Moriyama Y and De Robertis EM (2018), Embryonic regeneration by relocalization of the...

XB-ART-54846

Proc Natl Acad Sci U S A 2018 May 22;11521:E4815-E4822. doi: 10.1073/pnas.1802749115.

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Embryonic regeneration by relocalization of the Spemann organizer during twinning in Xenopus.

Moriyama Y , De Robertis EM .

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The formation of identical twins from a single egg has fascinated developmental biologists for a very long time. Previous work had shown that Xenopus blastulae bisected along the dorsal-ventral (D-V) midline (i.e., the sagittal plane) could generate twins but at very low frequencies. Here, we have improved this method by using an eyelash knife and changing saline solutions, reaching frequencies of twinning of 50% or more. This allowed mechanistic analysis of the twinning process. We unexpectedly observed that the epidermis of the resulting twins was asymmetrically pigmented at the tailbud stage of regenerating tadpoles. This pigment was entirely of maternal (oocyte) origin. Bisecting the embryo generated a large wound, which closed from all directions within 60 minutes, bringing cells normally fated to become Spemann organizer in direct contact with predicted ventral-most cells. Lineage-tracing analyses at the four-cell stage showed that in regenerating embryos midline tissues originated from the dorsal half, while the epidermis was entirely of ventral origin. Labeling of D-V segments at the 16-cell stage showed that the more pigmented epidermis originated from the ventral-most cells, while the less-pigmented epidermis arose from the adjoining ventral segment. This suggested a displacement of the organizer by 90°. Studies with the marker Chordin and phospho-Smad1/5/8 showed that in half embryos a new D-V gradient is intercalated at the site of the missing half. The displacement of self-organizing morphogen gradients uncovered here may help us understand not only twin formation in amphibians, but also rare cases of polyembryony.

