Comparison of two related lines of tauGFP transgenic mice designed for lineage tracing.

BACKGROUND: The tauGFP reporter fusion protein is produced nearly ubiquitously by the TgTP6.3 transgene in TP6.3 mice and its localisation to microtubules offers some advantages over soluble GFP as a lineage marker. However, TgTP6.3 Tg/Tg homozygotes are not viable and TgTP6.3 Tg/- hemizygotes are smaller than wild-type. TP6.4 mice carry the TgTP6.4 transgene, which was produced with the same construct used to generate TgTP6.3, so we investigated whether TgTP6.4 had any advantages over TgTP6.3. RESULTS: Although TgTP6.4 Tg/Tg homozygotes died before weaning, TgTP6.4 Tg/- hemizygotes were viable and fertile and only males were significantly lighter than wild-type. The TgTP6.4 transgene produced the tauGFP fusion protein by the 2-cell stage and it was widely expressed in adults but tauGFP fluorescence was weak or absent in several tissues, including some neural tissues. The TgTP6.4 transgene expression pattern changed over several years of breeding and mosaic transgene expression became increasingly common in all expressing tissues. This mosaicism was used to visualise clonal lineages in the adrenal cortex of TgTP6.4 Tg/- hemizygotes and these were qualitatively and quantitatively comparable to lineages reported previously for other mosaic transgenic mice, X-inactivation mosaics and chimaeras. Mosaicism occurred less frequently in TP6.3 than TP6.4 mice and was only observed in the corneal epithelium and adrenal cortex. CONCLUSIONS: Mosaic expression makes the TgTP6.4 transgene unsuitable for use as a conventional cell lineage marker but such mosaicism provides a useful system for visualising clonal lineages that arise during development or maintenance of adult tissues. Differences in the occurrence of mosaicism between related transgenic lines, such as that described for lines TP6.3 and TP6.4, might provide a useful system for investigating the mechanism of transgene silencing.

Lessons from mouse chimaera experiments with a reiterated transgene marker: revised marker criteria and a review of chimaera markers.

Recent reports of a new generation of ubiquitous transgenic chimaera markers prompted us to consider the criteria used to evaluate new chimaera markers and develop more objective assessment methods. To investigate this experimentally we used several series of fetal and adult chimaeras, carrying an older, multi-copy transgenic marker. We used two additional independent markers and objective, quantitative criteria for cell selection and cell mixing to investigate quantitative and spatial aspects of developmental neutrality. We also suggest how the quantitative analysis we used could be simplified for future use with other markers. As a result, we recommend a five-step procedure for investigators to evaluate new chimaera markers based partly on criteria proposed previously but with a greater emphasis on examining the developmental neutrality of prospective new markers. These five steps comprise (1) review of published information, (2) evaluation of marker detection, (3) genetic crosses to check for effects on viability and growth, (4) comparisons of chimaeras with and without the marker and (5) analysis of chimaeras with both cell populations labelled. Finally, we review a number of different chimaera markers and evaluate them using the extended set of criteria. These comparisons indicate that, although the new generation of ubiquitous fluorescent markers are the best of those currently available and fulfil most of the criteria required of a chimaera marker, further work is required to determine whether they are developmentally neutral.

Re-evaluation of the causes of variation among mouse aggregation chimaeras.

The composition of adult mouse aggregation chimaeras is much more variable than X-inactivation mosaics. An early theoretical model proposed that almost all the extra variation in chimaeras arises, before X-inactivation occurs, by spatially constrained, geometrical allocation of inner cell mass (ICM) cells to the epiblast and primitive endoderm (PrE). However, this is inconsistent with more recent embryological evidence. Analysis of published results for chimaeric blastocysts and mid-gestation chimaeras suggested that some variation exists among chimaeric morulae and more variation arises both when morula cells are allocated to the ICM versus the trophectoderm (TE) and when ICM cells are allocated to the epiblast versus the PrE. Computer simulation results were also consistent with the conclusion that stochastic allocation of cells to blastocyst lineages in two steps, without the type of geometrical sampling that was originally proposed, could cause a wide variation in chimaeric epiblast composition. Later allocation events will cause additional variation among both chimaeras and X-inactivation mosaics. We also suggest that previously published U-shaped frequency distributions for chimaeric placenta composition might be explained by how TE cells are allocated to the polar TE and/or the subsequent movement of cells from polar TE to mural TE.

Selection against BALB/c strain cells in mouse chimaeras.

