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First published online 19 December 2007
doi: 10.1242/dev.014894

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Constitutive PtdIns(3,4,5)P₃ synthesis promotes the development and survival of early mammalian embryos

¹ Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK.
² Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.

View larger version (52K): [in this window] [in a new window]   Fig. 1. Constitutive PtdIns(3,4,5)P3 synthesis in preimplantation embryos. Confocal sections of mouse embryos expressing GFP-PHGRP1. The following stages are shown (left to right, top to bottom): one-cell, two-cell, four-cell, compacted eight-cell, 16-cell morula and early blastocyst. Embryos were cultured at high density (one embryo/µl). Confocal images are representative of at least 12 similar observations. Scale bar: 20 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 2. Apical PtdIns(3,4,5)P3 is promoted by high-density culture. (A) PtdIns(3,4,5)P3 distribution in two-cell embryos cultured in high- or low-density conditions. PtdIns(3,4,5)P3 was detected with GFP-PHGRP1 or GFP-PHAkt, and fluorescence linescans (right) were aquired across the apical membrane, avoiding the nucleus (blue and red bars). Cytosolic fluorescence was normalised to 1 and apical PtdIns(3,4,5)P3 was considered to be present when fluorescence in the cortex reached a value of 1.2 (dotted line on the linescan graphs). Arrowheads indicate the position of the second polar body. (B) Analysis of the data collected using linescan analysis, as shown in A. The proportion of embryos with (blue) or without (red) apical PtdIns(3,4,5)P3 is expressed as a percentage. Culture conditions and the number of embryos examined are indicated on the x-axis. *P<0.0001; #P<0.001.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Junctional PtdIns(3,4,5)P3 synthesis is due to E-cadherin signalling. (A) Junctional PtdIns(3,4,5)P3 synthesis in a 16-cell morula (left) is inhibited upon removal of extracellular Ca2+ (middle; note the decompaction of the embryo). Readmission of extracellular Ca2+ allowed for PtdIns(3,4,5)P3 re-synthesis at cell-cell contacts (right; note embryo re-compaction). (B) Two-cell embryo (from a low-density culture; top) and 16-cell embryo (bottom) cultured in the presence of ECCD-1. The anti-E-cadherin antibody prevented junctional PtdIns(3,4,5)P3 synthesis and embryo compaction. (C) PtdIns(3,4,5)P3 synthesis upon embryo aggregation. The arrowhead points to the new contact region between two aggregated two-cell embryos. PtdIns(3,4,5)P3 was monitored with GFP-PHGRP1 (A,B) or GFP-PHAkt (C). Confocal images are representative of at least 12 similar observations.

View larger version (41K): [in this window] [in a new window]   Fig. 4. PI3K activity promotes early embryo development and survival. (A) Chronic PI3K inhibition with LY294002 prevents development beyond the two-cell stage. One-cell embryos were cultured continuously in the presence of LY294002 (10 µM, grey) or the equivalent amount of DMSO (black), in 3 ml of medium. Over 100 embryos were scored in each experimental condition. *P<0.0001; #P<0.01. (B) Developmental competence of embryos injected with 0.18 µg/µl (grey bar, 1x) or 1.8 µg/µl (dark green, 10x) of GFP-PHGRP1 cRNA, or GFP cRNA (2.0 µg/µl, light green). Control, non-injected embryos are shown in black. Culture was performed in high-density conditions. The number of embryos examined is indicated on the graph. *P<0.0001. (C) PI3K inhibition with LY294002 prevents the morula-blastocyst transition. Embryos were cultured in high-density conditions until they reached a given stage, before being exposed to LY294002 (without mineral oil). Embryos were checked the next day to examine their progression to the next developmental stage (indicated on the x-axis). Thirty to 50 embryos were scored for each developmental transition. *P<0.0001. (D) 16-32 cell morula recovered from a high-density culture and exposed to 10 µM LY294002 for 20 hours, without mineral oil. TUNEL staining reveals apoptosis in virtually all blastomeres (right panel). The arrowhead indicates a small blastocoel-like cavity. (E) Expanded blastocyst obtained after culturing a 16-32 cell morula in the presence of DMSO for 20 hours, without mineral oil. Little apoptosis was detectable (TUNEL staining, right). (F) Eight-cell embryo cultured in the presence of ECCD-1 from the one-cell stage, in high-density conditions. All blastomeres were negative for TUNEL staining (right). (G) 16-32 cell embryo cultured in the presence of ECCD-1 from the one-cell stage, in high-density conditions, exhibiting extensive TUNEL staining (right). All TUNEL images (D-G) are projections of multiple confocal sections across the whole embryo, and are representative of 12-26 similar observations.