Jean H. Flockhart

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.

DEATH OF MOUSE EMBRYOS THAT LACK A FUNCTIONAL GENE FOR GLUCOSE PHOSPHATE ISOMERASE

A null allele of the Gpi-1s structural gene, that encodes glucose phosphate isomerase (GPI-1; E.C. 5.3.1.9), arose in a mutation experiment and was designated Gpi-1sa-m1H. The viability of homozygotes has been investigated. No offspring homozygous for the null allele were produced by intercrossing two heterozygotes, so the homozygous condition was presumed to be embryonic lethal. Embryos were produced by crossing Gpi-1sa/null heterozygous females and Gpi-1sb/null heterozygous males. Homozygous null embryos were identified at different stages of development by electrophoresis and staining either for GPI-1 alone or GPI-1 plus phosphoglycerate kinase (PGK) activity. At 6 1/2 and 7 1/2 days post coitum homozygous null embryos were present at approximately the expected 25% frequency (37/165; 22.4% overall) although at 7 1/2 days the homozygous null embryos tended to be small. By 8 1/2 days most homozygous null embryos were developmentally retarded and had not developed significantly further than at 7 1/2 days; some were dead or dying. By 9 1/2 days the homozygous null conceptus was characterised by a small implantation site that contained trophoblast and often a small amount of extraembryonic membrane. Surviving trophoblast tissue was also detectable at 10 1/2 days. Previous studies have shown that oocyte-coded GPI-1 persists only until 5 1/2 or 6 1/2 days. Survival of homozygous null embryos to 7 1/2 or 8 1/2 days and survival of certain extraembryonic tissue to 10 1/2 days suggests that the homozygous null condition may not be cell-lethal although it is certainly embryo-lethal. Mutant cells that are deficient in glycolysis may use the pentose phosphate shunt to bypass the block in glycolysis created by the deficiency of glucose phosphate isomerase, and/or might be rescued by the transport, from the maternal blood, of energy sources other than glucose (such as glutamine). Either strategy may only permit slow cell growth that would not be adequate to support normal embryogenesis. Transport of maternal nutrients would be more efficient to the trophoblast and extraembryonic membranes and this may help to explain why these tissues survive for longer than the embryo itself. The morphological similarity between homozygous nulls and androgenetic conceptuses, where the trophoblast also survives better than the embryo, is discussed.

A maternal genetic effect on the composition of mouse aggregation chimaeras

Two series of 12 1/2 day mouse chimaeric conceptuses were produced by aggregating (C57BL x CBA)F2 strain preimplantation embryos with embryos that differed at the Gpi-1s locus that encodes glucose phosphate isomerase, GPI-1. The composition of individual issues was evaluated by quantitative electrophoresis to estimate the % GPI-1A in the chimaeric tissue containing GPI-1A and GPI-1B. In one series of chimaeras, the GPI-1A cells were derived from a backcross between inbred BALB/c strain females and (BC x BALB/c)F1 males, where BC is the partly congenic strain C57BL/Ola.AKR-Gpi-lsa,c/Ws. In the other series of chimaeras, the GPI-1A cells were derived from the reciprocal backcross between (BC x BALB/c)F1 females and inbred BALB/c strain males. The [(BC x BALB/c)F1 female x BALB/c male]<==>(C57BL x CBA)F2 series of chimaeras was reasonably balanced so that GPI-1A and GPI-1B cells were fairly equally represented in the foetuses, placentas and extraembryonic membranes (tissue means: 37-51% GPI-1A). This series did not differ significantly in composition from an earlier series of (BC x BALB/c)F2<==>(C57BL x CBA)F2 chimaeras. However, the [BALB/c female x (BC x BALB/c)F1 male]<==>(C57BL x CBA)F2 series of chimaeras was unbalanced, with mean tissue compositions (28-33% GPI-1A) that were intermediate between the above two balanced series and the unbalanced (BALB/c x BALB/c)<==>(C57BL x CBA)F2 series (tissue means: 14-22% GPI-1A), that was studied previously. Thus, both (BALB/c x BALB/c) and [BALB/c x (BC x BALB/c)F1] embryos contributed less to the tissues of chimaeric conceptuses than either (BC x BALB/c)F2 or [BC x BALB/c)F1 x BALB/c] embryos. This implies that embryos from BALB/c mothers contributed less to the tissues of chimaeric conceptuses than embryos from (BC x BALB/c)F1 mothers. We, therefore, conclude that a maternal genetic effect is responsible for some of the differences in composition among the four groups of chimaeras. This maternal effect must act before the 8-cell stage but it is not yet known whether it is mediated via cytoplasmic inheritance, genomic imprinting or by the reproductive tract. Evidence that a maternal effect retards preimplantation development of embryos from BALB/c females is reviewed and the possibility that this might cause them to contribute poorly to chimaeric conceptuses when aggregated with more precociously developing embryos is discussed.

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

AbstractMouse chimaeras were produced by aggregating eight-cell embryos from two different F2 matings, abbreviated to AF2 and BF2 respectively: (C57BL/ OIa.AKR-Gpi-1sa, 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

High activity of an unstable form of glucose phosphate isomerase in the mouse

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

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

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

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Non-additive inheritance of glucose phosphate isomerase activity in mice heterozygous at the Gpi-1s structural locus

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