It has been shown previously that BALB/c strain embryos tend to contribute poorly to mouse aggregation chimaeras. In the present study we showed that BALB/c cells were not preferentially allocated to any extraembryonic lineages of mouse aggregation chimaeras, but their contribution decreased during the early postimplantation period and they were significantly depleted by E8.5. The development of BALB/c strain preimplantation embryos lagged behind embryos from some other strains and the contribution that BALB/c and other embryos made to chimaeras correlated with their developmental stage at E2.5. This relationship suggests that the poor contribution of BALB/c embryos to aggregation chimaeras is at least partly a consequence of generalised selection related to slow or delayed preimplantation development. The suitability of BALB/c embryos for maximising the ES cell contribution to mouse ES cell chimaeras is also discussed.

Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras.

The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1(-/-) null mouse embryos die but a previous study showed that some homozygous Gpi1(-/-) null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1(-/-)↔Gpi1(c/c) chimaera with functional Gpi1(-/-) null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1(-/-) null cells in adult Gpi1(-/-)↔Gpi1(c/c) chimaeras and determine if Gpi1(-/-) null germ cells are functional. Analysis of adult Gpi1(-/-)↔Gpi1(c/c) chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1(-/-) null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1(-/-) null oocytes in one female Gpi1(-/-)↔Gpi1(c/c) chimaera were functional and provided preliminary evidence that one male putative Gpi1(-/-)↔Gpi1(c/c) chimaera produced functional spermatozoa from homozygous Gpi1(-/-) null germ cells. Although the male chimaera was almost certainly Gpi1(-/-)↔Gpi1(c/c), this part of the study is considered preliminary because only blood was typed for GPI. Gpi1(-/-) null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1(-/-) null germ cells, it successfully identified functional Gpi1(-/-) null oocytes and revealed that some Gpi1(-/-) null cells could survive in many adult tissues.

Evaluation of triploid<-->diploid and trisomy-3<-->diploid mouse chimeras as models for investigating how lineage restriction occurs in confined placental mosaicism.

Human confined placental mosaicism (CPM), where the placental trophoblast is mosaic for a chromosome abnormality but the fetus is chromosomally normal, can cause problems for prenatal diagnosis, but its causes are poorly understood. Tetraploid<-->diploid chimeras provide a model for the development of one type of CPM, but animal models for other types of restricted mosaicism are needed. The objective of the present study was to evaluate triploid<-->diploid and trisomy-3<-->diploid chimeric mouse conceptuses as new models for investigating the development of restricted mosaicism. Novel stocks of mice were generated to produce triploid and trisomy-3 embryos that could be identified by DNA in situ hybridisation to a chromosome 3 transgenic marker. Triploid<-->diploid and trisomy-3<-->diploid mouse chimeras were produced by embryo aggregation, and the contribution of triploid or trisomy-3 cells was analysed in the fetus and extraembryonic tissues. Only two trisomy-3<-->diploid chimeras were analysed but trisomy-3 cells contributed well to all lineages, so these chimeras did not show restricted mosaicism. In contrast, triploid cells usually contributed poorly to all lineages in the ten 3n<-->2n chimeras analysed. They contributed more to the primitive endoderm derivatives than other lineages and were present in the primitive endoderm derivatives of all ten chimeras, but excluded from fetuses and trophectoderm derivatives in some cases. This pattern of restricted mosaicism differs from that reported for tetraploid cells in tetraploid<-->diploid chimeras, and triploid<-->diploid chimeras may provide a useful model for the development of some types of restricted mosaicism in human conceptuses.

Evaluation of the mouse TgTP6.3 tauGFP transgene as a lineage marker in chimeras.

The mouse TgTP6.3 transgene, encoding a tauGFP fusion protein, is becoming widely used but has yet to be fully characterized and evaluated as suitable lineage marker. The aim of the present study was to investigate the phenotype of TgTP6.3(+/+) homozygotes and TgTP6.3(+/-) hemizygotes, characterize the expression of the TgTP6.3 transgene in different tissues and critically evaluate its use as a lineage marker. TgTP6.3(+/+) homozygotes died between embryonic day 14.5 and weaning, whereas TgTP6.3(+/-) hemizygotes were mostly viable and fertile but smaller than non-transgenic siblings. TgTP6.3 expression began in the late two-cell stage, persisted in most fetal and adult tissues and was uniformly expressed in many (but not all) tissues. TgTP6.3(+/-) cells were readily identified in many chimeric tissues and their contribution appeared to be quantitatively and spatially normal. Overall, tauGFP expression in hemizygous TgTP6.3(+/-) cells fulfils the main criteria of a good lineage marker for many tissues. It provides a useful lineage marker, which should be particularly suitable for axons, blood vessels and pre-implantation embryos.

Quantitative analysis of mid-gestation mouse aggregation chimaeras: non-random composition of the placenta.

