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GATA6 regulates differentiation of distal lung epithelium

¹ Department of Medicine, Molecular Cardiology Research Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
² Department of Pulmonary Biology, Children’s Hospital Medical Center, Cincinnati, Ohio 45229, USA

View larger version (9K): [in a new window]   Fig. 1. Schematic of the SP-C/G6en and SP-C/en transgenic constructs. (A) The 3.7 kb human SP-C promoter/enhancer was used to drive expression of a fusion cDNA consisting of the DNA binding domain of mouse GATA6 (aa233-352) fused to the repression domain of the Drosophila Engrailed protein (aa1-298). This was followed by the SV40 poly(A)+ sequence. (B) The 3.7 kb human SP-C promoter/enhancer was also used to drive expression of the Drosophila Engrailed repression domain (aa1-298) containing a nuclear localization sequence at the amino-terminus (MKRKKK). This was followed by the SV40 poly(A)+ sequence.

View larger version (83K): [in a new window]   Fig. 2. Expression of the SP-C/G6en and SP-C/en transgenes during lung development. The SV40 poly(A)+ sequence was used as an in situ hybridization probe to detect the expression pattern of the SP-C/G6en (A-D,F,G) and SP-C/en (E) transgenes at the following time points during lung development: E17.5 (A,B), E19.5 (C-E), and P0 (F,G). There was no hybridization of the sense probe to the SV40 sequence (data not shown). Notice the restriction of transgene expression to the distal airway epithelium during lung development as has been reported previously with the human SP-C promoter (Wert et al., 1993). Scale bar: 250 µm.

View larger version (96K): [in a new window]   Fig. 3. SP-C/G6en mice display abnormal lung morphology. Histological sections through E17.5 (A-D), E19.5 (E-H,M-P), or P0 (I-L) SP-C/G6en (A-L) and SP-C/en (M-P) transgenic embryos and neonates. At E17.5, the distal airways of SP-C/G6en embryos are lined with abnormal cuboidal epithelium (D, yellow arrowheads) while the distal airways of wild-type littermates are lined primarily with squamous epithelium (C, black arrowheads). Similar morphological differences are observed at E19.5 (H, yellow arrowheads) and P0 (L, yellow arrowheads). Increased ratio of mesenchyme to airway space is also noted in SP-C/G6en embryos (compare G and H, K and L). However, SP-C/en mice display normal distal epithelial morphology and branching as in the wild-type littermates (M-P). Scale bar: 500 µm (E, F,I,J,M,N), 250 µm (A,B), 125 µm (G,H,K,L,O,P) and 67.5 µm (C,D).

View larger version (91K): [in a new window]   Fig. 4. Abnormal ultrastructure of distal airway epithelium in SP-C/G6en mice. Transmission electron microscopy was used to determine the ultrastructural morphology of the distal airway epithelial cells in SP-C/G6en and SP-C/en mice at E19.5. Wild-type littermates show normal distal airway structure including AEC-1 (A, white arrowheads) and AEC-2 (A, black arrowhead) cells lining the airways, with small blood vessels closely adjoining the thin epithelial barrier (A, asterisk). In addition, numerous interalveolar septa were observed (B, bracket). In contrast, SP-C/G6en mice had AEC-2 cells lining all of their distal airways (C-E, black arrowheads). These AEC-2 cells contained large vacuoles reminiscent of the glycogen storage vesicles observed in immature AEC-2 cells in early lung development (Ten Have-Opbroek, 1991). However, many of these cells still contained lamellar bodies (F, black arrow) and secreted surfactant was observed in the airways (F, white arrow) showing that these cells were not arrested at an earlier stage of AEC-2 differentiation. Also, SP-C/G6en mice contained normal blood vessel development underlying the distal airway epithelium suggesting that pulmonary vascular development was not perturbed (C,D, asterisks).

