Overexpression of lunatic fringe does not affect epithelial cell differentiation in the developing mouse lung
Minke van Tuyl,1,2
Freek Groenman,1,2
Maciek Kuliszewski,1
Ross Ridsdale,1
Jinxia Wang,1
Dick Tibboel,2 and
Martin Post1
1Lung Biology Research Program, Hospital for Sick Children Research Institute, University of Toronto, Toronto, Canada; and 2Department of Pediatric Surgery, Sophia Children's Hospital, Erasmus Medical Centre, Rotterdam, The Netherlands
Submitted 1 July 2004
; accepted in final form 4 December 2004
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ABSTRACT
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The Notch/Notch-ligand pathway regulates cell fate decisions and patterning in various tissues. Several of its components are expressed in the developing lung, suggesting that this pathway is important for airway cellular patterning. Fringe proteins, which modulate Notch signaling, are crucial for defining morphogenic borders in several organs. Their role in controlling cellular differentiation along anterior-posterior axis of the airways is unknown. Herein, we report the temporal-spatial expression patterns of Lunatic fringe (Lfng) and Notch-regulated basic helix-loop-helix factors, Hes1 and Mash-1, during murine lung development. Lfng was only expressed during early development in epithelial cells lining the larger airways. Those epithelial cells also expressed Hes1, but at later gestation Hes1 expression was confined to epithelium lining the terminal bronchioles. Mash-1 displayed a very characteristic expression pattern. It followed neural crest migration in the early lung, whereas at later stages Mash-1 was expressed in lung neuroendocrine cells. To clarify whether Lfng influences airway cell differentiation, Lfng was overexpressed in distal epithelial cells of the developing mouse lung. Overexpression of Lfng did not affect spatial or temporal expression of Hes1 and Mash-1. Neuroendocrine CGRP and protein gene product 9.5 expression was not altered by Lfng overexpression. Expression of proximal ciliated (
-tubulin IV), nonciliated (CCSP), and distal epithelial cell (SP-C, T1
) markers also was not influenced by Lfng excess. Overexpression of Lfng had no effect on mesenchymal cell marker (
-sma, vWF, PECAM-1) expression. Collectively, the data suggest that Lunatic fringe does not play a significant role in determining cell fate in fetal airway epithelium.
Notch signaling pathway; lung development; epithelial differentiation
PATTERN FORMATION during embryonic development depends largely on specific cell-cell interactions and the regulated expression of numerous signaling molecules. The exact coordination of pattern formation within the developing lung remains an enigma. During early respiratory development, trachea and lung primordia are determined by interactions between the foregut endoderm and surrounding splanchnic mesenchyme (50). Later during lung development, epithelial-mesenchymal interactions program branching morphogenesis of the primitive respiratory epithelium as well as cell growth and differentiation (30, 48). Some of the morphogenic signaling pathways in the lung have been identified (53, 54), while others remain to be discovered.
The Notch signaling pathway is an evolutionary conserved pathway involved in cell fate control through cell-cell interactions (3). The generally accepted function of Notch signaling is to inhibit differentiation to prevent two neighboring cells from taking the same fate, thereby creating heterogeneity between cells (3). Notch is a transmembrane receptor that is activated through direct contact with a cell-surface ligand on a neighboring cell (37). Two vertebrate transmembrane ligands have been described, Delta and Jagged. Vertebrate CBF1/rJBk (suppressor of Hairless in Drosophila) is a downstream transcription factor in the Notch signaling pathway, repressing the genes of the Enhancer of split locus, Hes1 and Hes5 (3, 20). Both Hes1 and Hes5 encode basic helix-loop-helix (bHLH) proteins that are upregulated in response to Notch activation and subsequently affect downstream targets. One of the targets is the Achaete-Scute complex (Mash-1 in vertebrates), which is involved in the segregation of neuronal and epidermal lineages (3). In flies, fringe has been identified as a secreted signaling protein with a key role in dorsal-ventral aspects of wing formation (16). There is genetic and biochemical evidence that the fringe gene in flies modulates the Notch signaling pathway at the extracellular level (40, 57). At the posttranscriptional level, fringe protein positively regulates signaling via Delta and negatively regulates signaling via Serrate in the wing imaginal disc of flies (40). In vertebrates, three fringe genes have been identified: Manic, Lunatic, and Radical fringe (7, 21, 26).
