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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02720

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GATA and Nkx factors synergistically regulate tissue-specific gene expression and development in vivo

Yuzhen Zhang^1,*, Nibedita Rath^1,*, Sridhar Hannenhalli², Zhishan Wang¹, Thomas Cappola¹, Shioko Kimura³, Elena Atochina-Vasserman¹, Min Min Lu¹, Michael F. Beers¹ and Edward E. Morrisey^1,4,

¹ Department of Medicine and University of Pennsylvania, Philadelphia, PA 19104, USA.
² Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104, USA.
³ Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MA 20892, USA.
⁴ Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.

View larger version (107K): [in this window] [in a new window]   Fig. 1. Expression of Gata6 and Nkx2.1 during lung development. In situ hybridization for Gata6 (A) and Nkx2.1 (B) expression was performed at E16.5. Immunofluorescent staining for Gata6 and Nkx2.1 expression reveals extensive overlap between the expression of both of these proteins at E16.5, including in the distal airway epithelium (C-E, arrowheads) and more proximal bronchiolar airways (C-E, asterisks). Expression of Gata6 at E18.5 in proximal bronchiolar airways is confirmed by co-staining with the Clara cell marker gene CC10 (F-H, asterisks). Scale bars: 500 µm in A,B; and 100 µm in C-H.

View larger version (87K): [in this window] [in a new window]   Fig. 2. Defects in lung epithelial differentiation in G6-Nkx DH embryos. (A-P) G6-Nkx DH lungs exhibit defects in airway development and differentiation. (A,D,G,J) Hematoxylin and Eosin (H+E) staining of lung sections. Notice the increased mesenchymal thickness and reduced sacculation in G6-Nkx DH lungs (J). Wild-type (A) and single heterozygous (D,G) lungs appear normal. SP-C protein expression is more focal in G6-Nkx DH lungs (B,E,H,K). Both aquaporin-5 and SP-C are expressed in a tight, focal pattern that is less evenly distributed throughout the distal alveolar airspaces in G6-Nkx DH lungs compared with wild-type lungs (M-P, arrowheads). CC10 protein expression is reduced in G6-Nkx DH lungs compared with wild-type or single heterozygous lungs (C,F,I,L, arrowheads). Data are from either the left lung or the cranial lobe of the right lung. All histological data are representative of at least five embryos of each genotype. (Q) Microscopic measurements were made to determine the mesenchymal thickness in G6-Nkx DH lungs, as described in the Materials and methods section. These data show a significant thickening in the inter-alveolar mesenchyme of G6-Nkx DH lungs at E18.5. P values *<0.001 versus wild-type, Gata6+/- or Nkx2.1+/- lungs. Scale bars: 500 µm in C,F,I,L; and 100 µm in A,B,D,E,G,H,J,K,M-P.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Quantitative measurements of gene expression changes in G6-Nkx DH lungs. (A) Q-PCR expression levels of CC10, SP-B, SP-C and Wnt7b in wild-type and G6-Nkx DH lungs. (B) Q-PCR expression of Gata6 and Nkx2.1 in wild-type, Gata6+/-, Nkx2.1+/- and G6-Nkx DH lungs. Q-PCR was performed on RNA extracted from whole lungs of the indicated genotypes at E18.5. All values were compared with expression in wild-type lungs, which was arbitrarily set at 1. Values are the average of four lung samples from each genotype performed in triplicate ±s.e.m. *P<0.001 versus wild-type, Gata6+/- or Nkx2.1+/- lungs. **P<0.05, non-significant changes compared to wild type.

View larger version (61K): [in this window] [in a new window]   Fig. 4. Increased glycogen content in G6-Nkx DH lung epithelium. G6-Nkx DH lungs have increased glycogen deposits, as measured by transmission electron microscopy (A-F), and increased PAS staining (G,H, red staining and arrowheads). Lamellar bodies were observed in wild-type and single heterozygous lung epithelium (B-D, arrowheads) but were absent or only rarely found in G6-Nkx2.1 DH lung epithelium (E,F). Scale bars: 10 µm in A,E; 2 µm IN B-D,F; and 50 µm in G,H.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Decreased phospholipid levels in the lungs of adult G6-Nkx DH mutants. Phospholipids levels, measured as described in Materials and methods, were decreased by approximately 60% in G6-Nkx DH mice (A). Phospholipid levels were decreased by a small but significant amount in Nkx2.1+/- mice. Lung-to-body weight ratios were not significantly different among all genotypes tested (B). *P<0.05; **P<0.005.

