Film archive : From oocyte to 16 cell stage: the cytoplasmic and cortical  reorganizations which pattern the ascidian embryo.

1 - Maturation and
Polarization

                                      FILMS 1-9

Reorganizations between fertilization and first cleavage in ascidians.
The cortical endoplasmic reticulum domain (cER: red) and subcortical mitochondria-rich domain (myoplasm : green) are relocalized in 2 major phases which depend on microfilaments and microtubules (blue lines). Black dots represent putative germ plasm granules (Flash animation by Nico Sardet)
2 - Fertilization and Reorganizations
                                      FILMS 10-23
3 - Cleavages and
Asymmetric  Divisions

                                     FILMS 24-29
4 - Development to
Tadpoles

                                      FILMS 30-32

 

Maturation and Polarization                  
ZOOM

FILM 1

630 Ko

Germinal Vesicle BreakDown (GVBD) and elaboration of the chorion in a spontaneously maturing oocyte of Ciona . Small spherical test cells individualize and then move inside the vitelline space which forms during GVBD. At the end of GVBD, a central light zone corresponding to the meiotic apparatus can be distinguished in the center of the oocyte. Follicle cells enlarge progressively from the beginning to the end of this sequence. Time lapse: images were acquired every 30 s for 12 h.Transmitted light microscopy.
Prodon,et al. (2006) Dev.Biol, 290, 297-311 PDF

FILM 2

470 Ko

GVBD and migration of the meiotic apparatus towards the cell cortex in a dechorionated oocyte of Ciona .The light zone corresponding to the meiotic apparatus is formed in the center of the oocyte and migrates upward to the cortex over a 2 hour period. DIC optics.
Prodon et al. (2006) Dev.Biol, 290, 297-311 PDF

FILM 3

337 Ko

GVBD in three dechorionated oocytes of Ciona exposed to sea water. Oocyte in the upper right corner displays a transient movement just before the breakdown of the GV. Transmitted light microscopy, Time lapse : images were acquired every 45 s for 2 h 11 min.
Prodon et al. (2006) Dev.Biol, 290, 297-311 PDF

FILM 4

490 Ko

Polarization of the subcortical domain rich in mitochondria ( myoplasm: green) in Ciona during maturation.
Mitochondria labelled with DiOC2(3) accumulate progressively in the vegetal hemisphere (lower left corner) in this oocyte. Fluorescence microscopy, Time lapse : images were acquired every 2 min in the same confocal plane for 3 h 30 min starting after the complete disruption of the Germinal Vesicle.
Prodon et al.
(2006) Dev.Biol., 290, 297-311 PDF

FILM 5

400 Ko

Polarity of mitochondria-rich myoplasm in a mature Ciona oocyte.
Confocal z sections of an oocyte whose mitochondria are labelled with DiOC2(3). Images were acquired at 2 µm intervals from the surface to the equator. Individual mitochondria are seen as bright green dots or rods. Larger vesicles in light green correspond to yolk platelets. The vegetal pole is on the lower left.
Prodon et al. (2005) J.Cell Science, 119, 1592-1603 PDF

FILM 6

790 Ko

Polarity of the endoplasmic reticulum (ER) network in a mature Ciona oocyte.
Confocal z sections of an oocyte whose ER is labelled with a droplet of DiIC16(3) injected into the oocyte. Images were acquired at 1 µm intervals from the surface to the equator. The vegetal pole is on the lower left.
Prodon et al. (2005) J.Cell Science, 119, 1592-1603 PDF

FILM 7

780 Ko

Cortical endoplasmic reticulum (cER) network under the surface of a mature Ciona oocyte.
Transition zone between the animal (upper right) and vegetal(lower left) hemispheres showing the distribution of mitochondria (green) and ER (red) under the surface . Sequential confocal z sections were acquired at 0.5µm intervals.
Prodon et al. (2005) J.Cell Science, 119, 1592-1603 PDF

FILM 8

1 Mo
The ER network in a mature Phallusia oocyte.
The ER network(white) is labelled in a mature oocyte injected with a droplet of DiIC(16)3. Confocal z sections (28) were acquired in the vegetal hemisphere at intervals of 0.5 microns from the surface into the myoplasm (frame is 10 microns on a side).
Speksnijder et al. (1993) J. Cell Biology, 120, 1337-1346 PDF
FILM 9