Notch signaling has been implicated in epithelial-mesenchymal interactions guiding tooth and kidney development (31, 34). In Drosophila, the Notch signaling pathway is essential for the development of the tracheal system (29). In zygotic Notch null mutants, tracheal cells are converted into neuroblasts, leaving only rudimentary branches with abnormalities in fusion and terminal branching, implying that Notch signaling is required for tracheal fusion and terminal branching (29). Recent studies have demonstrated that several members of the Notch signaling pathway are expressed in the developing mouse (24, 42) lung. Together, these data suggest that the Notch signaling pathway may be important for mammalian lung development. However, the exact role of Notch signaling and its regulation during lung morphogenesis is unknown. In this study we investigated spatial and temporal expression of Notch-regulated basic bHLH genes such as Hes1 and Mash-1. Both genes have been implicated in pulmonary neuroendocrine development (18). Because Notch signaling is modulated by fringe (35), we also investigated whether overexpression of Lunatic fringe (Lfng) in the distal epithelium of the developing mouse lung would affect lung development.
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MATERIALS AND METHODS
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Transgene construction.
The SPC-Lfng vector was constructed using the full-length mouse Lfng cDNA (generous gift of Dr. S. Egan, Hospital for Sick Children, Toronto, ON, Canada). The 1.2-kb Lfng cDNA was subcloned 3' of the 3.7-kb human surfactant protein C (SP-C) promoter (25) and 5' of the SV40 small T intron and polyadenylation sequences. The expression cassette was excised with NdeI and NotI, purified using Glass Milk (Gene Clean Kit Bio101, BioCan) and Elutip-D columns (Schleier and Schuell), and ethanol precipitated.
Production of transgenic mice.
Transgenic embryos were generated according to Hogan et al. (14). DNA injections into the pronuclei of (C57BL/6 x SJL) F2 embryos were carried out at a concentration of 3 ng/µl. The genotype was established by PCR analysis of genomic DNA extracted from the embryonic tail and confirmed by Southern blot analysis. The primers used were 5'-TCACCTCTGTCCCCTCTCCCTAG-3' and 5'-TGGGCCGAGGAGCAGTTGGTGAGC-3'. Annealing temperature was 62°C, and 35 cycles were used for amplification.
Probes.
The murine cDNA used as template for riboprobe generation was a 0.9-kb Hes1 clone and a 2.8-kb full-length Mash-1 clone (provided by S. Egan) and PCR cloned fragments for SP-C (330 bp), CC10 (315 bp), and Lfng (756 bp). Riboprobes were digoxigenin (DIG)-labeled according to a protocol provided by the manufacturer (Roche, Montreal, Qc, Canada).
Whole mount in situ hybridization.
Whole mount in situ hybridization was performed essentially as described before (45). Lungs were dissected from E11.5E13.5 control embryos and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at 4°C. Lungs were washed in PBS-T (PBS containing 0.1% Tween 20), dehydrated in graded series of methanol in PBS-T, and stored in 100% methanol. After rehydration, lungs were bleached in 6% H2O2, treated with proteinase K (20 µg/ml), postfixed in 4% PFA and 0.2% glutaraldehyde, and prehybridized for 1 h at 70°C. Lungs were then hybridized with the appropriate DIG-labeled riboprobe (1 µg/ml) overnight at 70°C. After washes in 50% formamide, 5x SSC, pH 4.5, and 1% SDS at 70°C followed by 50% formamide, 2x SSC, pH 4.5, at 65°C, and Tris-buffered saline (TBS)-T (TBS containing 1% Tween 20) at room temperature, lungs were preblocked with sheep serum in TBS-T and subsequently incubated with anti-DIG alkaline phosphatase 1:5,000 in blocking solution (Roche) at 4°C. The next day, lungs were washed in PBS-T followed by washes in NTM-T (100 mM NaCl, 100 mM Tris, pH 9.5, 50 mM MgCl2, and 0.1% Tween) and then incubated with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) chromogen (Roche) at room temperature until a purple color appeared. After color development, lungs were washed in NTM-T and PBS-T, dehydrated in a graded series of methanol in PBS-T, and stored at 4°C in PBS-T. Pictures were taken with a Leica digital imaging system.
Tissue section in situ hybridization.
