two positive clones were identified. Initial restriction analyses of the two clones were identical, so one clone was selected for more detailed analysis.

Southern Blot Analysis

DNA from human lung adenocarcinoma line H441-4 and from the cosmid clone was digested with BamHI, EcoRI, HindIII, and KpnI, electrophoresed through an agarose gel, transferred to Hybond (Amersham), and probed with the labeled rat TTF-1 cDNA. Filters were washed at a final stringency of 0.2 times saline/sodium phosphate/EDTA, 0.1% SDS at 65 °C and exposed to Kodak XAR film at -80 °C. In addition, the cosmid clone DNA was digested with additional restriction enzymes, subjected to Southern analysis, and probed under less stringent conditions with labeled oligonucleotide probes made to various regions of the rat TTF-1 cDNA.

DNA Sequence Analysis

A 5.7-kb XhoI-HindIII fragment and a 4.6-kb BamHI fragment containing the human TTF-1 gene were subcloned into pUC18 and -19 and into M13 mp 18 and 19. The TTF-1 gene was sequenced using the U. S. Biochemical Corp. sequenase kit, using either single-stranded or double-stranded DNA. Human TTF-1-specific oligonucleotides were synthesized and used as primers as the sequence was generated. The resulting DNA sequence was stored and analyzed on a MacIntosh IIs, using the program DNA Star.

RNA Extraction and Northern Analysis

Cell lines were maintained in standard tissue culture prior to harvest including HeLa cervical epithelial cells, 3T3 fibroblasts, A549, H441, H820, 9/HTEo, and BEAS-2B pulmonary adenocarcinomas. H446 and H345 small cell carcinomas were obtained from ATCC and maintained as suggested prior to harvest. Total RNA was isolated by an adapted method of Chirgwin et al.(8) . Tissue was homogenized in 4 M guanidine thiocyanate, 0.5% N-lauroylsarcosine, 25 mM sodium citrate, and 0.1 M beta-mercaptoethanol. Cells grown in culture were lysed directly on the plate using the same buffer. Thereafter, Phase Lock gels (5 Prime 3 Prime, Inc., Boulder, CO) were used to prepare RNA. RNA quantity was determined by absorbance at 260 nm.

Total RNA (20 µg) was electrophoresed through a 1.0% agarose, 7% formaldehyde gel, transferred to Hybond (Amersham) or Nytran (Schleicher & Schuell), and bound to the filter by UV cross-linking. Filters were hybridized overnight at 42 °C in 50% formaldehyde plus standard sodium phosphate-EDTA solution as recommended, using P-random primer-labeled rat TTF-1 cDNA as probe. Filters were washed to a final stringency of 0.2 times saline/sodium/phosphate/EDTA, 0.1% SDS at 60 °C and exposed to Kodak XAR-2 film.

Luciferase Assays

The pGL2 vector, a luciferase reporter vector, was purchased from Promega. Two human TTF-1 gene fragments, HindIII/SspI and SmaI/SspI, were cloned into the multiple cloning site of the pGL2 basic construct to generate pGL2-2.7 kb and pGL2-0.55 kb, respectively, as seen in Fig. 2B.


Figure 2: Organization of the human TTF-1 gene and TTF-1 promoter-luciferase constructs. A, restriction map of the human TTF-1 gene showing position of exons (black), intron (hatched), translation start (ATG) and stop (TGA), and the homeodomain. B, representation of the luciferase constructs used in transfection assays.



Human NCI-H441-4 (H441) and mouse MLE-15 cells were maintained as described previously(5, 9) . NIH-3T3 cells (3T3) were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated bovine serum. Transfections were performed by the calcium phosphate co-precipitation method as described by Rosenthal (10) except that glycerol shock was not used. Luciferase reporter plasmid (5 pmol) and 2.5 pmol of the internal control plasmid, pCMV-betagal(11) , were co-transfected. Cells were incubated for approximately 18 h, washed once with Hanks' balanced salt solution (Life Technologies, Inc.), and returned to culture in original media for an additional 24 h for MLE-15 cells, 72 h for H441 cells, and 48 h for 3T3 cells. Cells were harvested with reporter lysis buffer (Promega) followed by a rapid single freeze-thaw cycle. The lysates were prepared, and aliquots were assayed for beta-galactosidase activity (5) and for luciferase activity using a luminometer (Analytical Luminescence Laboratory, San Diego, CA). To correct for variations in transfection efficiency, assays were normalized to beta-galactosidase activity.

