Pulmonary Histopathology.

<p>Light photomicrographs of hematoxylin and eosin stained lung sections from control (<b>A</b>), <i>Hif-1αΔ/Δ</i> (<b>B</b>), <i>Hif-2αΔ/Δ</i> (<b>C</b>) and <i>Hif-1/2αΔ/Δ</i> (<b>D</b>) pups. Analysis of septal thickness of control mice (<b>white bars</b>) and <i>Hif-1αΔ/Δ</i> mice (<b>black bars</b>) was measured morphometrically as described in materials and methods (<b>E</b>). Results are presented as mean alveolar thickness with error bars representing the standard error. Alveolar airspace labeled as (a), septae labeled as (S). * = P ≤ 0.05.</p

Periodic acid Schiff (PAS) staining.

<p>Periodic acid Schiff (PAS) stained lung sections from control (<b>A, C, E</b>) and <i>Hif-1αΔ/Δ</i> (<b>B</b>), <i>Hif-2αΔ/Δ</i> (<b>D</b>), <i>Hif-1/2αΔ/Δ</i> (<b>F</b>) pups. Cuboidal epithelium lining alveolar septa in <i>Hif-1αΔ/Δ</i> pups (<b>B</b>) has greater PAS-stained glycogen (arrows) compared to that of the control pup.</p

Merged Transcriptional Network with <i>c-Myc</i>.

<p>The linkage between the <i>Hif-1α</i> transcriptional network and <i>c-Myc</i> were merged and common genes in both networks were identified. <i>Hif-1α</i>-specific (blue circles), <i>c-Myc</i>-specific (green circles), and common genes (half blue, half green circles) are separated by function.</p

Merged Transcriptional Network with <i>C/ebpα</i>.

<p>The linkage between the <i>Hif-1α</i> transcriptional network and <i>C/ebpα</i> were merged and common genes in both networks were identified. <i>Hif-1α</i>-specific (green circles), <i>C/ebpα</i>-specific (blue circles), and common genes (half blue, half green circles) are separated by function.</p

Differential gene expression in lungs of <i>Hif-1αΔ/Δ</i>, <i>Hif-2αΔ/Δ</i> and <i>Hif-1/2αΔ/Δ</i> mice.

<p>Process for identifying differentially regulated genes across all three genotypes (<b>A</b>). Comparative gene expression analysis between <i>Hif-1αΔ/Δ</i>, <i>Hif-2αΔ/Δ</i>, and <i>Hif-1/2αΔ/Δ</i> pups. The Venn diagram illustrates common and unique differentially expressed genes (<b>B</b>).</p

QRTPCR verification of selected microarray gene expression responses.

<p>The same RNA used for Agilent microarrays was examined by QRTPCR. All fold changes were calculated relative to controls. Fold changes of the 22 most differentially expressed genes in the microarray are compared with fold changes from qRT-PCR verification (<b>A</b>). Three represented genes shows correlation of the fold changes between microarray and qRT-PCR verification (<b>B</b>); bars (left y-axis) and lines (right y-axis) represent the fold-change values relative to <i>Hprt</i> for QRTPCR and microarray data, respectively; “a” indicates a statistical difference from <i>2αΔ/Δ</i>, “b” indicates a statistical difference from <i>1αΔ/Δ</i>, “c” indicates a statistical difference from <i>1/2αΔ/Δ</i>. Statistical significance indicates a p-value ≤ 0.05.</p

Survivability Plot.

<p>Viability of neonatal <i>Hif-1αΔ/Δ</i>, <i>Hif-2αΔ/Δ</i> and <i>Hif-1/2αΔ/Δ</i> pups. (<b>A</b>) All <i>Hif-1αΔ/Δ</i> pups showed signs of respiratory distress whereas all <i>Hif-2αΔ/Δ</i> pups were viable with no obvious phenotype. Simultaneous removal of both <i>Hifα</i> forms resulted in approximately 79% survival. (<b>B</b>) Time-course of various <i>Hif-1α</i> mice with doxycycline (DOXY) exposure E14.5 compared with No DOXY. All mice had <i>SPC-rtTA</i>^<i>-/tg</i><i>/(TetO)</i>_<i>7</i><i>-CMV-Cre</i>^<i>tg/tg</i>; white bars indicate homozygous floxed <i>Hif-1α</i> (h1h1), grey bars indicate heterozygous floxed <i>Hif-1α</i> (H1h1), and black bars indicate wt<i>Hif-1α</i> (H1H1).</p

Loss of <i>Hif-2α</i> Rescues the <i>Hif-1α</i> Deletion Phenotype of Neonatal Respiratory Distress In Mice

<div><p>Hypoxia is a state of decreased oxygen reaching the tissues of the body. During prenatal development, the fetus experiences localized occurrences of hypoxia that are essential for proper organogenesis and survival. The response to decreased oxygen availability is primarily regulated by hypoxia-inducible factors (HIFs), a family of transcription factors that modulate the expression of key genes involved in glycolysis, angiogenesis, and erythropoiesis. HIF-1α and HIF-2α, two key isoforms, are important in embryonic development, and likely are involved in lung morphogenesis. We have recently shown that the inducible loss of <i>Hif-1α</i> in lung epithelium starting at E4.5 leads to death within an hour of parturition, with symptoms similar to neonatal respiratory distress syndrome (RDS). In addition to <i>Hif-1α</i>, <i>Hif-2α</i> is also expressed in the developing lung, although the overlapping roles of <i>Hif-1α</i> and <i>Hif-2α</i> in this context are not fully understood. To further investigate the independent role of <i>Hif-2α</i> in lung epithelium and its ability to alter <i>Hif-1α</i>-mediated lung maturation, we generated two additional lung-specific inducible <i>Hif-α</i> knockout models (<i>Hif-2α</i> and <i>Hif-1α+Hif-2α</i>). The intrauterine loss of <i>Hif-2α</i> in the lungs does not lead to decreased viability or observable phenotypic changes in the lung. More interestingly, survivability observed after the loss of both <i>Hif-1α</i> and <i>Hif-2α</i> suggests that the loss of <i>Hif-2α</i> is capable of rescuing the neonatal RDS phenotype seen in <i>Hif-1α</i>-deficient pups. Microarray analyses of lung tissue from these three genotypes identified several factors, such as <i>Scd1</i>, <i>Retlnγ</i>, and <i>Il-1r2</i>, which are differentially regulated by the two HIF-α isoforms. Moreover, network analysis suggests that modulation of hormone-mediated, NF-κB, C/EBPα, and c-MYC signaling are central to HIF-mediated changes in lung development.</p></div