Mouse embryos were obtained from timed-pregnant mice [morning of plug is designated as embryonic day 0.5 (E0.5)]. Whole embryos and isolated lungs were fixed in 4% PFA in PBS at 4°C for 418 h, dehydrated in ethanol, and embedded in paraplast. Sections of 12 µm were cut and mounted on Superfrost slides (Fisher Scientific, Unionville, ON, Canada). The lungs were then assayed for nonradioactive RNA in situ hybridization according to Ref. 36. Briefly, after dewaxing and rehydrating, tissue sections were permeabilized with proteinase K (20 µg/ml), postfixed in 4% PFA/0.2% glutaraldehyde, and prehybridized for 1 h at 70°C. The sections were hybridized overnight at 70°C with DIG-labeled riboprobes for Lfng, SP-C, Clara cell secretory protein (CCSP), Hes1, or Mash-1 (1 µg/ml). The next day, sections were washed in 50% formamide in 2x SSC, pH 4.5, at 65°C followed by PBS-T washes. Subsequently, the sections were incubated with anti-DIG alkaline phosphatase 1:1,000 in blocking solution (Roche) at 4°C. The next day, sections were washed in PBS-T followed by washes in NTM (100 mM NaCl, 100 mM Tris, pH 9.5, 50 mM MgCl2) and then incubated with NBT/BCIP (Roche) at room temperature until a purple color appeared (45 h). All slides for one probe were stopped at the same time to make comparison over different stages of development possible. After color development, sections were washed in distilled water, dehydrated in a graded series of ethanol and xylene, and mounted with coverslips using Permount (Fisher Scientific). Pictures were taken with a Leica digital imaging system.
Immunocytochemistry.
E11.518.5 SPC-Lfng transgenic and wild-type control embryos were killed, and lungs were dissected, fixed overnight in 4% PFA in PBS, dehydrated, and embedded in paraplast. Immunostaining was performed by the avidin-biotin (ABC) immunoperoxidase method as described by Hsu et al. (15). Seven-micrometer sections were deparaffinized and rehydrated in a graded series of ethanol. Antigen retrieval was achieved with heating in sodium citrate, pH 6.0. Sections were washed in PBS containing 0.1% Triton and endogenous peroxidase was blocked in 2% H2O2 in methanol. Sections were incubated at 4°C overnight with either mouse monoclonal anti-rat Ttf-1 (Labvision/Neomarkers, Fremont, CA) [1:50], hamster monoclonal anti-mouse T1
(hybridoma #8.1.1, Developmental Studies Hybridoma Bank, University of Iowa) [1:50], rat monoclonal anti-mouse CD31 [platelet endothelial cell adhesion molecule (PECAM)-1; BD Biosciences Pharmingen, Mississauga, ON, Canada] [1:60], rabbit polyclonal antifactor VIII-related antigen (Labvision/Neomarkers) [1:80], mouse monoclonal anti-mouse
-smooth muscle actin (
-sma, Labvision/Neomarkers) [1:1,000], mouse monoclonal anti-
-tubulin IV (Biogenix, San Ramon, CA) [1:100], polyclonal rabbit anti-human protein gene product (Pgp) 9.5 (UltraClone, Wellow, UK) [1:10,000], polyclonal rabbit anti-rat CGRP (Sigma, St. Louis, MO) [1:3,000], or polyclonal goat Lnfg (Santa Cruz Biotechnology, Santa Cruz, CA) [1: 400] antibody, all diluted in blocking solution (5% normal goat serum and 1% BSA in PBS). Sections were subsequently incubated with biotinylated secondary antibodies, and color detection was performed according to instructions in the Vectastain ABC and diaminobenzidine (DAB) kit (Vector Laboratories, Burlingame, CA). For Ttf-1, nickel was added to the DAB solution to enhance black nuclear staining. Sections were counterstained with hematoxylin, except for Ttf-1, which was counterstained with fast red, and mounted in Permount (Fisher Scientific). Digital images were captured with a Leica microscope and a Leica imaging system.
Transmission electron microscopy.
Lung tissue removed from fetuses was rinsed in 1 U/ml heparin in PBS to remove blood, minced in
1-mm pieces. The tissue was fixed for 1 h in 4% (wt/vol) PFA and 1% (wt/vol) glutaraldehyde in PBS. Tissues were then rinsed three times in PBS and exposed to 1% (wt/vol) osmium tetroxide for 1 h followed by another three rinses with PBS. The samples were then dehydrated through an ascending alcohol series ending in propylene oxide. Propylene oxide was then exchanged with an increasing concentration of Epon (Marivac, St. Laurent, Qc, Canada) until the samples were fully infiltrated with 100% Epon. Samples were placed in molds, and the Epon was polymerized at 70°C overnight. Ultrathin sections of the resulting blocks were cut with a diamond knife on a Reichert Ultracut microtome and placed onto 400-mesh copper grids. All samples were stained 10 min in 3% (wt/vol) uranyl acetate in double-distilled water and 5 min in 1% (wt/vol) lead citrate followed by double wash distilled water to remove excess stain. Samples were examined on a Philips 430 electron microscope.