Immunohistochemical Localization of Human TTF-1

Immunohistochemistry was performed on post-mortem samples of formalin-fixed tissues of human fetal and neonatal or adult lung obtained under protocols approved by the Human Research Committee, Vanderbilt University, Nashville, TN. Immunoperoxidase methods using a streptavidin-biotin kit (Biogenex) or an avidin biotin kit (Vectastain Elite ABC kit, Vector Laboratories) were used for immunolocalization of the antigen(12) . Antigen retrieval systems, using microwave heating, markedly enhanced TTF-1 staining and were routinely used. Anti-rat TTF-1 serum, produced in rabbits, was kindly provided by Dr. R. Di Lauro and used at a dilution of 1:1000 to 1:2000. Specificity was established by replacing the specific TTF-1 antibody with nonimmune rabbit antisera. Staining was completely blocked by preadsorption of the antisera with recombinant TTF-1 (data not shown). Sections were counterstained with hematoxylin or nuclear fast red prior to photography. The staining represents data from more than 20 distinct samples obtained at post mortem at ages 11 weeks of gestation through adulthood.


RESULTS

Cloning and Nucleotide Sequence Analysis of the Human TTF-1 Gene

Two identical genomic TTF-1 clones were isolated from an amplified human genomic library by hybridization screening with the rat TTF-1 cDNA under stringent conditions. Restriction fragment analysis of the cosmid clone was similar to that of DNA from human adenocarcinoma cell line H441 (Fig. 1), indicating the presence of only one human TTF-1 gene. The TTF-1 locus was contained within a 4.6-kb BamHI fragment consisting of two exons and one intron (Fig. 2). The predicted human TTF-1 peptide of 371 amino acids shared close identity with the amino acid sequence predicted by the rat TTF-1 cDNA sequence and 92.4% identity with the nucleotide sequence of the rat TTF-1 cDNA. The human TTF-1 gene consisted of two exons interrupted by a single exon of approximately 1 kb flanked by consensus splice donor acceptor sites that fit splice-acceptor donor rules. The restriction map, location of the exons, and nucleotide sequence are provided in Fig. 2and Fig. 3. The cosmid clone included the transcriptional start site previously identified for rat TTF-1 and termination signals consistent with the size of the 2.3-kb mRNA detected by Northern blot analysis of RNA from rat lung tissue (data not shown) and mouse and human pulmonary adenocarcinoma cells (H441) (Fig. 4). TTF-1 mRNA was detected in human pulmonary adenocarcinoma cells H441 and H820 (data not shown) and small cell carcinoma H345 but was not detected in 9/HTEo or BEAS-2B (tracheal-bronchial epithelial cell lines), A549, HeLa, or 3T3 cells, demonstrating the cell selectivity of TTF-1 expression. The size of TTF-1 mRNA was similar to that previously described in the rat thyroid and thyroid carcinoma cells(1) . The start of transcription was mapped by S1 analysis of mRNA from MLE-15 and H441 cells demonstrating three closely apposed transcriptional start sites located approximately -196 base pairs from the ATG initiator methionine in both species (data not shown).


Figure 1: Southern blot analysis of the human TTF-1 gene. 20 µg of DNA from the cosmid clone (A) or from H441-4 cells (B) was digested with BamHI (lane1), EcoRI (lane2), HindIII (lane3), or KpnI (lane4) and subjected to Southern analysis, using the rat TTF-1 cDNA as probe.




Figure 3: Nucleotide sequence and predicted amino acid sequence of human TTF-1 gene. The major start of transcription is marked (+1), and the polyadenylation signal (AATAAA) is underlined.




