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Transient pluripotent cell populations during primitive ectoderm formation: correlation of in vivo and in vitro pluripotent cell development.

T. A. Pelton^*, S. Sharma^*,, T. C. Schulz^*,, J. Rathjen and P. D. Rathjen^¶

Department of Molecular Biosciences, University of Adelaide, Adelaide 5005, South Australia
^* Contributed equally to this work
Current address: Department of Ophthalmology, Flinders University of South Australia, Bedford Park, SA 5042, Australia
Current address: Laboratory of Cellular and Developmental Biology, Section of Mammalian Development, NIDDK, NIH, Bethesda, MD 50892, USA

View larger version (58K): [in a new window]   Fig. 1. IdPCR and ddPCR analysis of ES and EPL cell RNA. (A) Cytoplasmic RNA was isolated from ES cells and EPL cells grown for 4 or 6 days in the presence of 50% MEDII + LIF (+). Then, 2 µg of this RNA were analysed by idPCR using the 5' primer OPB-04 (GGACTGGAGT; Operon Technologies). PCR products were resolved by electrophoresis on a 6% (v/v) denaturing polyacrylamide gel. (B) Cytoplasmic RNA was isolated from ES cells and EPL cells grown for 2, 4 or 6 days in 50% MEDII in the presence (+) or absence (–) of LIF. Then, 2 µg of this RNA were analysed by ddPCR using the 5' primer OPA-01 (CAGGCCCTTC; Operon Technologies) PCR products were resolved by electrophoresis on a 6% (v/v) denaturing polyacrylamide gel. Arrows mark the indicated products.

View larger version (45K): [in a new window]   Fig. 2. Expression of cloned PCR products in pluripotent cells. (A) Northern blot analysis of 4 µg poly-A+ RNA, isolated from ES cells and EPL cells grown for 2, 4 or 6 days in 50% MEDII in the presence (+) or absence (–) of LIF, probed for Oct4, CRTR1, Rex1, Psc1, Fgf5 and mGAP. Transcript sizes were: Oct4, 1.6 kb; CRTR-1, 9.4 kb; Rex1, 1.9 kb; Psc1, 5.5 kb; Fgf5, 2.7 kb; and mGAP, 1.5 kb. (Bi) Northern blot analysis of 25 µg total RNA isolated from ES cells and EPL cells grown for 2, 4, 6 or 8 days in 50% MEDII in the presence of LIF (+) and probed for PRCE and mGAP expression. Transcript sizes were: PRCE, 6.6 kb; mGAP, 1.5 kb. (Bii) Quantitation of northern in (Bi). PRCE expression was normalized against the mGAP loading control and expressed as a ratio, with ES cells assigned a value of 1. (C) In situ hybridization of ES cells (i, iii) and EPL cells cultured for 2 days in the presence of MEDII + LIF (ii, iv) with Oct4 (i, ii) and PRCE (iii,iv) antisense digoxygenin-labelled riboprobes. Images were captured using Hoffmann interference contrast microscopy. Bar, 90 µm. (D) In situ hybridization of EPL cells cultured for 4 days in the presence of MEDII and LIF with CRTR-1 (i, ii) and PRCE (iii, iv) antisense digoxygenin-labelled riboprobes. (i, iii) Phase contrast microscopy; (ii, iv) bright field microscopy. Arrows denote differentiated cells. Bar, 25 µm. Equivalent cell populations to those illustrated in C and D were probed with digoxygenin-labelled sense probes and failed to develop colour (data not shown).

View larger version (50K): [in a new window]   Fig. 3. Sequence and analysis of PRCE and Psc1. (A) Alignment of the PRCE ddPCR product sequence with the KIAA0165 cDNA sequence. (B) Alignment of the Psc1 idPCR product nucleotide sequence and open reading frame (ORF) with the predicted C. elegans ORF CELB0336.3.

View larger version (65K): [in a new window]   Fig. 4. Definition of embryonic stages and identification of pluripotent cell populations by analysis of Oct4 expression. Whole mount in situ hybridization analysis of 4.5 d.p.c. (A), 4.75 d.p.c. (B), 5.0 d.p.c. (C), 5.25 d.p.c. (D) and 5.5 d.p.c. (E) mouse embryos using antisense digoxygenin-labelled Oct4 riboprobes. (F) A 5.0 d.p.c. embryo analysed by whole mount in situ hybridization using antisense digoxygenin-labelled PRCE riboprobes. (G) A 5.25 d.p.c. embryo and (H) a 5.5 d.p.c. embryo analysed by whole mount in situ hybridization using sense digoxygenin-labelled PRCE riboprobes. (F-H) These images are included to illustrate the formation of the proamniotic cavity (PAC) and pseudostratified epithelial layer of primitive ectoderm (PE). Abbreviations: EEE, extra-embryonic ectoderm; ICM, inner cell mass; TE, trophectoderm; TGC, trophoblast giant cells; VEn, visceral endoderm. Bar, 50 µm. The colouration in the trophectoderm of the 4.5 d.p.c. blastocyst is not cellular but can be attributed to a shadow rather than gene expression.

View larger version (90K): [in a new window]   Fig. 5. Expression of CRTR-1, Rex1 and Psc1 during early mouse embryogenesis. Analysis of CRTR-1 (A), Rex1 (B) and Psc1 (C) expression by whole mount in situ hybridization of 3.5-5.5 d.p.c. mouse embryos using antisense digoxygenin-labelled riboprobes. Bar, 50 µm. Display of two embryos at a given time point indicates inconsistent expression, interpreted as marking the time of downregulation.

View larger version (63K): [in a new window]   Fig. 6. Expression of PRCE and Fgf5 during early mouse embryogenesis Analysis of PRCE (A) and Fgf5 (B) expression by whole mount in situ hybridization of 3.5-5.5 d.p.c. mouse embryos using antisense digoxygenin-labelled riboprobes. Bar, 50 µm. Display of two embryos at a given time point indicates inconsistent expression, interpreted as marking the time of upregulation.