RNA isolation and real-time RT-PCR.
Lungs from E15.5 and E16.5 SPC-Lfng transgenic and wild-type embryos were dissected in ice-cold PBS, snap-frozen in liquid nitrogen, and stored at 70°C. Total RNA was extracted with the RNA easy kit (Qiagen, Mississauga, ON, Canada). One microgram of total RNA was reverse transcribed in a total volume of 50 µl with random hexamers (Applied Biosystems, Foster City, CA). The resulting templates (50 ng of cDNA for our target genes and 5 ng for 18S) were quantified by real-time PCR (ABI Prism 7700). Primers and TaqMan probes for Lfng, Hes1, and Mash-1 were purchased from ABI as Assays-on-Demand for murine genes. For each probe a dilution series determined the efficiency of amplification of each primer/probe set and the relative quantification method employed (28). All measurements were performed in triplicate. For the relative quantitation, PCR signals were compared between groups after normalization with 18S as an internal reference. Relative expression was calculated as 2(Ctgene of interestCt18S). Fold change was calculated according to Livak and Schmittgen (28). For comparison between two groups we used Student's t-test. P values <0.05 were considered significant.
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RESULTS
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Endogenous Lfng expression in murine lung.
Whole mount in situ hybridization demonstrated Lfng expression in E11.5 pulmonary epithelium of proximal airways (Fig. 1A). The trachea, stem bronchi, and lobar bronchi displayed equal intensity of expression. At E12.5, Lfng showed a similar expression pattern, i.e., expression in the epithelial lining cells of trachea and lobar bronchi (Fig. 1B). No expression was noted in the mesenchyme that surrounded the developing airways. Similar results have been reported by Post et al. (42). Whole mount in situ hybridization has limited discrimination value in older tissue, and, therefore, we performed section in situ hybridization for lungs older than E13.5. We were unable to detect any Lfng mRNA in E15.5 and E18.5 lungs (Fig. 1, C and E, respectively), suggesting low Lfng expression at later stages of lung development.

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Fig. 1. Lunatic fringe (Lfng) mRNA expression in wild-type (A-C, E) and SPC-Lfng (D, F) lungs. Whole mount in situ hybridization showed Lfng mRNA expression in the endodermal layer of the trachea and proximal airways of embryonic day (E) 11.5-E12.5 lungs (A, B). At later (E15.5 and E18.5) gestation, Lfng mRNA expression was undetectable by section in situ hybridization (C, E). In contrast, Lfng mRNA was rapidly detected by section in situ hybridization in the epithelial cells of distal airways of transgenic SPC-Lfng lungs (D and F for E15.5 and E18.5, respectively). Blue-purple color is positive staining. Bar: 100 µm in A and CF, 250 µm in B.
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Lfng expression in SPC-Lfng transgenic lungs.
To establish a role for Lfng in lung development, we overexpressed Lfng in distal lung epithelium using the human SP-C enhancer/promoter (55). In situ hybridization using an antisense DIG-labeled Lfng probe demonstrated strong Lfng expression in E15.518.5 transgenic lungs in an identical pattern as seen for endogenous SP-C mRNA (Fig. 1, D and F). As mentioned in the previous section, hardly any endogenous Lfng transcripts were detected in the wild-type E15.5 and E18.5 lungs (Fig. 1, C and E). No expression was detected when a control sense DIG-labeled Lfng probe was used (not shown). These results clearly demonstrate that Lfng is overexpressed in SPC-Lfng transgenic lungs compared with wild-type lungs. Immunohistochemical analysis confirmed the overexpression Lfng at the protein level (Fig. 2). Overexpression of Lfng in distal lung epithelial cells caused no embryonic or postnatal lethality; neither were the transgenic mice in obvious respiratory distress. No differences were observed in body or lung weights between wild-type and SPC-Lfng transgenic littermates. Hematoxylin/eosin-stained sections revealed no obvious differences in gross morphology between SPC-Lfng and wild-type lungs at E15.5 and E18.5 (not shown).