View larger version (48K): [in a new window]   Fig. 5. SP-C/G6en mice have increased glycogen content in the distal airway epithelium. PAS staining was performed on histological sections of E19.5 wild-type (A) and SP-C/G6en mice (B,C) with (C) or without (A,B) prior treatment with amylase. At E19.5, wild-type littermates contain little glycogen in the distal airway epithelium (A), while SP-C/G6en mice show a significant increase in glycogen as determined by PAS staining (B). Pre-treatment with amylase (which digests the glycogen and eliminates PAS staining) demonstrates the specificity of the PAS staining (C). Scale bar: 80 µm.

View larger version (75K): [in a new window]   Fig. 6. AEC-2 cell marker gene expression in SP-C/G6en mice. In situ hybridization using specific probes for TTF-1 (A-D) and SP-C (E-H) was performed on wild-type (A,C,E,G) and SP-C/G6en (B,D,F,H,I) sections from E17.5 (A,B,E-I) and E19.5 (C,D) embryos. Non-radioactive in situ hybridization was utilized for the SP-C probe to better analyze the specific expression pattern, whereas radioactive in situ hybridization was used for TTF-1. TTF-1 expression is observed throughout the airway epithelium in both wild-type (A,C) and SP-C/G6en embryos (B,D). SP-C expression, however, was reduced in SP-C/G6en mice and in some regions almost completely absent from the distal airway epithelium expressing the transgene as demarcated by the SV40 probe (compare adjacent sections in H and I). However, SP-C expression was observed in regions outside the area of transgene expression (F,H, arrowheads). There was no detectable hybridization with the sense probes for TTF-1 and SP-C (data not shown). Asterisks denote proximal airways in A-D. Scale bar: 250 µm (A-D), 125 µm (E,F), 67.5 µm (G,H,I).

View larger version (76K): [in a new window]   Fig. 7. SP-A gene expression in SP-C/G6en mice. Radioactive in situ hybridization was performed on E17.5 wild-type (A) and SP-C/G6en (B) embryos using a specific antisense riboprobe for SP-A. Note hybridization to distal airway epithelium in both wild-type and transgenic embryos (red arrowheads). Sense probe did not produce a detectable hybridization signal (data not shown). Scale bar: 67.5 µm.

View larger version (108K): [in a new window]   Fig. 8. Proximal airway development and gene expression in SP-C/G6en mice. In situ hybridization analysis was performed on wild-type (A,C,E) and SP-C/G6en (B,D,F) embryos at E17.5 (A-D) and E19.5 (E,F) using gene-specific riboprobes for Foxj1 (A,B) and CC10 (C-F). Foxj1 and CC10 are expressed at similar levels in the proximal airways of wild-type and SP-C/G6en mice (A-F). However, the number of individual airways positive for Foxj1 and CC10 hybridization was significantly reduced at both E17.5 and E19.5 (A-F, yellow arrowheads). This decrease was most extreme in the number of smaller CC10- and Foxj1-positive proximal airways while the number of larger, more central airways was similar (A-F, arrows). These data correlated with a decrease in the number of peripheral airways containing thick convoluted epithelium (G, black arrowheads) which is characteristic of proximal airway epithelium while the number of large centralized proximal airways was similar in H+E stained sections from of wild-type and SP-C/G6en E17.5 embryos (G,H, arrows). Scale bar: 250 µm (A-F), 150 µm (G,H).

View larger version (83K): [in a new window]   Fig. 9. Disrupted Aqp5 and Foxp2 gene expression in SP-C/G6en embryos. Radioactive in situ hybridization analysis was performed on wild-type (A,C,E,G,I,K), SP-C/G6en (B,D,H,J,L), and SP-C/en (F) embryos at E17.5 (A,B,G-J) and E19.5 (C-F,K,L). Aqp5 is expressed at low levels primarily in distal airway epithelium at E17.5 in wild-type embryos (A). However, Aqp5 expression is not observed at appreciable levels in the lungs of E17.5 SP-C/G6en embryos (B). By E19.5, Aqp5 is expressed at high levels in the airways of wild-type (C,E) embryos but its expression is significantly reduced in the airways of SP-C/G6en embryos (D). SP-C/en mice express normal levels of Aqp5 as compared to wild-type littermates (F). At E17.5, Foxp2 is expressed at similar levels in both wild-type (G and I) and SP-C/G6en (H,J) embryos in the distal airway epithelium. By E19.5, Foxp2 gene expression has decreased in wild-type (K) embryos but remains high in the distal airways of SP-C/G6en (L) embryos. Note the dilated nature of the distal airways in SP-C/G6en embryos as highlighted by Foxp2 gene expression at E17.5 (H,J, red arrows). Sense probes for Aqp5 and Foxp2 did not produce a detectable hybridization signal (data not shown). Asterisks denote proximal airways in A-L. Scale bar: 250 µm (A-H,K,L), 67.5 µm (G,H).