2,6 Mo

ER network and microtubules in an solated cortical fragment from a mature Phallusia oocyte.
The fluorescent images show alternately the cER network (labelled with DiOC(6)3) and microtubules (labelled with antitubulin antibody) in a fixed cortical fragmant. ER tubes and sheets are most abundant on the vegetal side (left). The second part of of the film shows a high resolution DIC image of a live cortical fragment in which a part of the cER network is attached to the underlying plasma membrane.
Sardet et al. (1992) Development, 115, 221-237. PDF

Fertilization and Reorganizations             
ZOOM

FILM 10

1,6 Mo

From fertilization to first cleavage in Phallusia.
Fertilization triggers a cortical contraction and meiosis completion. A transient contraction pole is formed (on left) followed by the emission of a first polar body (on right), meiotic oscillations and then emisson of the second polar body. Migration of male and female pronuclei follows. Large rotational movements of the posterior cytoplasm (top) with respect to the cortex take place after the pronuclei meet(DIC optics). Spindle formation and mitosis are then imaged using polarization optics and cleavage occurs at 60 minutes after fertilization. Time lapse sequence (DIC and polarized microscopy) recorded with S Inoue, L Jaffe and J.E.Speksnijder, speeded up 150 times.
Sardet, C., et al. (1989). Development 105, 237-249 PDF

FILM 11

230 Ko

Fertilization, contraction and reorganization of the mitochondria-rich myoplasm domain in Phallusia.
Two eggs with their mitochondria labelled with DiO(C2)3 (white) undergo an actomyosin-driven cortical contraction triggered by the fertilization calcium wave. This contraction concentrates the myoplasm around a vegetal/contraction pole which lasts about 5 minutes. The location of the vegetal/contraction pole corresponds to the future Dorsal pole of the ascidian embryo. Time lapse fluorescence optics, speeded up 60 times. Roegiers F.,et al. (1995) Development, 121, 3457-3466
PDF

FILM 12

512 Ko

Fertilization, contraction and reorganization of the cortical endoplasmic reticulum (cER) domain in Phallusia.
Here the continuous ER network in the egg is labelled with injected DiIC(16)3 (white). The actomyosin-driven cortical contraction triggered by the fertilization calcium wave leads to concentration of both the cER and the adjacent ER-poor mitochondria-rich myoplasm in the vegetal/contraction pole (bottom). After contraction, the egg undergoes repetitive contractions or pulses which correspond to meiotic oscillations. Time lapse confocal microscopy of a single equatorial section, speeded up 10 times.
Speksnijder J.E., et al. (1993) J. Cell Biol. 120, 1337-1346 PDF

FILM 13

370 Ko

Fertilization calcium wave and following waves emitted by pacemaker PM1 in Phallusia.
A wave of elevated calcium(yellow-red) starts from the sperm entry site (in animal hemisphere, top) and travels vegetally. The egg cortex contracts in response to the fertilization calcium wave. This large wave is followed by smaller calcium waves emitted by pacemaker PM1 which moves vegetally along the cortex with the sperm aster. After a pause all subsequent waves come from pacemaker PM2 in contraction pole (bottom). Time lapse confocal ratio imaging in an equatorial plane covering the first seven minutes after fertilization.
Dumollard R. & Sardet, C. (2001), J. Cell Science, 114, 2471-2481.PDF
McDougall, A. & Sardet, C. (1995), Current Biology. 5, 318-328. PDF

FILM 14

221 Ko

Meiotic calcium waves: Pacemaker PM2 in action (Phallusia).
Between meiosis I and meiosis II (5-30 minutes PF), 6 to 12 calcium waves (yellow) are generated by a calcium wave pacemaker (PM2) situated in the vegetal/contraction pole(bottom of image). These repetitive calcium waves traverse the egg and produce cycles of contraction and relaxation of the cortex. Time lapse confocal ratio imaging in an equatorial plane.