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Fig. 2. Immunohistochemical staining for Lfng in wild-type and SPC-Lfng transgenic lungs. Lfng immunoreactivity (brown color) was stronger in transgenic SPC-Lfng lungs (B, D) relative to wild-type control lungs (A, C). Lfng localized to the airway epithelium at E15.5 (A, B) but was mainly confined to the distal epithelial cells at E17.5 (B, D) lungs. Cells were counterstained with hematoxylin. Bar: 100 µm.
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Expression of Hes1 in control and SPC-Lfng transgenic lungs.
Hes1 plays a significant role in the Notch signaling pathway and is transcriptionally activated by Notch signaling (19, 20). Hes1 encodes a bHLH transcriptional repressor (47), capable of counteracting the activity of bHLH transcriptional activators (6, 39). Therefore, we first investigated expression of Hes1 in the developing murine lung. Whole mount in situ hybridization showed that Hes1 was predominantly expressed in the airway epithelium during early lung development (Fig. 3, A and B; E11.5 and E13.5 lungs, respectively). Some Hes1 expression was found in the mesenchyme of early embryonic lungs (Fig. 3, A and B; E11.5 and E13.5, respectively). Section in situ hybridization confirmed the epithelial localized expression pattern of Hes1 mRNA in early lung development with enhanced expression in larger airways and marginal expression in surrounding mesenchyme (Fig. 3, C and D; E11.5 and E15.5, respectively). At later gestation, Hes1 mRNA expression was confined to the epithelial lining cells of smaller airways (Fig. 3E, E16.5 lungs) and terminal bronchioles (Fig. 3F, E17.5 lungs). A similar expression of Hes1 in pulmonary nonneuroendocrine cells has been reported by Ito et al. (18). We then determined whether overexpression of Lfng would affect Hes1 expression. Section in situ hybridization showed that Hes1 was equally expressed in the bronchiolar epithelium of both wild-type (Fig. 3G) and SPC-Lfng transgenic lungs (Fig. 3H).

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Fig. 3. Hes1 mRNA expression in wild-type and SPC-Lfng lungs. Whole mount in situ hybridization showed that Hes1 mRNA was predominantly expressed in early (E11.5E13.5) airway epithelium, whereas some Hes1 expression was detected in the mesenchymal compartment (A, B). Section in situ hybridization confirmed the epithelial localized expression pattern of Hes1 mRNA in early (E11.5E15.5) lung development with enhanced expression in larger airways and weak expression in surrounding mesenchyme (C, D). At later gestation, Hes1 mRNA expression was confined to the epithelial lining cells of smaller airways (E, E16.5 lungs) and terminal bronchioles (F, E17.5 lungs). Section in situ hybridization of E17.5 lungs showed no difference in Hes1 expression between wild-type (G) and SPC-Lfng lungs (H). Blue-purple color is positive staining. Sections (CH) were counterstained with methyl green. Bar: 100 µm in A and CF, 250 µm in B, 40 µm in G and H.
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Expression of Mash-1 in control and SPC-Lfng transgenic lungs.
The proneural gene Mash-1 is a bHLH factor that activates neural differentiation in the ectoderm. In E18.5 Hes1-deficient mice, Mash-1 is significantly upregulated, suggesting that Hes1 represses Mash-1 (18). We investigated Mash-1 mRNA expression in control and SPC-Lfng transgenic lungs using section in situ hybridization. Mash-1-positive cells were detected in the mesenchyme surrounding the trachea and esophagus of E11.5 control mice (Fig. 4A). This specific pattern of expression most likely represents the developing neural progenitors invading the mesenchyme around the esophagus and trachea (12, 52). The cells are likely neural crest cells that in E11.5 mouse lungs have been found on the trachea as well as in the vagus and in processes extending from the vagus into the lung (52). Mash-1-positive cells within the lung were first seen at E13.5 (Fig. 4B). At this stage, Mash-1 was expressed in cell clusters or single cells among epithelial cells lining the larger airways. No Mash-1 expression was detected in the periphery of the lung (Fig. 4B). At E16.5 (Fig. 4C) and E17.5 (Fig. 4D), Mash-1-positive cell clusters were detected in larger airways, typically at branch points, suggestive of pulmonary neuroendocrine cells (PNECs). This proximal distal wave of PNEC development agrees with previously published immunohistochemical studies (8, 17, 49). At E15.5, SPC-Lfng transgenic lungs had a similar number of Mash-1-positive clusters and cells as control lungs (Fig. 4, F vs. E). Additional markers for PNECs were also normally expressed in both wild-type and SPC-Lfng transgenic lungs. Both CGRP (Fig. 5, A and B) and Pgp9.5 (Fig. 5, C and D) were expressed in PNECs of large airways of both wild-type (Fig. 5, A and C) and SPC-Lfng transgenic lungs (Fig. 5, B and D).

