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Hypoxia-inducible factor 1alpha (HIF-1alpha) plays a central role in regulating oxygen-dependent gene expression and is involved in a range of pathways implicated in cellular survival, proliferation, and development. While the posttranslational regulation of HIF-1alpha is well characterized, the relative importance of its control at the transcriptional level during development remains less clear. Although the mouse and human promoter regions have been analyzed in vitro, to date, there has been no in vivo analysis of any vertebrate HIF-1alpha promoter. To investigate the transcriptional regulation of HIF-1alpha during development of the amphibian Xenopus laevis, we have described the gene's expression pattern and isolated the xHIF-1alpha upstream regulatory regions. We show xHIF-1alpha mRNA to be constitutively expressed at low levels throughout embryogenesis, but with significant up-regulation during gastrula stages, and subsequently, in specific regions of the central nervous system and axial tissues. Our functional analysis using a series of truncated xHIF-1alpha promoter constructs demonstrates that a 173-bp region of the proximal promoter, which is 100% conserved among five allelic variants, is sufficient to drive correct expression in transgenic embryos. Although these results are corroborated by a parallel set of in vitro transfection experiments in a Xenopus cell line, some key differences suggest the importance of using transgenic methods in conjunction with in vitro assays.
Figure 3. Spatial analysis of endogenous xHIF-1 alpha expression in the Xenopus embryo. A: A representative whole-mount in situ hybridization of a stage 35 embryo using a digoxigenin-labeled xHIF-1 alpha antisense probe showing a ubiquitous expression pattern. B: Elevated expression is seen in the head, somitic tissue, and developing nervous system when compared with stage 35 embryos hybridized with a complementary sense probe. C-F: The observed expression pattern was confirmed through histological analysis of embryos hybridized with either the xHIF-1 alpha antisense (C,E) or sense (D,F) probe. NO, notochord; NT, neural tube; OV, otic vesicle; SO, somite; arrowheads indicate somitic tissue; an asterisk also indicates the otic vesicle. Scale bars = 1 mm in A,B, 0.5 mm in C-F.
Figure 5. xHIF-1 alpha upstream sequences direct tissue-specific expression of green fluorescent protein (GFP) in transgenic Xenopus embryos. A-E: Representative HIF(-1650)GFP (A), HIF(-500)GFP (B), HIF(-173)GFP (C), and HIF(UTR)GFP (D) transgenic embryos show elevated levels of GFP expression in the head and eye, neural tube, and somitic tissue when compared with negative control embryos (E) containing a promoterless pEGFP-1 transgene. F: Higher magnification of the anterior end (dorsal view) of a HIF(-1650)GFP transgenic embryo demonstrates that the neural tube expresses elevated levels of GFP. G,H: Arrows indicate the elevated GFP expression pattern in the neural tube and somitic tissue of a HIF(-1650)GFP transgenic (G) compared with the same region in a negative control embryo (H). I: Optical section of a HIF(-1650)GFP embryo showing increased GFP expression in somitic regions. J-L: Histological analysis of transgenic embryos using in situ hybridization with an antisense GFP probe demonstrates an expression pattern comparable to that of endogenous xHIF-1 alpha in the anterior (J), spinal cord (K), and tail (L). M: Negative control transgenic embryo hybridized with the corresponding sense GFP in situ hybridization probe. cg, cement gland; ey, eye; gu, gut; nt, neural tube; so, somite; tf, tailfin. Scale bars = 1 mm in A-E, 0.5 mm in F-M.