Figure 4: Northern blot analysis of TTF-1 mRNA in mouse lung and human and mouse pulmonary adenocarcinoma cells. A, Northern analysis of 20 µg of total RNA from MLE-15 (lane1), MLE F6 (lane 2), 3T3 (lane 3), and H441 cells (lane4). The probe used was the rat TTF-1 cDNA. B, Northern analysis of 15 µg of total RNA from human cell lines HeLa (lane 1), H441 (lane2), H345 (lane3), H446 (lane4), BEAS-2B (lane5), 9/HTEo (lane6), and A549 (lane7). The probe used was a SacII-Sau3AI fragment of rat TTF-1 cDNA, which did not contain any Nkx or homeobox domain homology.



Transcriptional Activity of the 5`-Region of the TTF-1 Gene

Genomic fragments of 2.7 and 0.55 kb of the 5`-region of the TTF-1 gene were ligated into a firefly luciferase plasmid and transfected into H441, MLE-15, and 3T3 fibroblast cell lines. The TTF-1-luciferase constructs expressed luciferase activity in pulmonary adenocarcinoma cells H441 and MLE-15; activity of these constructs was detected, albeit at lower levels, in 3T3 cells (Fig. 5). Activity of the TTF-1-luciferase constructs was approximately 10-20-fold higher in mouse lung epithelial cells (MLE-15) and H441-4 cells than in 3T3 cells. Luciferase activity was higher in the 2.7-kb TTF-1-luciferase construct than in the 0.55-kb TTF-1-luciferase constructs in all cell types.


Figure 5: Activity of TTF-1-luciferase constructs in pulmonary adenocarcinoma cells and 3T3 fibroblasts. Luciferase assays were performed after transfecting H441, MLE-15, and 3T3 cells using a promoterless plasmid, pGL2-Basic, a plasmid containing 0.55 kb of human TTF-1 promoter, and another containing 2.7 kb of human TTF-1 promoter. The cells were co-transfected with a CMV-beta-Gal construct as described under ``Materials and Methods.'' Results are plotted as units of luciferase activity per unit of beta-galactosidase and represent at least three separate experiments performed in quadruplicate. Values are means ± S.E. (n = 3 separate experiments).



Distribution of TTF-1 in the Developing Human Lung

TTF-1 was detected by immunohistochemistry in nuclei of the respiratory epithelium in human fetal lung as early as 11-12 weeks of gestation. Immunostaining was observed in the developing airways in a distribution pattern similar to that previously described for pro-SP-C (14) (Fig. 6). Thereafter, TTF-1 was detected in subsets of respiratory epithelial cells in the developing lung, including nonciliated bronchiolar, and rarely in nonciliated bronchial respiratory epithelial cells in the immature lung (Fig. 6). At the time of birth, TTF-1 was detected in alveolar Type II epithelial cells and in subsets of nonciliated bronchiolar epithelial cells. TTF-1 was not detected in alveolar Type I cells or ciliated epithelial cells. The distribution of cells expressing TTF-1 is consistent with the overlapping distribution patterns of surfactant proteins A, B, and C and CCSP(13, 14, 15) . In the adult lung, TTF-1 was readily detected in subsets of nonciliated bronchiolar epithelial cells and was most prominent in Type II epithelial cells but was excluded from Type I cells (Fig. 6).


Figure 6: Immunohistochemistry of TTF-1 in fetal, newborn, and adult lung. Immunoperoxidase staining was utilized to stain human lung samples from 12 weeks of gestation (A and B), 37 weeks of gestation (C and D), and adult (E and F). PanelF represents the control panel of adult lung without primary antibody. Nuclear staining was detected in airway epithelial cells. Slides were counterstained with hematoxylin (A, B, C, and D) or nuclear fast red (E and F). A, B, and C, magnification is times 530; D, E, and F, magnification is times 425.