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Species referenced: Xenopus laevis
Genes referenced: chrd ctnnb1 sia1 sia2
GO keywords: somitogenesis
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	Fig. 1. Sagittal bisection of Xenopus blastulae with an eyelash knife generates twins. (A) Diagram showing that bisection by an eyelash knife (SI Appendix, Movie S1). Bisection leaves a large wound that heals by tissue movements from all directions, closing the gap within 1 h, and leaving only a small healing point where the dorsal and ventral-most cells become juxtaposed. Dorsal lip formation starts 3.5 h later and pigment asymmetry can be observed 1 d later in the epidermis of tailbud tadpoles. (B–E) Still images from SI Appendix, Movie S2 showing the process of convergence of blastula cell sheets toward a central healing point at which the original dorsal and ventral tissues become juxtaposed. Note that cell divisions continue in the blastula as the healing process is completed, but cell division near the wound proceeds at the same rate as in the rest of the embryo. The 1.3× objective lens of Zeiss stereomicroscope, which was connected to a DCR-PC350 Sony Handicam, was used for images.
	Fig. 2. Xenopus embryos bisected sagittally develop into twins with an intriguing asymmetric epidermal pigmentation. (A) Experimental diagram. Half embryos were cultured in the same dish until tailbud stage. (B) Whole embryo; note that pigmentation of the epidermis is uniform on both sides. All of this pigment is inherited maternally from the oocyte. h, head; t, tail. (C and D) Phenotype of left and right half embryos from the same egg. Note that one side of epidermis is strikingly more pigmented than the other (n = 204 half embryos with pigment asymmetry). The search for the cause of this asymmetry was the starting point for the investigations reported here. The 40× digital magnification zoom of Axio Zoom V.16 Stereo Zoom Zeiss was used for images.
	Fig. 3. Fate of dorsal and ventral cells marked at the four-cell embryos: dorsal cells give rise to the midline even in half embryos. (A) Diagram of lineage tracing in whole embryos. A mixture of GFP mRNAs was microinjected into the two ventral blastomeres while RFP mRNAs were injected into dorsal ones, embryos cultured until tailbud, and sectioned with a Vibratome. (B–F) Sections showing that the entire epidermis (except for a few cells in the dorsal midline) and most of the lateral somite was derived from the ventral side (in green), while midline trunk organs, such as the CNS, notochord, dorsal endoderm, and medial somite were derived from the dorsal side (in red) (n = 28). (G) Experimental diagram of lineage tracing in half embryos bisected at midblastula. (H– L) Section of a right-side half embryo showing that tissues in the dorsal midline derived from the dorsal side, while the epidermis and lateral somite were still of ventral origin (n = 20). The 5× objective lens of LSM880 Zeiss confocal microscope was used for images. En, endoderm; epi, epidermis; L, left; L-So, lateral somite; m-So, medial somite; No, notochord; R, right.
	Fig. 4. The origin of medial somite in Xenopus. The medial region of the Xenopus somite is normally derived from the dorsal half of the embryo while more lateral regions of the somite migrate over and under it during movements of convergence and extension in the tailbud embryo. Embryos were lineage-traced by injecting the two dorsal or two ventral blastomeres at the four-cell stage and sectioned at neurula or tailbud stages. (A and B) Diagram showing that the region of the somite next to the notochord originates from dorsal cells (in red), and during development is surrounded by converging more lateral cells originating from the ventral half of the embryo (in green). (C) Stage 16 neurula embryo before CNS closure labeled ventrally with Cascade blue dextran amine (CsBl-DA) and dorsally with fluorescent Alexa 568 dextran amine (F1568-DA) at the four-cell stage. Note that the medial somite, notochord, and neural plate are of dorsal origin (n = 3). (D) Xenopus sibling sectioned at tailbud stage 22 showing that the medial somite, ventral spinal cord, notochord and dorsal endoderm are of dorsal origin, while the rest of the somite and all of the epidermis are ventral (n = 2). Images were acquired with the 5× objective lens of LSM880 Zeiss confocal microscope. D, dorsal; V, ventral.
	Fig. 5. Experiment showing that the maternal pigment asymmetry in twins results from the splitting of ventral segments 3 and 4 during gastrulation; this suggests that Spemman organizer formation is displaced about 90° from its original site in the intact embryo. (A and B) Diagram indicating that segment 4 can be labeled by four injections of nLacZ mRNA at 16-cell stage in ventral and dorsal views; note that segment 3 is less pigmented than segment 4. All segments were analyzed in this way. (C) Sagittal bisection splits the embryo in two halves, allowing one to follow the fate of each segment (in the case shown here the ventral-most segment 4). (D) Diagram of a half embryo showing that during the healing process, segment 4 is juxtaposed to the dorsal-most segment 1. The healing scar is outlined in the center of half embryo. (E and F) Diagram indicating how a displacement of organizer formation (in blue) would result in the splitting of segments 3 and 4 by the involuting mesoderm (gray arrow). (G) In half embryos, the more pigmented side is derived from the segment 4 after regeneration (n = 36). (H) In bisected twins, the less pigmented epidermis is derived from segment 3 (n = 24). Note that the epidermis without lineage tracer is lighter in G than in H; both half embryos are from the same clutch and therefore the pigmentation of the epidermis is comparable. The diagrams in D–F and the embryos in G and H all correspond to right half embryos. The 40× digital magnification zoom of Axio Zoom V.16 Stereo Zoom Zeiss was used for images. h, head; L, left; R, right t, tail.
	Fig. 6. Displacement of organizer formation can lead to pigment asymmetry in whole embryos. (A) Diagram of experiment in which organizer formation is blocked by βCat antisense MO and subsequently a new organizer displaced by 90° (dark blue) is induced by injecting Siamois mRNA. Gray arrow indicates the direction of gastrulation. (B) Control embryo; note uniform pigmentation of the epidermis (n = 46). (C) Embryo injected with βCat MO showing a ventralized phenotype lacking all axial structures due to loss of Spemann’s organizer (n = 50). (D) Embryo injected with βCat MO and rescued by injection of Siamois mRNA into the vegetal tier of segments 2 and 3 injected embryo at the 16-cell stage (n = 62, of which 60 showed pigment asymmetry). Note that the pigmentation of the epidermis is asymmetrical after inducing a new organizer displaced by 90° from its original site. The 40× digital magnification zoom of Axio Zoom V.16 Stereo Zoom Zeiss was used for images.
	Fig. 7. Formation of the organizer and the ventral center was displaced in half embryos arising by sagittal bisection. (A and A′) The organizer marker chd was expressed within the progeny of segment 1 (n = 65). Embryos were lineage labeled at 16-cell and cultured until gastrula. (B and B′) In half embryos the chdexpressing region only partially overlapped with segment 1, indicating a displacement of the organizer from its normal location (n = 101, 9 independent experiments). (C and C′) The progeny of blastomere C1 at the 32-cell stage also overlapped with chd (n = 4). (D and D′) In half embryos the chd+ organizer region was displaced at a distance from the original dorsal C1 region (n = 13); in this particular embryo the healing point at which the original dorsal- and ventral-most cells were juxtaposed was still visible, underscoring the extent of the displacement. (E) Staining with antiphospho-Smad1/5/8 antibody in whole embryos showing the ventral BMP gradient (n = 2). (F) The ventral center (high BMP signaling) was regenerated in half embryos; staining with Chd antibody (weaker because the much stronger pSmad1/5/8 signal required stopping the staining) indicated that both centers were formed opposite each other (n = 2). (G) Phospho-Smad1/5/8 signaling in sagittal sections of stage 12 gastrula embryos (n = 6). (H) Regenerated half embryo showing that the ventral (V) center was formed directly opposite the dorsal (D) blastopore lip (n = 4). Right-side half embryos are shown in B, D, and H, while a left-side half embryo is shown in F. (I) Hypothetical diagram indicating how the dorsal organizer and ventral center are displaced in opposite directions in half embryos. The original dorsal center region (light blue) is displaced 90° to the boundary between segment 1 and 2 (dark blue). The ventral center is also displaced from the region in segment 4 next to the healing point (light red) to the boundary between segments 3 and 4 (dark red). Early after healing at blastula the prospective regions of high BMP and high Chd signaling are juxtaposed in the half embryo. However, by the gastrula stage the dorsal and ventral centers are formed at a distance from their original location by the intercalation of a new D-V morphogen gradient. The 5× objective lens of LSM880 Zeiss confocal microscope was used for A–D′ and the 40× digital magnification zoom of Axio Zoom V.16 Stereo Zoom Zeiss for E–H.

References [+] :

Agius, Endodermal Nodal-related signals and mesoderm induction in Xenopus. 2000, Pubmed, Xenbase