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Dynamics of anterior-posterior axis formation in the developing mouse embryo.

Morris SA, Grewal S, Barrios F, Patankar SN, Strauss B, Buttery L, Alexander M, Shakesheff KM, Zernicka-Goetz M - Nat Commun (2012)

Bottom Line: These 'older' and 'younger' AVE domains coalesce as the egg cylinder emerges from the blastocyst structure.Importantly, we show that AVE migration is led by cells expressing the highest levels of AVE marker, highlighting that asymmetry within the AVE domain dictates the direction of its migration.Ablation of such leading cells prevents AVE migration, suggesting that these cells are important for correct establishment of the AP axis.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge CB2 1QN, UK.

ABSTRACT
The development of an anterior-posterior (AP) polarity is a crucial process that in the mouse has been very difficult to analyse, because it takes place as the embryo implants within the mother. To overcome this obstacle, we have established an in-vitro culture system that allows us to follow the step-wise development of anterior visceral endoderm (AVE), critical for establishing AP polarity. Here we use this system to show that the AVE originates in the implanting blastocyst, but that additional cells subsequently acquire AVE characteristics. These 'older' and 'younger' AVE domains coalesce as the egg cylinder emerges from the blastocyst structure. Importantly, we show that AVE migration is led by cells expressing the highest levels of AVE marker, highlighting that asymmetry within the AVE domain dictates the direction of its migration. Ablation of such leading cells prevents AVE migration, suggesting that these cells are important for correct establishment of the AP axis.

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In vitro culture of blastocysts on collagen-coated polyacrylamide matrices allows successful development to the egg cylinder stage.(a, b) DIC time-lapse observations of in vitro development of two representative blastocysts on collagen-coated polyacrylamide gels covalently bound to a glass-bottomed dish. Each panel shows a single embryo continuously observed from the second day of in vitro culture (ivc), until the fifth day. Panels (a) and (b) show selected frames from Supplementary Movies 1 and 2, respectively. Elapsed developmental time from the point at which the blastocyst was placed on the gel is shown at the top of each frame. Scale bars represent 50 μm. (c, d) Development of two further representative embryos in vitro and observed using a ×40 objective. DIC images are shown alongside schematics showing different tissues: epiblast, blue; primitive/visceral endoderm, yellow; trophectoderm/ExE, grey; the pro-amniotic cavity is highlighted by an asterisk. The overlying trophectoderm is omitted from the schematics of the third and fourth day of in vitro culture, for clarity. Development of the embryo shown in (c) is presented in Supplementary Movie 3. (e) On day 5 of in vitro culture, the embryo shown in (d) was removed from the gel and stained with the lipophilic dye FM4-64. DIC, selected confocal stacks and projected images are shown. (f) DIC time-lapse observations during 4 days of development in vitro. A representative embryo is shown for each day of culture. Elapsed developmental time from the point at which the blastocyst was placed on the gel is shown at the top of the first and the last frame presented for each day. Time points at the bottom of each still represent individual time frames from time-lapse observations on the indicated day. Embryonic and extra-embryonic structures are indicated as follows: primitive endoderm (blastocyst) and visceral endoderm (egg cylinder), yellow-dashed line; trophectoderm (blastocyst) and ExE (egg cylinder), white-dashed line; epiblast, blue-dashed line. Scale bars represent 50 μm.
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f1: In vitro culture of blastocysts on collagen-coated polyacrylamide matrices allows successful development to the egg cylinder stage.(a, b) DIC time-lapse observations of in vitro development of two representative blastocysts on collagen-coated polyacrylamide gels covalently bound to a glass-bottomed dish. Each panel shows a single embryo continuously observed from the second day of in vitro culture (ivc), until the fifth day. Panels (a) and (b) show selected frames from Supplementary Movies 1 and 2, respectively. Elapsed developmental time from the point at which the blastocyst was placed on the gel is shown at the top of each frame. Scale bars represent 50 μm. (c, d) Development of two further representative embryos in vitro and observed using a ×40 objective. DIC images are shown alongside schematics showing different tissues: epiblast, blue; primitive/visceral endoderm, yellow; trophectoderm/ExE, grey; the pro-amniotic cavity is highlighted by an asterisk. The overlying trophectoderm is omitted from the schematics of the third and fourth day of in vitro culture, for clarity. Development of the embryo shown in (c) is presented in Supplementary Movie 3. (e) On day 5 of in vitro culture, the embryo shown in (d) was removed from the gel and stained with the lipophilic dye FM4-64. DIC, selected confocal stacks and projected images are shown. (f) DIC time-lapse observations during 4 days of development in vitro. A representative embryo is shown for each day of culture. Elapsed developmental time from the point at which the blastocyst was placed on the gel is shown at the top of the first and the last frame presented for each day. Time points at the bottom of each still represent individual time frames from time-lapse observations on the indicated day. Embryonic and extra-embryonic structures are indicated as follows: primitive endoderm (blastocyst) and visceral endoderm (egg cylinder), yellow-dashed line; trophectoderm (blastocyst) and ExE (egg cylinder), white-dashed line; epiblast, blue-dashed line. Scale bars represent 50 μm.

Mentions: We set out to use these collagen-coated polyacrylamide matrices to follow the cellular dynamics of an egg cylinder emergence by spinning-disc, confocal time-lapse microscopy. Typically, when zona-free E3.5 blastocysts were seeded onto the culture gels, embryos developed over 5 days following the indicated time course (Fig. 1a–d; Supplementary Movies 1–3). We confirmed the normal morphology of the egg cylinders that had developed from the blastocysts in vitro by recovering them from the gel after time-lapse imaging and staining them with the vital lipophilic dye FM4-64 to reveal individual cells by optical sectioning (Fig. 1e).