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Fig. 4. Mash-1 mRNA expression in wild-type and SPC-Lfng lungs using section in situ hybridization. Mash-1-positive cells were detected in the mesenchyme surrounding the trachea and esophagus of E11.5 wild-type lungs (A). This specific pattern of expression most likely represents the developing neural progenitors invading into the mesenchyme around the esophagus and trachea. Mash-1-positive cells within the lung were first seen at E13.5 (B). At this stage, Mash-1 was expressed in cell clusters or single cells among epithelial cells lining the larger airways. No Mash-1 expression was detected in the periphery of the lung. At E16.5 (C) and E17.5 (D), Mash-1-positive cell clusters were detected in larger airways, typically at branch points, suggestive of pulmonary neuroendocrine cells (PNECs). At E15.5, SPC-Lfng transgenic lungs (F) had a similar number of Mash-1-positive clusters and cells as control lungs (E). Blue-purple spots are positive staining PNECs. Sections were counterstained with methyl green. Bar: 100 µm.
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Fig. 5. Immunohistochemical staining for CGRP (E18.5) and protein gene product 9.5 (Pgp9.5, E17.5) in wild-type and SPC-Lfng transgenic lungs. Both markers for PNECs (arrows) were normally expressed in larger airways of both wild-type (A, C) and SPC-Lfng transgenic lungs (B, D). Dark brown spots are positive staining PNECs. Sections were counterstained with hematoxylin. Bar: 40 µm in A and B, 50 µm in C and D.
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Quantitative RT-PCR for Lfng, Hes1, and Mash-1.
To quantify Lfng overexpression we performed real-time RT-PCR. At E15.5, Lfng mRNA levels were significantly (more than ninefold) increased in SPC-Lfng transgenic lungs compared with lungs of wild-type littermates (Fig. 6A). The increase in Lfng mRNA expression in the transgenic lungs was even more pronounced (>44-fold) at E16.5 (Fig. 6A). Neither Hes1 nor Mash-1 mRNA expression was altered in SPC-Lfng lungs compared with control lungs (Fig. 6B), in agreement with the qualitative in situ hybridization results.

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Fig. 6. Quantitative assessment of Lfng, Hes1, and Mash-1 mRNA expression in wild-type and transgenic lungs. Real-time RT-PCR showed dramatic increased Lfng expression in E15.5E16.5 SPC-Lfng lungs (A). Hes1 and Mash-1 expression, both downstream components of the Notch signaling pathway, were unchanged in SPC-Lfng transgenic lungs (B). Data are means ± SD, n = 4 separate experiments. *P < 0.05.
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Expression of Ttf-1, CCSP, SP-C, T1
, and
-tubulin IV in control and SPC-Lfng transgenic lungs.
To investigate whether overexpression of Lfng affected lung cell differentiation along the proximal-distal axis, we immunohistochemically analyzed the expression of Ttf-1, T1
, SP-C, CCSP, and
-tubulin IV in control and SPC-Lfng lungs. Ttf-1 is normally expressed in epithelial cells of the trachea and bronchial tubules and is required for lung formation (33, 59). Moreover, Ttf-1 regulates the expression of pulmonary genes like SP-B and CCSP (56) and works synergistically with GATA-6 to influence the activity of the SP-C promoter (27). In the control littermates, at E17.5, Ttf-1 was detected in the bronchial epithelium and in the epithelial cells lining the peripheral acinar tubules and buds (Fig. 7A). In the lungs of SPC-Lfng transgenic mice, Ttf-1 immunoreactivity was found in a similar pattern as control littermates (Fig. 7B). Likewise, SP-C mRNA (Fig. 7, C and D) and CCSP mRNA (Fig. 7, E and F), markers of distal (type II) and proximal (Clara) pulmonary epithelial cells, respectively, showed a similar pattern of expression in E18.5 SPC-Lfng transgenic and wild-type lungs. Also, T1
, a marker for distal type I epithelial cells (44), was equally expressed in lungs of E17.5 control and SPC-Lfng transgenic mice (Fig. 7, G and H). Immunostaining for
-tubulin IV, a marker for ciliated cells, was not different between lungs of SPC-Lfng transgenics and control littermates (Fig. 8, A vs. B). To investigate whether overexpression of Lnfg had any ultrastructural effects on epithelial cell differentiation, E17.5 mouse lungs were fixed, processed, and examined by transmission electron microscopy (TEM). TEM did not reveal any significant differences in upper airway cell differentiation between control and SPC-Lfng transgenic mice (Fig. 8, C and E vs. D and F).