Roegiers, F. et al. (1999). Development, 126, 3101-3117
McDougall, A. & Sardet, C. (1995), Current Biology. 5, 318-328. PDF
FILM 15

560 Ko
Fertilization calcium wave triggers surface movements (Phallusia).
The calcium wave (red) propagates from the sperm entry point. When the wave reaches the antipode, nile blue particles (black) attached to the surface begin to move with the cortical contraction.Time lapse microscopy of the first 5 minutes after fertilization.
Roegiers et al. (1999) Development, 126, 3101-3117.PDF

FILM 16

440 ko

Meiotic contractions, Vegetal Button formation, and the second major phase of reorganization (Phallusia).
The sequence corresponds to the period between the end of Meiosis I (10 minutes after fertilization) and first cleavage ( 60 minutes after fertilization). Observe: periodic contractions, second polar body formation (upper right), vegetal button protrusion ( lower left), the meeting of male and female pronuclei, and a large translocation corresponding to the second major phase of reorganization. Posterior is up. Time lapse DIC microscopy : this sequence is speeded up 100 times
Sardet C., et al. (1989). Development, 105, 237-249 PDF
Roegiers et al. (1999).   Development, 126, 3101-3117. PDF

FILM 17

516 Ko
Vegetal button formation and resorption (Phallusia ).
The vegetal button forms 30 minutes after fertilization and resorbs 10 minutes later in the vegetal pole region (bottom). It is rich in microvilli. Sperm aster microtubules can be discerned upper left. Time lapse, DIC microscopy.
Roegiers et al. (1999) Development, 126, 3101-3117. PDF

FILM 18

550 Ko

Close up of male aster centrosomal region during the second major phase of reorganization (Phallusia):
35 minutes after fertilization,
the male and female pronuclei , centrosomal and mitochondria-rich domains (granular area on the rightof the nucleus ) move together from the posterior cortex (on the right) to the egg center . The rotational movement is driven by aster Microtubules located in the ER-rich domain (smooth regions) sliding against Cortex (Phallusia mammillata).
Sardet, C., et al. (1989).   Development, 105, 237-249 PDF
Roegiers F.,et al. (1999) Development, 126, 3101-3117
PDF

FILM 19

1,1 Mo

Microtubules in the posterior-vegetal region at the time of the second major phase of reorganization (Phallusia).
Posterior pole region of an egg fixed 35 minutes after fertilization and immunolabelled for tubulin (white). The male pronucleus is seen as a non labelled circle and the microtubule-poor region near the cortex is the mitochondria-rich myoplasm.
Confocal z sections were acquired at 1 micron increments; the stack plays once slowly then several times faster.
Roegiers F. et al. (1999) Development 126, 3101-3117.PDF

FILM 20

100 Ko

Vibrations of the posterior-vegetal region at the time of the second major phase of reorganization (Phallusia).
The posterior cortex vibrates at high frequency due to microtubules contacting and coursing along the surface.DIC optics. Time lapse recording covering the period 30-35 minutes after fertilization.
Roegiers F. et al. (1999) Development 126, 3101-3117. PDF

FILM 21

1,3 Mo

The ER network at the time of the second major phase of reorganization (Phallusia).
A fertilized egg whose ER network is labelled with DiIC16(3) (white) . The bright spot at the top is the site of injected DiI. Five series of confocal z sections were acquired at one minute intervals over the period 30-35 minutes after fertilization. The cER network (bottom part of the image) translocates towards the sperm aster (left part of the image) .
Speksnijder J.E., et al. (1993) J. Cell Biol. 120, 1337-1346 PDF

FILM 22

985 Ko

Reorganizations of ER network (red) and mitochondria-rich myoplasm (green) in Phallusia.
This film covers the period from fertilization to mitosis. It shows - the first major phase of reorganization (contraction concentrating myoplasm and cER vegetally) - the second major phase of reorganization (posterior translocation of the bulk of myoplasm and cER). The animal pole is in the upper right corner, posterior is left. The bright red droplet corresponds to the site of injection for DiIC16 (3) to label ER (red). Time-lapse confocal sequence(equatorial sections) .
Prodon et al. (2005) J.Cell Science 119, 1592-1603. PDF