View larger version (71K): [in this window] [in a new window]   Fig. 6. Genes involved in phospholipid production and processing are downregulated in the lungs of G6-Nkx DH embryos. Q-PCR was performed on E18.5 lung tissue from wild-type, Gata6+/-, Nkx2.1+/- and G6-Nkx DH embryos using the oligonucleotides listed in Table 3. (A) Scd1, Dpp4, RBP-L and napsin are all downregulated to a small extent in Nkx2.1+/-, but not Gata6+/-, lungs. However, all four genes are severely downregulated in G6-Nkx DH lungs. (B) Comparison of changes in gene expression of Scd1, Dpp4, RBP-L and napsin from microarray and Q-PCR experiments. (C) In situ hybridization performed on wild-type and G6-Nkx DH lungs using riboprobes for RBP-L, Dpp4, napsin and Scd1 shows reduced expression of all four genes in G6-Nkx DH lungs at E18.5. Scale bars: 500 µm in C.

View larger version (58K): [in this window] [in a new window]   Fig. 7. Bmp4 expression is downregulated in G6-Nkx DH lungs. Immunohistochemistry using a Bmp4 antibody was performed to determine the expression of Bmp4 in the developing lungs of E12.5 wild-type (A), Gata6+/- (B), Nkx2.1+/- (C) and G6-Nkx DH (D) embryos. Bmp4 expression was significantly reduced in G6-Nkx DH lungs, but was unchanged in Gata6+/- and Nkx2.1+/- lungs. Inset for each panel is a higher magnification of a single distal airway. Q-PCR was performed using oligonucleotides listed in Table 3 to quantitatively determine the decrease in expression of Bmp4 in G6-Nkx DH lungs at E12.5 (E). Three samples from each indicated genotype were evaluated in these assays. *P<0.001 wild-type versus G6-Nkx DH samples. Scale bars: 100 µm in A-D.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Gata6 and Nkx2.1 interact at specific regions within the upstream regulatory regions of Scd1 and Dpp4. (A) mVista analysis was used to determine conserved regions in the upstream regulatory sequences of Scd1 and Dpp4. Regions probed using ChIP analysis are highlighted in brackets. Red underlined regions are genomic enhancers used in transactivation reporter assays. (B) ChIP analysis demonstrates an interaction of Gata6 with regions A and B in Scd1 and region A in Dpp4, whereas Nkx2.1 interacts with regions A and B in Scd1 and regions A and C in Dpp4. (C) Q-PCR of ChIP reactions on the same regions shown in A and B demonstrating quantitative differences in binding of Gata6 and Nkx2.1 to the identified conserved regions in the Scd1 and Dpp4 regulatory regions. *P<0.01 Gata6 and Nkx2.1 antibodies versus normal IgG.

View larger version (13K): [in this window] [in a new window]   Fig. 9. Activation of Scd1 and Dpp4 enhancers by Gata6 and Nkx2.1. Region A in Scd1 and Dpp4, and region B in Scd1 were all cloned into the pGL3promoter plasmid. These regions contain both Gata6 (stars) and Nkx2.1 (circles) DNA-binding sites. (A,B) In NIH-3T3 cells, expression of either Gata6 or Nkx2.1 transactivated region A in Scd1 and Dpp4 in a dose-dependent manner. However, co-expression of Gata6 and Nkx2.1 did not synergistically activate either of these enhancer regions. (C) Gata6 and Nkx2.1 synergistically activated Scd1 region B, even though this region lacks Gata6 DNA-binding sites. Data represent the average of three assays ±s.e.m.