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Dynamic changes in the association between maternal mRNAs and endoplasmic reticulum during ascidian early embryogenesis

Axis formation is one of the most important events occurring at the beginning of animal development. In the ascidian egg, the antero-posterior axis is established at this time owing to a dynamic cytoplasmic movement called cytoplasmic and cortical reorganisation. During this movement, mitochondria, endoplasmic reticulum (ER), and maternal mRNAs (postplasmic/PEM RNAs) are translocated to the future posterior side. Although accumulating evidence indicates the crucial roles played by the asymmetrical localisation of these organelles and the translational regulation of postplasmic/PEM RNAs, the organisation of ER has not been described in sufficient detail to date owing to technical difficulties. In this study, we developed three different multiple staining protocols for visualising the ER in combination with mitochondria, microtubules, or mRNAs in whole-mount specimens. We defined the internally expanded “dense ER” using these protocols and described cisterna-like structures of the dense ER using focused ion beam-scanning electron microscopy. Most importantly, we described the dynamic changes in the colocalisation of postplasmic/PEM mRNAs and dense ER; for example, macho-1 mRNA was detached and excluded from the dense ER during the second phase of ooplasmic movements. These detailed descriptions of the association between maternal mRNA and ER can provide clues for understanding the translational regulation mechanisms underlying axis determination during ascidian early embryogenesis. Supplementary Information The online version contains supplementary material available at 10.1007/s00427-021-00683-y.


Fig. S1
Vegetal or posterior views of Fig. 2c. A 3D model of approximately 4 µm thickness was rendered from optical sections of high-magnification images of dense ER regions at PNfo, PNfu, and Meta (as indicated on the top). Mitochondria, ER, and merged fluorescence channels are separately shown (as indicated on the left). PNfo: Vegetal view of vegetal pole region is indicated with posterior side (Pos↑) up. Dense ER is visible; however, MRC cannot be observed in this 3D model. PNfu and Meta: Posterior views of posterior pole region (Pos) are indicated with animal pole side (A↑) up. The animal pole side borders of dense ER (arrowheads), which was peeled off from the egg cortex and started to intrude into cytoplasm, are obvious. Bright dots in the ER channel can be seen just beneath the plasma membrane. Scale bar: 5 µm. The animal pole side borders of the dense ER (arrowheads), peeled off from the egg cortex and starting to intrude into the cytoplasm, are obvious. Bright dots in the ER channel are visible just beneath the plasma membrane. The parallel array of microtubules in CAMP is obvious and shortened at the Meta stage. Scale bar: 10 µm.

Fig. S3
Spatio-temporal pattern of dense ER and pem-1 mRNA. (a) Doubleimmunostaining of ER (red) and MRC (green) and in situ hybridisation of another example of type I postplasmic/PEM mRNAs (blue; pem-1), were performed on the same embryo during the first cell cycle (developmental stages are indicated on the top; unfertilised, Telo I, PNfo, PNfu, and Meta). The optical sections of the mid-plane are shown. Animal pole (A) is up and vegetal pole (V) is down in all photographs. As the antero-posterior axis becomes evident from the PNfo stage, posterior pole (Pos) is at the right from this stage onward. Upper tier: Merged images of entire egg (no label) and enlarged dense ER region (Enlarged) are shown. The nucleus (white in the merged images) was counterstained with 4',6-Diamidino-2-phenylindole dihydrochloride. Middle and lower tiers: Mitochondria (Mito), ER, and in situ hybridisation (pem-1) fluorescence channels of each enlarged images are separately represented (as indicated in the upper-left corner). Outlines of the densely stained ER region (red lines) were superimposed on in situ hybridisation signals (ER/pem-1). Most of the pem-1 mRNA signals overlapped with the dense ER region in the unfertilised egg and Telo I stage (yellow arrowheads); however, they were excluded from the dense ER region and extruded into the MRC region after the PNfo stage (light blue arrowheads). Scale bar: 50 µm (entire egg image) and 10 µm (enlarged image). (b) Localisation patterns of pem-1 mRNAs (blue) at the 32-cell stage were co-stained for ER (red) and mitochondria (green) using our new method. Enlarged images of the CAB (arrowheads) show blotchy staining of ER (red) and pem-1 mRNA signals (blue) within the CAB. Scale bars: 50 µm (left image) and 10 µm (right image).

Fig. S4
Spatial pattern of ER and macho-1 mRNA in the isolated cortices. Cortices isolated from embryos at 45 mpf were quadruple-stained for ER (red), macho-1 (green), mitochondria (blue), and microtubule (MT; white) by the conventional or our new experimental method (as indicated on the top). The 3D models of approximately 4 µm thickness are rendered from optical sections. Merged images (Merge) and microtubule fluorescent channel (MT) are shown. These cortices were assumed to be sheared by the water flow from upper-left corner; thus, the dislocated MRCs were located at the lower-right side of each isolated cortex (dotted line). Enlarged images of solid and dashed rectangles area are shown (Enlarged image; as indicated on the left side). Upper-left and lower-right halves of both solid rectangle areas represent posterior-vegetal cortex and MRC region, respectively. The dotted rectangle represents the rim of ER-rich region, which is assumed to correspond with the dense ER. With the conventional method, posterior-vegetal cortex was rich in ER, whereas the signals of macho-1 mRNA were more evident in the MRC region. With our method, the signals of macho-1 mRNA predominantly reside in the MRC region and are rarely observed on the posterior-vegetal cortex. Scale bars: 50 µm (upper tier) and 10 µm (enlarged images).

Fig. S5
Spatio-temporal pattern of vasa mRNA during the cleavage stages. Embryos from 2-to 32-cell stages were stained for ER (red) and vasa (blue) with our new method and counterstained with DAPI. Cell cycle of 2-to 16-cell stages were interphase and those of 32-cell stage was metaphase. Enlarged images of the CAB-forming regions are shown (Merge). Outlines of the densely stained ER region (red lines) were superimposed on the vasa signals (white: ER/vasa). Most of the vasa mRNA (type II postplamic/PEM mRNA) signals were colocalised with dense ER from the 4-cell stage, similar to the type I postplasmic/PEM RNA. Scale bar: 10 µm.

Fig. S6
Spatio-temporal pattern of macho-1 mRNA from the first to third cleavage stage. To reveal when the macho-1 mRNA was relocalised with dese ER, embryos in the first mitotic phase (first cleavage), interphase and mitotic phase of the 2-cell stage (second cleavage), and interphase and mitotic phase of the 4cell stage (third cleavage) were stained for ER (red) and macho-1 (blue), and counter stained with DAPI (white). Upper tier: Merged images of equatorial plane are shown. Posterior pole (Pos) is at the right. Lower tier: Enlarged images of presumptive CAB-forming regions are represented in two ways: simply enlarged image (Merge) and image with outlines of the densely stained ER region (red lines) on macho-1 signals (white: ER/macho-1). Most of the macho-1 mRNA signals were excluded from dense ER region until interphase of 2-cell stage, whereas at the metaphase of second mitosis (2-to 4-cell stage), most of the mRNA signals were colocalised with the dense ER at the posterior pole. Scale bars: 50 µm (upper tire) and 10 µm (lower tier).