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Fig. 7. Immunohistochemical staining for Ttf-1 and T1 (E17.5) and in situ hybridization for surfactant protein C (SP-C) and Clara cell secretory protein (CCSP) mRNA (E18.5) in wild-type and transgenic lungs. Expression of the distal type II cell markers Ttf-1 and SP-C was not different between SPC-Lfng (B and D, respectively) and wild-type (A and B, respectively) lungs. Similarly, expression of the distal type I cell marker T1 (G, H) and proximal epithelial Clara cell marker CCSP (E, F) were unaltered in the SPC-Lfng transgenic lungs (H, F) compared with the wild-type (G, E) lungs. Black (A, B), blue-purple (CF), and brown (G, H) staining is positive. Sections were counterstained with fast red (A, B) and hematoxylin (G, H). Bar: 50 µm in A and B, 100 µm in CH.
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Expression of von Willebrand factor and
-sma in control and SPC-Lfng transgenic lungs.
To explore whether Lfng overexpression would influence endothelial and smooth muscle cell development, we immunohistochemically analyzed the expression of factor VIII-related antigen [von Willebrand factor (vWF)] and
-sma. vWF is normally expressed in the endothelial cells of larger blood vessels within the lung. Immunostaining for vWF was not different between lungs of E17.5 SPC-Lfng transgenics and control littermates (Fig. 9, B vs. A). Another endothelial marker, PECAM-1/CD-31, which marks the peripheral capillary complex of the lung, was also not differently expressed in transgenic and control lungs (results not shown). Thus vascular development appears not to be affected by overexpression of Lfng. Immunopositive
-sma reactivity, a marker of smooth muscle cells, was equally detected in the mesenchyme surrounding the bronchi, bronchiolar tubules, and larger vessels of E15.5 control and SPC-Lfng lungs (Fig. 9, C vs. D), suggesting that mesenchymal smooth muscle cell differentiation was not influenced by excess of Lfng.
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DISCUSSION
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Fringe proteins that modulate the Notch signaling pathway (57) have been shown to play a crucial role in defining borders in invertebrates and vertebrates. In flies, fringe is involved in determination of the dorsal/ventral border of the Drosophila wing disc (23) and eye (41). In vertebrates, fringe genes play roles in the formation of the apical ectodermal ridge at the dorsal/ventral border in the limb bud (46), the boundary of the enamel knot (38), midline precursor cell fate (2), and in the segmentation of body plan (11, 43). Herein, we demonstrate that fringe proteins do likely not define epithelial morphogenic boundaries along the anterior-posterior axis of the airways. Overexpression of Lfng in distal lung epithelial cells did not influence the expression of downstream bHLH factors, such as Hes1 and Mash-1. Moreover, epithelial differentiation of distal and proximal airways as assessed by cell marker analysis was not altered by overexpression of Lfng.
Several Notch signaling components, including Notch1, Notch2, and Notch3 receptors and their ligands, Jagged-1, Jagged-2, and Dll1, have been reported to be expressed in the developing murine lung (24, 42), suggesting a role for this conserved signaling pathway in lung development. In early murine lung, Notch1 and Dll1 are expressed within the distal respiratory epithelium (42). Notch2 and Notch3 are expressed throughout the mesenchyme (42), whereas Jagged-1 is found in undifferentiated distal epithelial and mesenchymal cells (24). Jagged-2 is present in epithelial and differentiated mesenchymal cells, but not in airway-associated smooth muscle cells (24). Also, Notch1 and Jagged-1 were localized on endothelial cells lining the lung vasculature (51). Antisense gene silencing experiments in murine lung explants have revealed functional diversity of Notch and Jagged family members during development with only partial redundancy (24). Overexpression of the activated domain of Notch3 throughout the peripheral lung epithelium caused abnormal tubular formation with diffuse metaplasia and perinatal death (9). Type I alveolar epithelial cells did not develop in these lungs (9). In agreement with previous findings (42) we found that Lfng is solely expressed in the epithelial cells lining the airways. Interestingly, Lfng expression was absent at the time of pulmonary epithelial cell diversification suggesting a potential role in this process. However, overexpression of Lfng in the developing lung did not influence proximal and distal epithelial cell differentiation.