FILM 23

980 Ko

Surface movements after meiosis and before first cleavage (Phallusia).
The egg was labelled on its surface with nile blue particules. The film starts just after meiosis completion. Note the transient appearance of the vegetal button (lower left)and the surface relaxation wave which spreads surface particles over the vegetal region between mitosis and first cleavage. DIC time lapse microscopy.
Roegiers et al. (1999) Development 126, 3101-3117. PDF

Cleavages and Asymmetric
Divisions
    
    
ZOOM
FILM 24

290 Ko
First cleavage and equal partitionning of myoplasm (Phallusia).
The mitochondria-rich myoplasm is labelled with DIOC2(3). Note the empty space in the middle of myoplasm representing the cER domain. Time lapse fluoresence microscopy
Roegiers et al. (1999) Development 126, 3101-3117. PDF

FILM 25

1,5 Mo

Asymmetric cleavage in posterior blastomeres of 16 cell stage embryo (Phallusia).
In the first part of the film the centrosome can be observed as a clear zone in front of the interphase nucleus . Led by the centrosome, the nucleus stretches and migrates near the posterior cortex. In the second part of the movie (from 11 seconds), the nuclei are already positioned near the cortex. After nuclear breakdown two CABs are visible as a smooth moustache-shaped zone. One spindle pole in each cell approaches the CAB and unequal cleavage ensues. DIC time lapse microscopy. Frames were collected every 10 seconds and are displayed at a rate of 30 per second.
Patalano et al. (2006) J.Cell Science, 119, 1592-1603. PDF

FILM 26

704 Ko

Asymmetric cleavages in posterior blastomeres (Phallusia).
The film shows divisions of the posterior pair of cells as the embryo passes from the 8 to the 32 cell stages. ER is labeled with DiIC16 (3) (red) and mitochondria with DiOC2(3) (green). Arrow shows the cER-rich CABs. Time-lapse sequence of a single confocal plane. Images were acquired every 2 minutes.
Prodon et al. (2005) J. Cell Science, 118, 2393-2404. PDF

FILM 27

380 Ko

Multilayerd structure of the Centrosome Attracting Body (CAB) (Phallusia).
Posterior blastomeres of a 16 cell embryo fixed and labelled for aPKC polarity protein (green) and mitochondria (red). Sequential confocal z sections show the unlabelled space occupied by the cER/mRNA domain between the cortical patch of aPKC and the mitochondria-rich myoplasm.
Patalano et al. (2006) J.Cell Science, 119, 1592-1603. PDF

FILM 28

200 Ko

aPKC protein and microtubules in the CAB region (Phallusia).
This B4.1 blastomere in prophase is part of an 8-cell stage embryo which has been fixed and labelled for aPKC(green) and microtubules (red) . Plus ends of microtubules contact the cell surface at the aPKC-rich domain in the CAB and also reach cortical regions adjacent to the CAB. The film traverses the blastomere via confocal z sections at intervals of 0.4 µm.
Patalano et al. (2006) J.Cell Science, 119, 1592-1603. PDF

FILM 29

880 Ko

aPKC particles in the CAB (Phallusia).
An 8 cell embryo fixed and labelled for aPKC (green). The film shows sequential confocal z sections 1.2 microns apart of the posterior vegetal surface of B4.1 blastomeres (posterior view) . aPKC-rich particles line the blastomere surface and are densely packed at the position of the CABs. The field measures 45 µm on each side.
Patalano et al. (2006) J.Cell Science, 119, 1592-1603. PDF

Development to Tadpoles
FILM 30

1,85 Mo

Development from egg to tadpole in 12 hours (Phallusia).
Mitochondria-rich myoplasm domain (labelled with DIOC2(3) (green) is partitioned into the posterior pair of blastomeres which give rise to primary muscle cells in the tail of the tadpole . Time lapse DIC and fluorescence microscopy.


FILM 31

860 Ko

Development from 8 cell stage to tadpoles(Phallusia):
This sequence of 2 embryos held in a microchamber was recorded overnight using DIC optics and time lapse microscopy. The field is about 300 microns wide.
FILM 32

850 Ko

Mesenchyme cells migrate within the tadpole head (Phallusia).
Note the population of large flattened cells which migrate anteriorly (to the left) and then ingress inside the tadpole head. DIC optics, time lapse microscopy

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