mycHIF3α induces the expression of proximal differentiation markers.

<p>mycHIF3α induces an expansion of the Foxp2 positive cells in the double transgenic lungs at gestational age E18.5 (A, D), as well as an expansion towards the distal parts of the lungs of Sox2 (B, E) and p63 (C, F). Sox2 was expressed in both proximal airways and alveolar epithelial cells in mycHIF3α transgenic lungs (<i>arrows</i>, E) at PN1. Basal cells are absent in control lungs (C), but are expressed in basal cells of trachea (C, insert). However, p63 is expressed in the proximal airways and alveolar epithelial cells in mycHIF3α transgenic lung (<i>arrows</i>, F). <i>Scale bar</i>: 200 µm (A and D) and 100 µm (B, C, E, F). (G) <i>Foxp2</i> and <i>Rarβ</i> are significantly upregulated in Hif3α transgenic lungs at gestational age E18.5 as shown by quantitative PCR. (Foxp2: 1.25 <u>+</u> 0.1 versus control 0.87 <u>+</u> 0.1, n = 3, P = 0.007; Rarβ: 1.55 <u>+ </u>0.1 versus control 0.87 <u>+</u> 0.1, n = 3, P = 0,009). White bars represent control lung samples, black bars represent mycHIF3α double transgenic lung samples. (H) Hif2α (black bars) and Hif3α (white bars) induce the 9*HRE-Luc (HRE) and Sox2-Luc (Sox2) as measured by the amount of luciferase activity. The fold induction of the HRE promoter is higher with Hif2α (20,3 fold and 24,5 fold under hypoxic conditions-CoCl₂) than with Hif3α (2,4 fold and 13,4 fold under hypoxic conditions-CoCl₂). The induction of the Sox2 promoter is higher with Hif2α than with Hif3α under normoxic conditions (4,8 versus 2,5), but equally strong under hypoxia mimicking conditions (8,8 versus 7,3). Data are presented as the induction (n-fold) relative to cells transfected with the corresponding reporter plasmid and control vector (pcDNA3). The values are the average of two duplicates, and standard deviations are: 0,04 (HRE-Hif2α), 0,02 (Sox2-Hif2α), 0,03 (ΔSox2-Hif2α), 0,08 (HRE-Hif3α), 0,24 (Sox2-Hif3α), 0,06 (ΔSox2-Hif3α), 0,53 (HRE-Hif2α+CoCl2), 0,007 (Sox2-Hif2α+CoCl2), 0,03 (ΔSox2-Hif2α+CoCl2), 0,88 (HRE-Hif3α+CoCl2), 0,02 (Sox2-Hif3α+CoCl2), 0,1 (ΔSox2-Hif3α+CoCl2). (I) Chromatin immunoprecipitation (ChIP) using anti-HIF3α antibody and chromatin isolated from A549 cells. Graph represents the fold enrichment of the HIF3α-specific binding to the conserved HRE of the SOX2 promoter compared to the IgG control ChIP. HIF3α also bound the ARRDC3 HRE region, and the enhancer region D of the EGLN3 gene served as negative control (EGLN3-D).</p

Significant downregulated genes in the mycHIF3α expressing lungs.

<p>Significant downregulated genes in the mycHIF3α expressing lungs.</p

Normal differentiation of distal epithelial cells in mycHIF3α transgenic lungs.

<p>The site and expression pattern of T1α (A and B), Lpcat1 (C and D) and Ki67 (E and F) are comparable between control and mycHIF3α double transgenic lungs at gestational age E18.5. Scale bars: 100 µm</p

Significant upregulated genes in the mycHIF3α expressing lungs.

<p>Significant upregulated genes in the mycHIF3α expressing lungs.</p

Normal differentiation of proximal epithelial cells in mycHIF3α transgenic lungs.

<p>The site and expression pattern of α-Sma (A and B), Ttf1 (C and D), β-tubulin (E and F) and cGRP (arrows in G and H) are comparable between control and mycHIF3α double transgenic lungs at gestational age E18.5. Scale bars: 100 µm.</p

mycHIF3α reduces the number of Clara cells.