DISCUSSION

Human TTF-1 was detected in respiratory epithelial cells from as early as 11 weeks of gestation to adulthood, marking distinct subsets of nonciliated respiratory epithelial cells in the conducting airway and Type II epithelial cells within the alveoli postnatally. Transcriptional elements from the 5`-flanking region of the human TTF-1 gene provide, at least in part, for the cell-selective expression of TTF-1 in the respiratory epithelium. The temporal-spatial pattern of human TTF-1 polypeptide is consistent with its importance in the regulation of surfactant protein synthesis and perhaps with a more general role in the early differentiation of the foregut epithelium during fetal lung development. The structure and function of TTF-1 have been highly conserved during evolution, providing further support for its potential role in mammalian lung development and in surfactant homeostasis.

The polypeptide sequences of human TTF-1 have been strongly conserved among mammalian species. TTF-1 is a member of the Nkx2 class of homeodomain-containing proteins, initially isolated by screening of thyroid cDNA library with oligonucleotides based on a partial amino acid sequence of the bovine TTF-1 protein(1) . TTF-1 contains a highly conserved homeodomain DNA binding domain distinct from the Antenapedia class of transcriptional proteins and is also distinguished by the presence of a highly conserved 17-amino acid peptide domain characteristic of genes of the Nkx2 gene family(1, 16) . The predicted TTF-1 polypeptide shares 98-99% identity with that predicted from the TTF-1 cDNA from canine^2 and rat(1) , with conservation of the homeodomain and the 17-amino acid Nkx2 domain. While the gene structures of other mammalian TTF-1 genes have not been reported, the 5`-flanking region of the TTF-1 gene has also been highly conserved between the rat (17) and the human TTF-1 genes, supporting the likelihood of shared regulatory characteristics of the TTF-1 genes.

The temporal-spatial pattern of TTF-1 expression in the developing human lung recapitulates the findings in the developing rat lung previously described by Lazzaro et al.(4) . In the rat, TTF-1 is expressed in the primordial thyroid epithelium between postconceptional days 10 and 11 and at a similar time in the primordial lung buds, being expressed at high levels in the distal tips of the developing airways. In the human fetal lung, the distribution of TTF-1 staining is similar to the pattern of SP-C mRNA in the distal respiratory epithelium(14) , a finding consistent with the observation that TTF-1 strongly activated lung cell-specific SP-C gene transcription(5) . However, SP-C was nearly completely excluded from the conducting airway epithelium in the more mature human (14) and rodent lung(18) , while TTF-1 was detected in both nonciliated respiratory epithelial cells in the conducting airways and in alveolar Type II cells. The distribution of TTF-1 in the conducting airway in human lung included regions of the respiratory epithelium expressing SP-A, SP-B, and CCSP genes. The promoters of each of these genes are transactivated by TTF-1 in vitro(5) . Thus TTF-1 expression includes distinct subsets of epithelial cells that express the lung epithelial cell-specific markers; however, the presence of TTF-1 alone is not sufficient to explain the heterogeneous distribution of these markers in the developing fetal and mature lung.

In the human, TTF-1 was expressed at the highest levels in the tips of the fetal lung buds and in the alveolar Type II epithelial cells in the postnatal lung. TTF-1 staining was excluded from the ciliated respiratory epithelial cells and from terminally differentiated Type I epithelial cells, neither of which contain detectable levels of SP-A, SP-B, SP-C, or CCSP. It is therefore not surprising that TTF-1 mRNA was detected in several differentiated cell lines including mouse MLE-15 and human H441-4 adenocarcinoma cells that express surfactant proteins. Surprisingly, TTF-1 mRNA was also detected in a small cell carcinoma cell line H345 and was detected in 12 of 12 human small cell carcinomas in clinical pathology specimens, (^3)a tumor type that generally does not express surfactant proteins. TTF-1 mRNA was not present in HeLa, 3T3 fibroblasts, or human pulmonary adenocarcinoma cell line A549 that does not express surfactant proteins. In the present study, TTF-1 was also detected in the human fetal thyroid epithelium by immunohistochemistry (data not shown), consistent with previous findings in the rat thyroid and in thyroid carcinoma cells wherein TTF-1 is known to activate thyroperoxidase and thyroglobulin synthesis (1, 2, 4) . Surfactant proteins are not detected in the thyroid gland or in thyroid carcinoma cells, suggesting that in addition to TTF-1, other factors must further distinguish lung and thyroid-specific gene transcription. Whether thyroid and lung cell specificity is mediated by differences of the binding sites for TTF-1 or by the interactions of TTF-1 with other transcriptional proteins (for example, PAX-8 in the thyroid) is unclear at present.

