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Dev Dyn
2007 Apr 01;2364:1115-25. doi: 10.1002/dvdy.21106.
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Kidney development and gene expression in the HIF2alpha knockout mouse.
Steenhard BM
,
Freeburg PB
,
Isom K
,
Stroganova L
,
Borza DB
,
Hudson BG
,
St John PL
,
Zelenchuk A
,
Abrahamson DR
.
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The hypoxia-inducible transcription factor-2 (HIF2), a heterodimer composed of HIF2alpha and HIF1beta subunits, drives expression of genes essential for vascularization, including vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2, Flk-1). Here, we used a HIF2alpha/LacZ transgenic mouse to define patterns of HIF2alpha transcription during kidney development and maturation. Our results from embryonic heterozygotes showed HIF2alpha/LacZ expression by apparently all renal endothelial cells. At 4 weeks of age, glomerular mesangial and vascular smooth muscle cells were also positive together with endothelial cells. These expression patterns were confirmed by electron microscopy using Bluo-gal as a beta-galactosidase substrate. Small numbers of glomerular and tubular epithelial cells were also positive at all stages examined. Light and electron microscopic examination of kidneys from HIF2alpha null embryos showed no defects in renal vascular development or nephrogenesis. Similarly, the same amounts of Flk-1 protein were seen on Western blots of kidney extracts from homozygous and heterozygous HIF2alpha mutants. To examine responsiveness of HIF2alpha null kidneys to hypoxia, embryonic day 13.5 metanephroi were cultured in room air or in mild (5% O(2)) hypoxia. For both heterozygous and null samples, VEGF mRNA levels doubled when metanephroi were cultured in mild hypoxia. Anterior chamber grafts of embryonic HIF2alpha knockouts were morphologically indistinguishable from heterozygous grafts. Endothelial markers, platelet endothelial cell adhesion molecule and BsLB4, as well as glomerular epithelial markers, GLEPP1 and WT-1, were all expressed appropriately. Finally, we undertook quantitative real-time polymerase chain reaction of kidneys from HIF2alpha null embryos and wild-type siblings and found no compensatory up-regulation of HIF1alpha or -3alpha. Our results show that, although HIF2alpha was widely transcribed by kidney endothelium and vascular smooth muscle, knockouts displayed no detectable deficits in vessel development or VEGF or Flk-1 expression.
Figure 1. Hypoxia-inducible transcription factor (HIF) 2 alpha expression in kidney. A: Embryonic day (E) 13.5 HIF2 alpha +/- metanephros stained for beta -galactosidase histochemistry shows a branching, vessel-like pattern. B: E14HIF2 alpha -/- kidney showing beta -galactosidase reaction product in glomerular endothelial cells migrating into the vascular cleft (VC), and in capillary loop stage glomeruli (G). Also, small arrows denote individual cells in the metanephric mesenchyme containing beta -galactosidase. C: The same slide in B was also labeled with BsLB4, an endothelial marker. D: Colocalization of beta -galactosidase and BsLB4 shows complete overlap. E: Some visceral epithelial cells (developing podocytes) in early glomeruli express HIF2 alpha (*). F: Intense vascular expression (arrowhead) of HIF2 alpha is seen in newborn kidney, and within glomeruli (G). G: (Higher magnification of boxed area in F): Some podocytes express HIF2 alpha (arrows), whereas others do not (arrowheads). H: Frozen section of a 4-week HIF2 alpha +/- mouse showing intense beta -galactosidase product in an endothelial pattern, and in smooth muscle cells of arteries (arrowheads). I: Frozen section of a 4-week HIF2 alpha +/- mouse showing a small artery. Reaction product is seen in both endothelial cells (arrow) and smooth muscle cells (arrowheads). J: Localization of HIF2 alpha /LacZ to peritubular capillary endothelial cell (arrow) and (rarely) to a tubular epithelial cell (arrowhead).
Figure 3. Normal glomerular capillary development in hypoxia-inducible transcription factor (HIF) 2 alpha -/- mice. A-D: Toluidine blue staining of thin sections from an embryonic day (E) 18.5 HIF2 alpha -/- kidney. A: Ureteric bud (UB) and condensing mesenchyme (CM) appear normal. B: Endothelial cells are migrating into the vascular cleft of S-shaped bodies (arrow). C,D: Red blood cells are seen in capillary loop stage glomeruli (arrow in C), and open capillary loops with red blood cells are readily apparent in mature glomeruli (arrow in D). E-G: Frozen sections from E14HIF2 alpha -/- mutants show beta -galactosidase product in endothelial cells migrating into the vascular cleft (arrow) of comma-shaped nephric figures (E). F: Capillary loop stage glomeruli contain beta -galactosidase-positive endothelial cells. Afferent and efferent arterioles seen entering the glomerulus also contain reaction product (arrowhead). G: The arrowhead depicts the open capillary loop within a mature glomerulus. H: Ultrastructure of E18.5 HIF2 alpha -/- capillary loop shows normal podocyte (Po) foot process interdigitation and slit diaphragms, glomerular basement membrane, and fenestrated endothelium (arrow).
Figure 4. Flk-1 and GLEPP1 expression appear normal in hypoxia-inducible transcription factor (HIF) 2 alpha -/- mouse kidney. A: Western blot of HIF2 alpha wild-type (+/+) or knockout (-/-) kidney lysates at embryonic day (E) 13.5 was probed with anti-Flk-1, then stripped and re-probed with anti-HIF1 beta . B: Quantification of Flk-1 from two separate litters of HIF2 alpha mice showing no difference in the levels of Flk-1 in whole kidney lysates (n = 3 for each genotype). C: Double labeling with anti-Flk-1 (red) and anti-GLEPP1 (green) in a HIF2 alpha +/- mouse shows podocyte expression of GLEPP1 and endothelial labeling of Flk-1 as expected. D: Both GLEPP1 and Flk-1 expression appear normal in the HIF2 alpha -/- glomeruli.
Figure 6. Hypoxia-inducible transcription factor (HIF) 2 alpha -/- kidney development in anterior chamber grafts proceeds normally. A: Frozen section of a grafted HIF2 alpha -/- E13.5 kidney shows widespread vasculature development following beta -galactosidase histochemistry. B,C: Serial frozen sections from a HIF2 alpha -/- graft were either reacted for beta -galactosidase (B) or labeled (C) with the podocyte marker anti-WT-1 (green) and the endothelial marker anti- platelet endothelial cell adhesion molecule (PECAM, red). Both labels show normal distribution patterns. D-I: Double labeling of frozen sections with antibodies to the basement membrane-specific type IV collagen (ColIV( alpha 3, alpha 4, alpha 5), green) and the podocyte marker GLEPP1 (red). J-O: Double labeling of frozen sections with antibodies to the endothelial marker BsLB4 (green) and the podocyte marker WT-1 (red). The same expression pattern is seen for both heterozygous HIF2 alpha grafts (D-F and J-L) and knockout HIF2 alpha grafts (G-I and M-O). Merged images are shown in F, I, L, and O.