<p>The expression of the Clara cell marker, Ccsp, was strongly decreased in mycHIF3α transgenic lungs at gestational age E18.5 compared to controls (A and C versus B and D). (E) Alveolar epithelial cell markers are downregulated in Hif3α transgenic lungs at gestational age E18.5 as shown by quantitative PCR. <i>Epas1</i> (0,4 <u>+</u> 0.1 versus control 0.87 <u>+</u> 0.1, n = 3 each, P = 0.012), <i>Aqp5</i> (0.33<u>+</u> 0.1 versus control 0.96 <u>+</u> 0.1, n = 3 each, P = 0.005), <i>Abca3</i> (0.25 <u>+</u> 0.1 versus control 0.92 <u>+</u> 0.1 n = 3 each, P = 0.002), <i>Scd1</i>(0.35 <u>+</u> 0.1 versus control 0.92 <u>+</u> 0.1, n = 3 each, P = 0.001). (F) There is no significant change in the mRNA expression of Hif1α gene (0,8 <u>+</u> 0.1 versus control 0.7 <u>+</u> 0.1, n = 3 each, P>0.05). Quantification of the number of (G) type II pneumocytes (<i>Sftpd</i> over <i>Ttf1</i>, 0.36 <u>+</u> 0.1 versus control 0.9 <u>+</u> 0.1; n = 3, P = 0.01) and (H) Clara cells (<i>Ccsp</i> over <i>Ttf1</i>, 0.3 <u>+</u> 0.1 versus control 0.82 <u>+</u> 0.1; n = 5, P = 0.01) showed a significant reduction of in the Hif3α double transgenic animals. White bars represent control lung samples, black bars represent mycHIF3α double transgenic lung samples. Scale bars: 100 µm (A, B) and 50 µm (C,D).</p

Transcriptome analysis of mycHIF3α expressing lungs.

<p>Treescape showing that the transcriptome of the lungs of the mycHIF3α expressing animals are significantly different from that of the control lungs (A). The red color indicates the upregulated genes and the blue color indicates downregulated genes. The expression of the genes presented in the treescape is at least 1.5 fold changed with a false discovery rate (FDR) of 10%. The top 10 biological processes (B) and molecular functions (C) of the differentially expressed genes are shown.</p

Enhanced expression of HIF3α results in late branching defect.

<p>Endogenous expression of Hif3α was detected in epithelial cells at gestational age E18.5 (A, arrows) and in type II pneumocytes in adult mice (B, arrows). (C) Graphic representation of the tet-inducible Hif3α/NEPAS cDNA construct used to generate transgenic mice. TRE is the Tet-responsive element containing minimal promoter, II and III refer to exon 2 and 3 of the ß-blobin gene and AAAAAA is the poly-adenylation signal. Indicated are the position of the myc-epitope, and the bHLH, PAS and NTAD domains (see text) (D) Quantification of the number of airspaces in the lung. Three independent samples of control and double-transgenic lungs at gestational age E18,5 were used to count the number of airspaces. External appearances of control (E, I) and double transgenic mycHIF3α (F, J) lungs at E18.5 days of gestation (E18.5) and post natal age 1 (PN1) do not show apparent differences. Histological analysis of control (G, K) and double transgenic lungs (H, L) showed decreased number of alveolar spaces and reduced branching in the double transgenic lungs (H and L). Anti-Myc epitope staining confirmed the expression of the mycHIF3α transgene in double transgenic lungs (H and L), which is absent in control lungs (G and K). Scale bars: Scale bars: 25 µm (A, B), 2 mm (E, F, I, J) or 200 µm (G, H, K, L).</p

Expression of genes involved in branching morphogenesis.

<p>Analysis of the distribution of mycHIF3α early in lung development in double transgenic animals at E11.5 (A), E12.5 (B), E13.5 (C) and E14.5 (D). Whole mount in situ hybridization to detect the expression and localization of Fgf10 (E and I), FgfR2 (F and J), Bmp4 (G and K) and Shh (H and L) in lungs isolated at gestational age E12.5 from control (E–H) and mycHIF3α double transgenic animals (I–L). Tr: Trachea; Es: Esophagus. Scale bars: 200 µm.</p