The start of transcription and the proximal 5`-flanking region of the TTF-1 gene were highly conserved in the rat and human. Lung-selective expression of luciferase reporter gene was conferred by the 5`-flanking regions in the TTF-1-luciferase constructs when transfected into cells in vitro. The 5`-flanking region of the human TTF-1 gene contains a TATA-like element, located approximately -30 base pairs from the start of transcription. The sequence at the start of transcription located at -196 base pairs from the ATG is similar to that recently reported for the TTF-1 gene in the rat(17) . The coding region of the human TTF-1 gene predicts a polypeptide of 371 amino acids (38.6 kDa). The size of the major human TTF-1 mRNA (approximately 2.3 kb) is similar to that of rat lung, mouse, and human pulmonary adenocarcinoma cells. The 5`-flanking region of the human and rat TTF-1 genes is also strongly conserved, sharing 98% identity in the region -142 to +103 base pairs relative to the start of transcription(17) . These findings are consistent with the shared temporal-spatial distribution of TTF-1 gene expression in both human and rat thyroid and lung. TTF-1 is also expressed in the rat diencephalon; however, in the present study we could not assess its distribution in the fetal human brain.

While the 5`-flanking region of the human TTF-1 gene was expressed at much higher levels in pulmonary adenocarcinoma cells, significant expression, albeit at lower levels, was also detected in 3T3 cells. The finding that elements between 2.7 kb and the start of transcription conferred higher level expression in lung cells than in 3T3 cells supports the presence of elements stimulating activity in lung cells in that region. Binding and activation of TTF-1 expression by Hox 2.7 was recently described by Guazzi et al.(17) . This region of the rat TTF-1 gene has been nearly completely conserved in the human gene. Hox 2.7 is expressed in the developing lung(7) ; however, its precise colocalization and potential role in TTF-1 expression have not been discerned. The 5`-region of the TTF-1 gene also contained elements consistent with SP-1 binding as well as potential sites for binding of TTF-1 to its own promoter. Whether the TTF-1 interacts with these elements to modulate its own expression is unclear. The finding that TTF-1-luciferase constructs were expressed at low levels in a non-lung cell supports the concept that critical lung-specific elements may not be included in the 2.7-kb TTF-1 promoter constructs or that cell-specific function of the TTF-1 gene is further influenced by the context of the sites that distinguish the endogenous TTF-1 gene locus but are lacking from the TTF-luciferase constructs tested.

In conclusion, the strong conservation of the TTF-1 gene and polypeptide among species, its temporal and spatial distribution in the developing respiratory epithelium, and the lung cell-selective expression of TTF-1-luciferase constructs support the important role of this nuclear transcription factor in lung differentiation and gene expression. The abundance of TTF-1 in alveolar Type II cells is consistent with its role in the modulation of surfactant protein gene expression and surfactant homeostasis that is critical to postnatal adaptation to air breathing.


FOOTNOTES

*
This work was supported by Center for Gene Therapy Grant HL51832, Center of Excellence in Molecular Biology Grant HL41496, the Cystic Fibrosis Foundation (RDP Center), and National Institutes of Health Grant HL14214 SCOR (to M. T. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U19816[GenBank].

§
To whom correspondence should be addressed: Children's Hospital Medical Center, Division of Pulmonary Biology, TCHRF, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Tel.: 513-559-4830; Fax: 513-559-7868.