Dynamics of anterior-posterior axis formation in the developing mouse embryo.

Morris SA, Grewal S, Barrios F, Patankar SN, Strauss B, Buttery L, Alexander M, Shakesheff KM, Zernicka-Goetz M - Nat Commun (2012)

In vitro culture of blastocysts on collagen-coated polyacrylamide matrices allows successful development to the egg cylinder stage.(a, b) DIC time-lapse observations of in vitro development of two representative blastocysts on collagen-coated polyacrylamide gels covalently bound to a glass-bottomed dish. Each panel shows a single embryo continuously observed from the second day of in vitro culture (ivc), until the fifth day. Panels (a) and (b) show selected frames from Supplementary Movies 1 and 2, respectively. Elapsed developmental time from the point at which the blastocyst was placed on the gel is shown at the top of each frame. Scale bars represent 50 μm. (c, d) Development of two further representative embryos in vitro and observed using a ×40 objective. DIC images are shown alongside schematics showing different tissues: epiblast, blue; primitive/visceral endoderm, yellow; trophectoderm/ExE, grey; the pro-amniotic cavity is highlighted by an asterisk. The overlying trophectoderm is omitted from the schematics of the third and fourth day of in vitro culture, for clarity. Development of the embryo shown in (c) is presented in Supplementary Movie 3. (e) On day 5 of in vitro culture, the embryo shown in (d) was removed from the gel and stained with the lipophilic dye FM4-64. DIC, selected confocal stacks and projected images are shown. (f) DIC time-lapse observations during 4 days of development in vitro. A representative embryo is shown for each day of culture. Elapsed developmental time from the point at which the blastocyst was placed on the gel is shown at the top of the first and the last frame presented for each day. Time points at the bottom of each still represent individual time frames from time-lapse observations on the indicated day. Embryonic and extra-embryonic structures are indicated as follows: primitive endoderm (blastocyst) and visceral endoderm (egg cylinder), yellow-dashed line; trophectoderm (blastocyst) and ExE (egg cylinder), white-dashed line; epiblast, blue-dashed line. Scale bars represent 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293425&req=5

f1: In vitro culture of blastocysts on collagen-coated polyacrylamide matrices allows successful development to the egg cylinder stage.(a, b) DIC time-lapse observations of in vitro development of two representative blastocysts on collagen-coated polyacrylamide gels covalently bound to a glass-bottomed dish. Each panel shows a single embryo continuously observed from the second day of in vitro culture (ivc), until the fifth day. Panels (a) and (b) show selected frames from Supplementary Movies 1 and 2, respectively. Elapsed developmental time from the point at which the blastocyst was placed on the gel is shown at the top of each frame. Scale bars represent 50 μm. (c, d) Development of two further representative embryos in vitro and observed using a ×40 objective. DIC images are shown alongside schematics showing different tissues: epiblast, blue; primitive/visceral endoderm, yellow; trophectoderm/ExE, grey; the pro-amniotic cavity is highlighted by an asterisk. The overlying trophectoderm is omitted from the schematics of the third and fourth day of in vitro culture, for clarity. Development of the embryo shown in (c) is presented in Supplementary Movie 3. (e) On day 5 of in vitro culture, the embryo shown in (d) was removed from the gel and stained with the lipophilic dye FM4-64. DIC, selected confocal stacks and projected images are shown. (f) DIC time-lapse observations during 4 days of development in vitro. A representative embryo is shown for each day of culture. Elapsed developmental time from the point at which the blastocyst was placed on the gel is shown at the top of the first and the last frame presented for each day. Time points at the bottom of each still represent individual time frames from time-lapse observations on the indicated day. Embryonic and extra-embryonic structures are indicated as follows: primitive endoderm (blastocyst) and visceral endoderm (egg cylinder), yellow-dashed line; trophectoderm (blastocyst) and ExE (egg cylinder), white-dashed line; epiblast, blue-dashed line. Scale bars represent 50 μm.
Mentions: We set out to use these collagen-coated polyacrylamide matrices to follow the cellular dynamics of an egg cylinder emergence by spinning-disc, confocal time-lapse microscopy. Typically, when zona-free E3.5 blastocysts were seeded onto the culture gels, embryos developed over 5 days following the indicated time course (Fig. 1a–d; Supplementary Movies 1–3). We confirmed the normal morphology of the egg cylinders that had developed from the blastocysts in vitro by recovering them from the gel after time-lapse imaging and staining them with the vital lipophilic dye FM4-64 to reveal individual cells by optical sectioning (Fig. 1e).

Bottom Line: These 'older' and 'younger' AVE domains coalesce as the egg cylinder emerges from the blastocyst structure.Importantly, we show that AVE migration is led by cells expressing the highest levels of AVE marker, highlighting that asymmetry within the AVE domain dictates the direction of its migration.Ablation of such leading cells prevents AVE migration, suggesting that these cells are important for correct establishment of the AP axis.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge CB2 1QN, UK.

ABSTRACT
The development of an anterior-posterior (AP) polarity is a crucial process that in the mouse has been very difficult to analyse, because it takes place as the embryo implants within the mother. To overcome this obstacle, we have established an in-vitro culture system that allows us to follow the step-wise development of anterior visceral endoderm (AVE), critical for establishing AP polarity. Here we use this system to show that the AVE originates in the implanting blastocyst, but that additional cells subsequently acquire AVE characteristics. These 'older' and 'younger' AVE domains coalesce as the egg cylinder emerges from the blastocyst structure. Importantly, we show that AVE migration is led by cells expressing the highest levels of AVE marker, highlighting that asymmetry within the AVE domain dictates the direction of its migration. Ablation of such leading cells prevents AVE migration, suggesting that these cells are important for correct establishment of the AP axis.

Show MeSH
Related in: MedlinePlus