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First published online October 22, 2003
doi: 10.1242/10.1242/dev.00805

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Maternal mRNAs of PEM and macho 1, the ascidian muscle determinant, associate and move with a rough endoplasmic reticulum network in the egg cortex

Christian Sardet1,*,{dagger}, Hiroki Nishida2,*, François Prodon1 and Kaichiro Sawada2

1 BioMarCell, UMR 7009, CNRS/UPMC, Station Zoologique, Observatoire Océanologique, Villefranche sur Mer, 06230, France
2 Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan



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  		Fig. 1. Localization of HrPEM and macho 1 mRNAs in eggs, zygotes and embryos. mRNAs are visualized in fluorescent (A-D,F), confocal (A1-A3,B1-B3,C1-C3) and DIC microscopy (E,G), by fluorescent (TSA) (A-D,F) and chromogenic (E,G) methods. (A) Unfertilized egg (Unfert.). (B,C) Zygotes after first (Fert.20, 20 minutes after fertization) and second (Fert.80, 80 minutes after fertilization) major relocalization phases. (D,E) Lateral views of 8-cell embryos. (F,G) Animal-posterior views of 8-cell embryos. (A1,B1,C1) Median sections, the thickness of the labelled zone is ~2 µm in unfertilized eggs and 5-8 µm in fertilized eggs. (A2-A3,B2-B3,C2-C3) Confocal tangential sections below the surface. Probes are indicated in the lower-left corner of each panel. a, animal; v, vegetal; p, posterior; CAB, centrosome-attracting body. Arrowheads in A,B,C show mRNA-rich cortical regions. Arrows in A2,B2 show that this mRNA-rich structure is reticulate.

 


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  		Fig. 2. Isolated cortices from zygotes and 8-cell-stage embryos retain HrPEM and macho 1 mRNA. Low magnification (A,C) and higher magnification (B,D) views of cortices isolated from zygotes attached and sheared 20 minutes after fertilization. HrPEM is visualized by the chromogenic (A,B) or fluorescent (TSA) methods (C,D). v, region of the isolated cortex corresponding to the vegetal pole; arrow in D indicates that mRNA-labelled network is reticulate. (E-I) Cortices isolated from 8-cell embryos. (E) Visualization of ER with the lipid dye DiIC16(3) (left) and of HrPEM mRNA with Alexa green 488 (right). Arrows show a stretched-out CAB. (F) Overexposed image of ubiquitous mRNA (arginyl-tRNA synthetase) visualized by the TSA method. In E and F, p indicates 2 posterior quartet patches with the characteristic mustache-shaped CAB (arrowhead) present in 2 of the 4 patches. (G-I) Characteristic mustache-shaped CAB in which HrPEM (G,H) and macho 1 (I) mRNAs are concentrated.

 


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  		Fig. 3. The cER network in cortices isolated from eggs and 8-cell-stage embryos. (A-D) ER networks from cortices isolated from live unfertilized eggs labelled with the lipophilic dye DiIC16(3) and viewed with fluorescence microscopy (A,B), or prepared for electron microscopy using the fast-freeze deep-etch method (C,D), revealing ribosomal particles (arrows in D) mixed with smaller or odd-shaped particles (arrowheads). (E-G) High-magnification confocal sections of the DiOC6(3)-labelled CAB region in live cortices isolated from 8-cell embryos. (E) Confocal section close to the plasma membrane. The ER network in the CAB (arrowhead) is contiguous with the surrounding cER network. Vesicles are present in the CAB. (F) Three, evenly-spaced confocal sections (bottom, middle and top) through a CAB isolated with the cortex. The third image is an enlarged view of the ER network. (G) The ER network vesiculates (arrowheads) when a live labelled cortex is exposed to hypotonic solution.

 


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  		Fig. 4. HrPEM and macho 1 mRNA localization on the cER network in eggs and zygotes. (A) Edge of a cortex isolated from an unfertilized egg, labelled to visualize the cER network (red, left panel), and the network of HrPEM (green, middle panel). The right panel shows an overlay of red and green images and the colocalization (yellow) of ER and mRNA on the cortex (arrowhead). Most ER tubes outside the cortex are devoid of HrPEM (er arrow). (B) Vegetal pole region (v) of cortex isolated from a fertilized egg 20 minutes after fertilization (this area corresponds to the densely labelled regions of isolated cortices seen in Fig. 2C,D. Microvillated region characteristic of the vegetal pole region is indicated (mv). (C) Edge of an unfertilized egg cortex treated with puromycin-KCl to detach particles from the cER. Only a weak signal of HrPEM remains, and partial colocalization (arrowheads) is shown. ER tubes outside the cortex are indicated (er arrow). (D) Edge of a fertilized egg cortex showing the continuity between the HrPEM-labelled cER network (upper part) and the nonlabelled cytoplasmic ER network projected outside the cortex during the shearing process (lower part). The arrows (er arrow) show the zone of transition between cER and cytoplasmic ER outside the cortex. (E) Colocalization of cER and HrPEM in the central region of an isolated unfertilized egg cortex. The yellow colour results from superposed green and red images as in panel A. (F) Edge of an unfertilized egg cortex showing the continuity between the cER network (arrowhead) and the cytoplasmic ER tubes projected outside the cortex (red). Note the lack of HrPEM signal outside the cortex. The arrows (er arrow) show the zone of transition between cER and cytoplasmic ER outside the cortex. (G) Edge of an isolated unfertilized egg cortex colabelled for ER (red signal) and macho 1 mRNA (green signal). Co-localization is seen on the cortex (yellow; arrowhead), but not outside the cortex (red, er arrow). (H) Fragment of isolated cortex exposed to hypo-osmotic shock showing colocalization (yellow) of ER and HrPEM. Most of the cER network is vesiculated (arrowheads).

 


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  		Fig. 5. HrPEM mRNA concentrates in the ER-rich CAB region. (A-E) Merged images of ER (red) and HrPEM (green) in cortices isolated from Halocynthia 8-cell-stage blastomeres. Areas where ER and HrPEM mRNA are perfectly colocalized appear yellow. (A) Quartet of cortices isolated from an 8-cell embryo attached by the posterior pole to a coverslip. Cortices of vegetal-posterior (vp) blastomeres are rich in cER and in HrPEM in and around the CAB (CAB arrowhead). Animal-posterior (ap) blastomeres have a cER network that lacks HrPEM. (B,C) Views of the CAB and of the cER network that surround the CAB. C is a higher magnification of the boxed region (edge of CAB) in B. In C, large patches of HrPEM mRNA localize on the cER network in the immediate vicinity of the CAB (CAB arrowhead), shown by arrows. The cER network away from the CAB (arrowheads in C) displays only occasional and small mRNA-rich patches. (D,E) These merged images show that the ER network, which is normally compacted in the CAB, has stretched away from the attached cortex under the force of shear. E is a higher magnification view of the boxed region in D. Large patches of HrPEM mRNAs are detected both on and inbetween stretched ER tubes (arrowheads show green RNA-rich patch). (F,G) Nonmerged images. Maximum magnification of confocal images of the inside of the CAB showing colocalization of ER [ER is labelled with DiIC(16)3 in F, and HrPEM mRNA labelled by Alexa green 488 in G] in some regions of the CAB (arrows). In other regions, differences in the relative intensity of mRNA and ER labelling can be seen.

 

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© The Company of Biologists Ltd 2003