Lfng has high homology to bacterial glycosyltransferases and acts in the Golgi (5). Lfng makes a complex with the Notch receptor before secretion to the cell-surface, thereby potentiating Dll-induced Notch signaling, while inhibiting Jagged-induced Notch signaling (13, 22). Thus Lfng need to be present in the same cell as the Notch receptor to have an effect on Notch signaling. In the present study, the SP-C promoter directed Lfng expression toward distal lung endoderm, which is same tissue layer where Notch1 is expressed (42). Antisense knockdown experiments have implicated Notch1 in regulating pulmonary neuroendocrine differentiation (24), and, therefore, we speculated that Lfng excess in the same cell as Notch1 would modulate Notch signaling via bHLH transcription factors (3). We observed that Hes1 was expressed in the early lung endoderm, but with advancing gestation its expression became restricted to epithelial cells lining terminal branches of bronchioles. However, this temporal-spatial expression pattern for Hes1 was not altered by overexpression of Lfng, suggesting that Notch signaling was not affected. Hes5, another bHLH repressor regulated by Notch (10), is not present in the murine lung (18), and overexpression of Lfng did not trigger its expression (not shown). It has been shown that Hes1 binds directly to the promoter of Mash-1, a bHLH transcriptional activator, and represses Mash-1 expression (6). In the lung, Mash-1 is expressed in neuroendocrine cells (18). Neuroendocrine cells are the first cells to differentiate morphologically before any other epithelial cell within the pulmonary epithelium (8, 58). Mash-1 seems to be specifically necessary for pulmonary neuroendocrine development, since Mash-1-deficient mice lack PNECs, whereas neuroendocrine cells in other organs are unaffected (4, 18). In lungs of Hes1-deficient mice, Mash-1 is upregulated, and the number of PNECs is markedly increased (18). In the present study, we found Mash-1-positive cells localized in isolated clusters in larger airways, typically at branch points, suggestive of PNECs. Overexpression of Lfng did not affect the temporal-spatial expression of Mash-1, in agreement with the observation that Hes1 is not altered by Lfng excess. In addition, expression of pulmonary neuroendocrine markers, such as Pgp9.5 and CGRP, was undisturbed by Lfng overexpression.
In Hes1-deficient mice, the number of Clara cells appears to be reduced (18). In line with the finding that Hes1 expression was not affected by Lfng overexpression, we observed that the temporal-spatial expression pattern of CCSP, a Clara cell marker, was not altered by Lfng overexpression. Also, the temporal appearance of
-tubulin VI-positive ciliated cells was not affected by Lnfg excess. Marker analyses for distal epithelial cells and mesenchymal cells revealed no change in their phenotypic expression in Lfng overexpressing lungs. In addition, TEM confirmed that Lnfg overexpression did not affect upper airway cell differentiation.
The lack of any response to Lfng excess was not due to limited overexpression, since we found that Lfng mRNA expression was upregulated 10- to 40-fold in the E1516 SPC-Lfng mice, although Lnfg protein expression appeared to be less upregulated. Radical fringe (Rfng) has been shown to be expressed in the lung endoderm (42), and it is possible that Rfng mediates the interaction with Notch1. However, we are not aware of any functional diversity between the fringe proteins.
In conclusion, Notch regulates cell fate in many branched organs (1, 32) including the developing lung (18, 24), but contrary to other developmental systems (57), fringe proteins do not play a crucial role in setting epithelial cell boundaries along the anterior-posterior axis of the airways.
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GRANTS
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This work was supported by the David Vervat Foundation and The Netherlands (M. van Tuyl) and the Canadian Institutes of Health Research (CIHR FRN-15273, M. Post).
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ACKNOWLEDGMENTS
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We thank Wahyuni Otmodo for technical assistance, tissue preparation, and immunohistochemistry; Pooja Agarwal for assistance with whole mount in situ hybridization; and Angie Griffin for animal care and handling. M. Post is the holder of a Canadian Research Chair (tier 1) in Fetal, Neonatal and Maternal Health.
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FOOTNOTES
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Address for reprint requests and other correspondence: M. Post, Prog. in Lung Biology Research, Hospital for Sick Children Research Inst., 555 Univ. Ave., Toronto, Ontario M5G1X8, Canada (E-mail: martin.post{at}sickkids.ca)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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