title: The Role of ELf5 in Mouse Trophoblast Stem Cells

authors: Redgate, Emma Louise

subjects: Trophoblast

abstract: Elf5 is a DNA transcription factor that has been identified as being involved in placentation of the early embryo. Elf5 is expressed in the extra embryonic ectoderm (ExE), a lineage that contributes to the placenta of the embryo. Pluripotent trophoblast stem (TS) cells can be derived from the ExE and can be cultured in vitro. Such cells can contribute to all the placental lineages when injected back into an embryo. Elf5 homozygous mutant embryos do not possess an ExE and trophoblast stem cells cannot be derived and the mutation is therefore embryonic lethal. When Elf5 is knocked out, the TS cells in the ExE are thought to differentiate into EPC/giant cells leading to the absence of the ExE. In previous experiments in this laboratory, potential Elf5 targets were identified by RNA interference in mouse trophoblast stem cells followed by global gene expression analysis using an Affymetrix array. In the present experiments these results have now been confirmed by siRNA induced knockdown of Elf5 in TS cells followed by quantitative real time PCR of potential target genes. Most of the target genes that were affected by Elf5 knockdown were changed in the same way by growth factor removal and therefore stem cell differentiation. This suggested that Elf5 usually acts to maintain the TS cells in a stem cell fate. As Elf5 is expressed in the ExE, whole mount In situ hybridisation was used to determine if the genes showed the correct spatial and temporal patterning to be in vivo relevant Elf5 targets. The targets that were down regulated upon Elf5 knockdown (and therefore positively regulated by Elf5) were expressed in the ExE as expected. Genes that were up-regulated upon Elf5 knockdown (and therefore usually repressed by Elf5) were expressed in the differentiated ectoplacental cone or giant cells. . Preliminary work was also carried out for the over-expression of Elf5 by a tamoxifen inducible Elf5. TS cells were stably transfected with a plasmid containing Elf5 fused to a tamoxifen inducible ERT2 receptor and a VP16 transcriptional activation domain to turn Elf5 into a potent activator, and were analysed for target gene expression.  Preliminary data showed that Elf5 is involved in a complex transcriptional network of events as the genes did not behave as expected when Elf5 was turned into an activator. The effect of Elf5 knockdown on TS cells has been studied closely in terms of morphology, proliferation, changes in DNA content and apoptosis. The knockdown of Elf5 caused an increase in the number of differentiated cells (as shown by changes in morphology and DNA content). This further supported the knockdown and in situ data. There was no change in proliferation or apoptosis.  These experiments which demonstrate which genes are regulated by Elf5 and the changes to TS cellular characteristics when Elf5 is knocked down, support the proposal that Elf5 acts as a trophoblast stem cell maintenance factor of the extraembryonic ectoderm of the mouse.

text: The Role of ELf5 in Mouse Trophoblast Stem Cells Elf5 is a DNA transcription factor that has been identified as being involved in placentation of the early embryo. Elf5 is expressed in the extra embryonic ectoderm (ExE), a lineage that contributes to the placenta of the embryo. Pluripotent trophoblast stem (TS) cells can be derived from the ExE and can be cultured in vitro. Such cells can contribute to all the placental lineages when injected back into an embryo. Elf5 homozygous mutant embryos do not possess an ExE and trophoblast stem cells cannot be derived and the mutation is therefore embryonic lethal. When Elf5 is knocked out, the TS cells in the ExE are thought to differentiate into EPC/giant cells leading to the absence of the ExE. In previous experiments in this laboratory, potential Elf5 targets were identified by RNA interference in mouse trophoblast stem cells followed by global gene expression analysis using an Affymetrix array. In the present experiments these results have now been confirmed by siRNA induced knockdown of Elf5 in TS cells followed by quantitative real time PCR of potential target genes. Most of the target genes that were affected by Elf5 knockdown were changed in the same way by growth factor removal and therefore stem cell differentiation. This suggested that Elf5 usually acts to maintain the TS cells in a stem cell fate. As Elf5 is expressed in the ExE, whole mount In situ hybridisation was used to determine if the genes showed the correct spatial and temporal patterning to be in vivo relevant Elf5 targets. The targets that were down regulated upon Elf5 knockdown (and therefore positively regulated by Elf5) were expressed in the ExE as expected. Genes that were up-regulated upon Elf5 knockdown (and therefore usually repressed by Elf5) were expressed in the differentiated ectoplacental cone or giant cells. . Preliminary work was also carried out for the over-expression of Elf5 by a tamoxifen inducible Elf5. TS cells were stably transfected with a plasmid containing Elf5 fused to a tamoxifen inducible ERT2 receptor and a VP16 transcriptional activation domain to turn Elf5 into a potent activator, and were analysed for target gene expression.  Preliminary data showed that Elf5 is involved in a complex transcriptional network of events as the genes did not behave as expected when Elf5 was turned into an activator. The effect of Elf5 knockdown on TS cells has been studied closely in terms of morphology, proliferation, changes in DNA content and apoptosis. The knockdown of Elf5 caused an increase in the number of differentiated cells (as shown by changes in morphology and DNA content). This further supported the knockdown and in situ data. There was no change in proliferation or apoptosis.  These experiments which demonstrate which genes are regulated by Elf5 and the changes to TS cellular characteristics when Elf5 is knocked down, support the proposal that Elf5 acts as a trophoblast stem cell maintenance factor of the extraembryonic ectoderm of the mouse.