Mouse chimaeras were produced by aggregating eight-cell embryos from two different F2 matings, abbreviated to AF2 and BF2 respectively: (C57BL/ OIa.AKR-Gpi-1s a, c/Ws female × BALB/c male)F2 and (C57BL/Ws female × CBA/Ca male)F2. Quantitative electrophoresis of glucose phosphate isomerase (GPI-1) was used to estimate the proportions of the two cell populations in different tissues of the 12[Formula: see text] day chimàeric conceptuses, with the % GPI-1A indicating the percentage of cells derived from the AF2 embryos. The % GPI-1A was found to be highly positively correlated within the primitive ectoderm lineage (between the fetus, amnion and yolk sac mesoderm) and within the primitive endoderm lineage (between the yolk sac endoderm and the parietal endoderm) but no correlation (either positive or negative) was seen between the two lineages. This confirms the results of a previous,study of chimaeras made between partially congenic strains and suggests the original conclusions have general validity. The % GPI-1A in the placenta was corrected for the expected contribution of maternal GPI-1, based on control experiments involving transfer of homozygous Gpi-1s b /Gpi-1s b embryos to the uteri of Gpi-1s a /Gpi-1s a pseudopregnant females. The corrected % GPI- lA in the placenta was positively correlated with that in each of the three primitive ectoderm derivatives. This suggests either (1) exchange of cells between the polar trophectoderm and the underlying part of the inner cell mass that forms the primitive ectoderm or (2) cells are incompletely mixed in the chimaeric blastocyst and patches of AF2 and BF2 cells straddle the boundary between the polar trophectoderm and the underlying primitive ectoderm. The second explanation does not imply the existence of shared developmental lineages between trophectoderm and primitive ectoderm in non-chimaeric embryos. Unlike that of any other tissue, the distribution of placental GPI-1A was U-shaped; in 17/28 placenta samples the proportion of the minor component was 10% or less. This suggests that the placental trophoblast is derived from a small number of coherenct clones of polar trophectoderm cells (either a small number of polar trophectoderm cells or a larger number if the two cell populations are not finely intermingled). Thus, although as a population the placentas of chimaeric conceptuses are balanced with respect to the % GPI-1A (mean close to 50%), individually most placentas are extremely unbalanced in their chimaeric composition (< 10% or > 90% GPI-IA). This non-random composition of the chimaeric placentas is in contrast to the widely held assumption that the distribution of cells in chimaeric conceptuses is normally random.

Fetal abnormalities produced after preimplantation exposure of mouse embryos to ammonium chloride.

BACKGROUND: The aims of this study were to determine whether preimplantation exposure of mouse embryos to ammonium resulted in abnormal fetal development and to evaluate similar risks to the outcome of human assisted conception. METHODS: Mouse embryos cultured from the 1-cell stage were exposed to 0.3 mmol/l ammonium chloride for 3 days. Embryos cultured from the 2-cell stage were exposed to 0.3 or 0.6 mmol/l ammonium for 2 days. After transfer to the uteri of pseudopregnant recipient females, post-implantation development was evaluated on embryonic day 15.5 (E15.5) or E18.5. RESULTS: There was no consistent effect of preimplantation exposure to ammonium chloride on fetal or placental weights. All 101 E18.5 fetuses were normal but 5/217 E15.5 fetuses were abnormal (three exencephalic and two polydactylous), which was significantly higher than the 0/363 for the pooled groups of E15.5 control fetuses (P = 0.007). The combined E15.5 and E18.5 frequency was also significantly higher than the controls (5/318 versus 0/433; P = 0.013). CONCLUSIONS: These results support the conclusion that abnormal preimplantation culture conditions can cause fetal abnormalities in mice, but the risks may be lower than previously suggested. Further work is needed to evaluate the risk more fully but this risk should be considered when designing new strategies for human assisted conception.

Clonal analysis of patterns of growth, stem cell activity, and cell movement during the development and maintenance of the murine corneal epithelium.

Patterns of growth and cell movement in the developing and adult corneal epithelium were investigated by analysing clonal patches of LacZ-expressing cells in chimeric and X-inactivation mosaic mice. It was found that cell proliferation throughout the basal corneal epithelium during embryogenesis and early postnatal life creates a disordered mosaic pattern of LacZ(+) clones that contrasts with patterns of proliferation and striping produced during the later embryonic stages of retinal pigmented epithelium development. The early mosaic pattern in the corneal epithelium is replaced in the first 12 postnatal weeks by an ordered pattern of radial stripes or sectors that reflects migration without mixing of the progeny of clones of limbal stem cells. In contrast to previous assumptions, it was found that maturation of the activity of limbal stem cells and the pattern of migration of their progeny are delayed for several weeks postnatally. No evidence was found for immigration of the progeny of stem cells until the 5th postnatal week. There are approximately 100 clones of limbal stem cells initially, and clones are lost during postnatal life. Our studies provide a new assay for limbal and corneal defects in mutant mice.