View larger version (81K): [in a new window]   Fig. 10. Northern blot analysis of Aqp5 and Foxp2 gene expression in the lungs of SP-C/G6en mice. Total RNA (10 µg) from E19.5 wild-type (lanes 1 and 2) and SP-C/G6en (lanes 3 and 4) dissected lung tissue was analyzed by northern blot analysis using radiolabeled probes specific for Aqp5, Foxp2, and the engrailed repression domain (to detect transgene expression). Bottom panel represents the ethidium bromide-stained gel before transfer showing equal loading of RNA in all the lanes. All RNAs represent individual and separate F0 E19.5 wild-type or SP-C/G6en transgenic littermates.

View larger version (16K): [in a new window]   Fig. 11. GATA6 activates the mouse Aqp5 promoter. (A) Representation of the two regions of the mouse Aqp5 promoter used in the trans-activation assays with the full length –1437 bp region containing four putative GATA DNA binding sites (ovals) and the –503 bp region lacking all four putative GATA binding sites. Homology of GATA DNA binding site 1 and site 2 between the mouse and rat Aqp5 promoters are also shown with the consensus GATA DNA binding sequence underlined. Note that site 2 is on the antisense strand. (B) NIH-3T3 cells were transfected with either the pGL3/Aqp5/–1.4 or pGL3/Aqp5/–0.5 reporter constructs, the pcDNA3 plasmid or the pcDNAG6 expression construct and the pMSVß-gal control plasmid as indicated. Background luciferase activity is normalized to either of the reporter vectors co-transfected with the pcDNA3 vector lacking the GATA6 cDNA. (C) The GATA6/En fusion protein represses GATA6-dependent trans-activation of the mouse Aqp5 promoter. Sub-maximal levels of pcDNA3G6 expression plasmid (1 µg) were transfected into NIH-3T3 cells along with increasing amounts of the pcDNA3G6/En, pcDNA3G6mut/En, or pcDNA3En expression plasmids as well as the pMSVß-gal control plasmid as indicated. Background luciferase activity was normalized to the reporter vectors co-transfected with the insertless pcDNA3 vector. Data is represented as fold activation above background and is the average of three experiments ±s.e.m.

View larger version (22K): [in a new window]   Fig. 12. Model of GATA6 function during lung development. A model for GATA6 function in AEC-2 differentiation (i.e. SP-C expression), AEC-1 differentiation (loss of squamous epithelium and Aqp5 gene expression), and proximal airway development (reduced number of proximal airways) during lung development. Lack of SP-C expression in both the SP-C/G6en embryos and in chimeric lung tissue generated from Gata6–/– ES cells shows that GATA6 is crucial for SP-C gene expression and thus for certain aspects of AEC-2 differentiation (Keijzer et al., 2001). Lack of AEC-1 cells and direct activation of the mouse Aqp5 proximal promoter in SP-C/G6en embryos indicate that GATA6 plays an important role in AEC-1 cell differentiation and gene expression (this report). Reduced number of proximal airway tubules indicates that disruption of distal airway differentiation in SP-C/G6en embryos leads to disrupted proximal airway development which correlates with a recent model of lung epithelial differentiation and development (Weaver et al., 1999). GATA6 may affect all of these processes through stage-specific requirements or by disrupting early lung epithelial differentiation, leading to affects observed later in development.