year: 2010

title: The stable carbon isotope ratios of fish and their invertebrate prey in four New Zealand rivers

authors: Rounick, J.S.

subjects: Stable carbon isotopes

abstract: 1. Sources of carbon utilized by invertebrates and fish in four New Zealand streams were investigated using stable carbon isotope analysis. Complementary analyses were made to determine σ13C alteration (fractionation) by fish reared on a known food and to examine the distribution of 13C/12C ratios in different tissues.
2.σ13C values of hatchery-reared rainbow trout (white muscle tissue) were 13C-enriched compared with their food (trout pellets) by an average of 1.7%0. σ13C values of different organs and tissues never differed by more than 1.8%o in any fish species; white muscle always had an intermediate value.
3. σ13C values of invertebrates ranged from-28.4 to –20.4%o– At the more open sites invertebrates were 13C-depleted, reflecting some use of algal carbon. However, invertebrates from the most heavily shaded stream site were unusually enriched in 13C. An undetected carbon source of terrestrial origin, perhaps 13C-enriched dissolved organic carbon in groundwater, may have been contributing indirectly to invertebrate biomass carbon.
4. σ13C values for fish were not closely linked to aquatic invertebrate ratios; instead, at all sites their carbon was isotopically similar to terrestrial organic carbon. The reasons for this apparent anomaly are unclear.

text: The stable carbon isotope ratios of fish and their invertebrate prey in four New Zealand rivers 1. Sources of carbon utilized by invertebrates and fish in four New Zealand streams were investigated using stable carbon isotope analysis. Complementary analyses were made to determine σ13C alteration (fractionation) by fish reared on a known food and to examine the distribution of 13C/12C ratios in different tissues.
2.σ13C values of hatchery-reared rainbow trout (white muscle tissue) were 13C-enriched compared with their food (trout pellets) by an average of 1.7%0. σ13C values of different organs and tissues never differed by more than 1.8%o in any fish species; white muscle always had an intermediate value.
3. σ13C values of invertebrates ranged from-28.4 to –20.4%o– At the more open sites invertebrates were 13C-depleted, reflecting some use of algal carbon. However, invertebrates from the most heavily shaded stream site were unusually enriched in 13C. An undetected carbon source of terrestrial origin, perhaps 13C-enriched dissolved organic carbon in groundwater, may have been contributing indirectly to invertebrate biomass carbon.
4. σ13C values for fish were not closely linked to aquatic invertebrate ratios; instead, at all sites their carbon was isotopically similar to terrestrial organic carbon. The reasons for this apparent anomaly are unclear.

year: 1985

title: Puna

authors: Apiata, Hāwea

abstract: Bilingual poem for issue 11 of Mayhem Literary Journal.

text: Puna Bilingual poem for issue 11 of Mayhem Literary Journal.

year: 2024-09-26