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Physiological rationale for responsiveness of mouse embryonic stem cells to gp130 cytokines

Jennifer Nichols1,*, Ian Chambers1, Tetsuya Taga2 and Austin Smith1

1 Centre for Genome Research, University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh EH9 3JQ, UK
2 Department of Cell Fate Modulation, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1, Honjo, Kumamoto, 860-0811, Japan



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  		Fig. 1. The number and genotypes of embryos dissected from 9.5d.p.c. implantation sites generated by transfer of blastocysts from Lifr and gp130 double heterozygous intercross matings that were in diapause for 6 days. The numbers for wild type (+/+) and heterozygous (+/-) genotypes are combined (+). *Two of the four embryos were abnormal.

 


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  		Fig. 2. SSEA-1 immunostaining on ICMs isolated from blastocysts delayed for 6 days. (A) phase contrast, (B) fluorescence (x40 objective). In situ hybridisation for Sparc mRNA on cultured ICMs from blastocysts delayed for 6 days. (C) gp130+/-, (D) gp130-/- (x10 objective). ICM, proliferating inner cell mass; VE, visceral endoderm; PE, parietal endoderm.

 


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  		Fig. 3. Comparison of cell numbers of ICMs of blastocysts generated from intercross matings of mice heterozygous for gp130, in diapause for 2 or 6 days. Wt/het, combined gp130+/+ and gp130+/- genotypes; null, gp130-/-. Error bars denote standard deviation. For 2 days of delay there is no significant difference in the mean inside cell number (P>0.1 by Student’s t-test). For 6 days of delay there is a highly significant difference between the mean numbers of inside cells by Student’s t-test (P<0.001). Comparing mean inside cell numbers from null embryos delayed for 2 days with those delayed for 6 days the difference is significant by Student’s t-test (P<0.002).

 


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  		Fig. 4. Composite bright-field and fluorescence image of blastocyst delayed for 3 days and stained for TUNEL. Dead cells fluoresce. Subsequent PCR analysis revealed that this embryo was gp130-/-. Images were collected using a x40 objective and Open Lab imaging software.

 


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  		Fig. 5. Number of dead cells detected by TUNEL analysis and Hoechst staining of blastocysts from gp130+/- intercross matings delayed for 3 days. Wt/het, combined gp130+/+ and gp1+/-; null, gp130-/-. Error bars denote standard deviation. Using Student’s t-test significantly more dead cells per embryo are detected in the gp130-/- category: P<0.01 using TUNEL analysis; P<0.002 by Hoechst staining.

 




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