(^1)
The abbreviations used are: TTF-1, thyroid transcription factor-1; SP, surfactant protein; CCSP, Clara cell secretory protein; kb, kilobase(s).

(^2)
P. H. G. Van Renterhen, G. Vassant, and D. Christophe, GenBank accession number X77910[GenBank].

(^3)
P. A. Bejarano, R. P. Baughman, P. W. Biddinger, M. A. Miller, C. Fenoglio-Preiser, and J. A. Whitsett, unpublished observations.


ACKNOWLEDGEMENTS

The rat TTF-1 cDNA and TTF-1 antibody was kindly provided by Dr. R. Di Lauro. We acknowledge the assistance of Ann C. Maher, the technical assistance of Sandra Olson and Sherri Profitt with immunohistochemistry, and Dr. Mary E. Gray and Dr. Susan E. Wert for interpretation of TTF-1 distribution.


REFERENCES

  1. Guazzi, S., Price, M., De Felice, M., Damante, G., Mattei, M., and Di Lauro, R. (1990) EMBO J. 9, 3631-3639 [Abstract]
  2. Civitareale, D., Lonigro, R., Sinclair, A. J., and Di Lauro, R. (1989) EMBO J. 8, 2533-2542
  3. Price, M., Lazzaro, D., Pohl, T., Mattei, M., Rüther, U., Olivo, J. C., Duboule, D., and Di Lauro, R. (1992) Neuron 8, 1-20
  4. Lazzaro, D., Price, M., De Felice, M., and Di Lauro, R. (1991) Development 113, 1093-1104 [Abstract]
  5. Bohinski, R. J., Di Lauro, R., and Whitsett, J. A. (1994) Mol. Cell. Biol. 14, 5671-5681 [Abstract]
  6. Glasser, S. W., Korfhagen, T. R., Wert, S. E., and Whitsett, J. A. (1994) Am. J. Physiol. 267, L489-L497
  7. Sham, M. H., Hunt, P., Nonchev, S., Papalopulu, N., Graham, A., Boncinelli, E., and Krumlauf, R. (1992) EMBO J. 11, 1825-1836 [Abstract]
  8. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299 [Medline] [Order article via Infotrieve]
  9. Wikenheiser, K. A., Vorbroker, D. K., Rice, W. R., Clark, J. C., Bachurski, C. J., Oie, H. K., and Whitsett, J. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11029-11033 [Abstract]
  10. Rosenthal, N. (1987) Methods Enzymol. 152, 704-720 [Medline] [Order article via Infotrieve]
  11. MacGregor, G. R., Nolan, G. P., Fiering, S., Roederer, M., and Herzenberg, L. A. (1989) Methods Mol. Biol. 7, 1-19
  12. Sternberger, L. A. (ed) (1979) Immunocytochemistry , 2nd Ed., pp. 104-114, John Wiley & Sons, Inc., New York
  13. Khoor, A., Gray, M. E., Hull, W. M., Whitsett, J. A., and Stahlman, M. T. (1993) J. Histochem. Cytochem. 41, 1311-1319 [Abstract/Free Full Text]
  14. Khoor, A., Stahlman, M. T., Gray, M. E., and Whitsett, J. A. (1994) J. Histochem. Cytochem. 42, 1187-1199 [Abstract/Free Full Text]
  15. Singh, G., Singh, J., Katyal, S. L., Brown, W. E., Kramps, J. A., Paradis, I. L., Dauber, J. H., Macpherson, T. A., and Squeglia, N. (1988) J. Histochem. Cytochem. 36, 73-80 [Abstract]
  16. Damante, G., and Di Lauro, R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5388-5392 [Abstract]
  17. Guazzi, S., Lonigro, R., Pintonello, L., Boncinelli, E., Di Lauro, R., and Mavilio, F. (1994) EMBO J. 13, 3339-3347 [Abstract]
  18. Wert, S. E., Glasser, S. W., Korfhagen, T. R., and Whitsett, J. A. (1993) Dev. Biol. 156, 426-443 [CrossRef][Medline] [Order article via Infotrieve]

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