Dirks RP et al. (2010), Manipulating heat shock factor-1 in Xenopus tad...

XB-ART-41441

PLoS One 2010 Apr 08;54:e10158. doi: 10.1371/journal.pone.0010158.

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Manipulating heat shock factor-1 in Xenopus tadpoles: neuronal tissues are refractory to exogenous expression.

Dirks RP, van Geel R, Hensen SM, van Genesen ST, Lubsen NH.

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The aging related decline of heat shock factor-1 (HSF1) signaling may be causally related to protein aggregation diseases. To model such disease, we tried to cripple HSF1 signaling in the Xenopus tadpole.Over-expression of heat shock factor binding protein-1 did not inhibit the heat shock response in Xenopus. RNAi against HSF1 mRNA inhibited the heat shock response by 70% in Xenopus A6 cells, but failed in transgenic tadpoles. Expression of XHSF380, a dominant-negative HSF1 mutant, was embryonic lethal, which could be circumvented by delaying expression via a tetracycline inducible promoter. HSF1 signaling is thus essential for embryonic Xenopus development. Surprisingly, transgenic expression of the XHSF380 or of full length HSF1, whether driven by a ubiquitous or a neural specific promoter, was not detectable in the larval brain.Our finding that the majority of neurons, which have little endogenous HSF1, refused to accept transgene-driven expression of HSF1 or its mutant suggests that HSF1 levels are strictly controlled in neuronal tissue.

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Species referenced: Xenopus
Genes referenced: hsbp1 hsf1 hsp70 hsp90aa1 hspa1l myc

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	Figure 1. Ubiquitous transgene-driven expression of XHSBP1 in Xenopus tadpoles.(A) ClustalW alignment of human HSBP1 (top) and HSBP1 sequences deduced from five different Xenopus laevis ESTs. The central coiled coil region is highly conserved between man and Xenopus. (B) Fluorescence microscope analysis of tadpoles carrying the indicated transgenes. Tadpoles with ubiquitous, high level expression of GFP-HSBP1 look normal. (C) Western blot analysis of lysates from whole tadpoles carrying the indicated transgenes. Proteins were separated by SDS-PAGE and analyzed with an anti-GFP antibody.
	Figure 2. The effect of exogenous expression of GFP-XHSBP1 on the HSR.(A) Heat shock induced expression of Hsp90. Xenopus A6 kidney epithelial cells were continuously cultured at room temperature (C) or exposed to a 1 h heat shock (33°C) and then cultured at room temperature for the indicated times. Cell lysates were subjected to SDS-PAGE and western blot analysis using anti-Hsp90 and anti-tubulin antibodies. (B) Western blot analysis of Hsp90 levels in tadpoles carrying the indicated transgenes. Lysates were made from whole tadpoles before heat shock (C) or after a 1 h heat shock at 33°C followed by 6 h recovery at room temperature (HS). The lysates were subjected to SDS-PAGE and western blot analysis using anti-GFP, anti-Hsp90 and anti-tubulin antibodies. (C) Reporter gene analysis of the effect of GFP-XHSBP1 on the HSR. A6 cells were transfected with mixtures of an Hsp70-luciferase reporter, a CMV-β-galactosidase reporter and the indicated plasmids. At 48 h after transfection, cells were exposed to a 1 h heat shock at 33°C. Control cells were left at room temperature. Cell lysates were made at 6 h after heat shock and used for reporter gene assays. Hsp70 promoter activities were determined by dividing luciferase values by the corresponding β-galactosidase values to correct for varying transfection efficiencies. The HSR is indicated as fold induction relative to the activity of the Hsp70 promoter in control cells, which was set at 1 (C). The results are the average of three independent transfections (standard deviations are indicated by error bars).
	Figure 3. The effect of exogenous expression of XHSBP1 on the HSR.(A) Fluorescence microscope analysis of reporter gene expression from bicistronic plasmids. A6 cells were transfected with the indicated dual reporter gene plasmids based on viral 2A peptides. At 24 h after transfection, GFP and DsRed expression was determined by fluorescence microscope analysis. (B) Western blot analysis of gene expression from bicistronic plasmids. A6 cells were transfected with the indicated 2A peptide-based plasmids. At 24 h after transfection, cell lysates were made and subjected to SDS-PAGE and western blot analysis using anti-GFP antibodies. (C) Western blot analysis of gene expression from bicistronic plasmids. A6 cells were transfected with the indicated 2A peptide-based plasmids. At 24 h after transfection, cell lysates were made and subjected to SDS-PAGE and western blot analysis using anti-GFP and anti-myc tag antibodies. (D) Reporter gene analysis of the effect of native XHSBP1 on the HSR. A6 cells were transfected with mixtures of an Hsp70-luciferase reporter, a CMV-β-galactosidase reporter and the indicated plasmids. Relative luciferase activities and -fold induction were determined as described in the legend to fig. 2C. The results are the average of three independent transfections (standard deviations are indicated by error bars).
	Figure 4. The effect of XHSF1 shRNAs on the HSR.(A,B) Reporter gene analysis of (A) the effect of HSF1 shRNAs expressed from the human or Xenopus H1 promoter, and (B) the effect of different shRNAs expressed from the Xenopus H1 promoter, on the HSR. A6 cells were transfected with mixtures of an Hsp70-luciferase reporter, a CMV-β-galactosidase reporter and the indicated plasmids. Relative luciferase activities and -fold induction were determined as described in the legend to fig. 2C. The results are the average of three independent transfections (standard deviations are indicated by error bars). (B) Western blot analysis of Hsp90 levels in tadpoles carrying the indicated transgenes. Lysates were made from whole tadpoles before heat shock (C) or after a 1 h heat shock at 33°C followed by 6 h recovery at room temperature (HS). The lysates were subjected to SDS-PAGE and western blot analysis using anti-Hsp90 and anti-tubulin antibodies.
	Figure 5. Transgene-driven expression of dominant-negative HSF1 in Xenopus larvae.(A) Reporter gene analysis of the effect of GFP-XHSF380 or XHSF380-GFP on the HSR. A6 cells were transfected with mixtures of an Hsp70-luciferase reporter, a CMV-Î²-galactosidase reporter and the indicated plasmids. Relative luciferase activities and -fold induction were determined as described in the legend to fig. 2C. The results are the average of three independent transfections (standard deviations are indicated by error bars). (B) Fluorescence microscope analysis of GFP-tagged XHSF380 constitutively expressed from either the CMV or the EF1Î± promoter in transgenic Xenopus larvae. Larvae with high constitutive expression of GFP-tagged XHSF380 develop poorly and never reach the feeding tadpole stage. Also shown are transgenic Xenopus larvae showing strong neuronal expression of GFP when driven by the EF1Î± or the Ntub promoter. (C) Fluorescence microscope analysis of transgenic Xenopus larvae expressing full length GFP-tagged XHSF1 from the CMV promoter. Fluorescence is pronounced in kidney, epiphysis, nasal epithelium, olfactory lobes, gills, retinal ganglion cell layer (RGC) and tail muscle nuclei. (D) Fluorescence microscope analysis of doxycycline-induced expression of GFP-tagged XHSF380 expressed in transgenic Xenopus larvae. A mixture of TetO-XHSF380-GFP and CS2+rtTA2A-M2 (CMV-TAM2) cassettes was used to generate transgenic Xenopus larvae. The larvae were allowed to develop in the absence of doxycycline until the feeding tadpole stage. GFP-negative larvae were exposed to doxycycline for 20 h and then monitored for GFP fluorescence.

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