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FIGURE 1
During Xenopus development the dorsal-ventral axis is specified sequentially. The maternal molecules VegT and Vg1 interact with the zygotic transcriptional activator β-catenin that becomes stabilized in a broad area of the dorsal side. An endodermal signal mediated by Xenopus-Nodal related growth factors (Xnr) of the TGF-β family induces the overlying cells to differentiate into ventral mesoderm at low levels. On the dorsal side, β-catenin is stabilized and in combination with VegT and Vg1 induces high levels of Xnr signals in dorsal endoderm, also called the Nieuwkoop signaling center, inducing the Spemann-Mangold organizer in overlying dorsal mesoderm. The Spemann organizer secretes a cocktail of growth factor inhibitors such as Chordin, Noggin, Dkk1, Frzb1, and Cerberus, which constitute the horizontal signal that inhibits BMP4 and xWnt8 originating form ventral regions. This generates a morphogenetic gradient that determines the D-V tissue differentiations of the embryo (blood, lateral plate, kidney, somite and notochord in mesoderm, as well as central nervous system, neural crest and epidermis in ectoderm). Diagram based on tissue recombination experiments from reference 7.
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FIGURE 2
The dorsal side is visible as a less pigmented region called the gray crescent from the earliest stages of development of the Xenopus embryo. (A) In the unfertilized egg the animal (top) hemisphere is pigmented, showing a clear spot where the large oocyte GV nucleus breaks down during meiotic maturation. (B) Fertilized egg close to the end of the first cell cycle. The sperm entry point on the bottom side of this photo is darker due to accumulation of pigment and the white spot of GV breakdown is erased by cortical movements. The cortical rotation generates a less pigmented side, or gray crescent, opposite the sperm entry point. (C) 2-cell stage 90 min after fertilization, showing clear D-V differences in pigmentation due to cortical rotation. (D) 4-cell stage showing the two dorsal blastomeres on the top of the photo. At this stage the dorsal blastomeres are larger in many, but not all, batches of eggs. Cleavage divisions are rapid and occur every 30 min until midblastula when zygotic transcription starts. (E) 8-cell stage. (F) 16 cell stage. The D-V contrast in pigmentation increases in subsequent divisions due cytoplasmic ingression associated with cleavage furrowing. These early asymmetries have made Xenopus a favorite material for embryology investigations. All embryos shown were from the same batch of eggs. Scale bar, 500 µm.
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FIGURE 3
The cortical rotation and internal cytoplasmic movements. (A) Diagram showing the rotation of the entire cortex by 30o to form the gray crescent on the dorsal side. Internal cytoplasmic movements are indicated by the asymmetry of the vegetal yolk and the curvature of the pigment trail left by sperm entry. Based on Xenopus histology and work on urodele eggs.[3, 11] (B) Xenopus fertilized egg at the end of the first cell cycle showing an unlabeled swirl of vegetal cytoplasm on the dorsal side separating the recently endocytosed yolk platelets of the animal pole. (C) Embryo at 64-cell stage; the arrowhead indicates a ring of recently deposited yolk flanked by unlabeled cytoplasm specifically in the dorsal marginal zone. The last two panels were provided by Prof. Mike Danilchik, Oregon, and reproduced with permission from the Company of the Biologists.[13]
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FIGURE 4
Dorsalization by Hwa mRNA, and its inhibition by co-injection of EIPA. (A) Uninjected control. (B) Microinjection of Hwa mRNA (5 pg one time ventrally at the four-cell stage) induces complete twinned axes in Xenopus 3-day tadpoles. Scale bar 1 mm. (C) Control at early tailbud. (D) 1-day embryos in which 10 pg Hwa mRNA was microinjected into each blastomere at the 4-cell stage become radially dorsalized, phenocopying the effects of LiCl treatment at 32-cell stage; note the circular cement gland. (E) Microinjection of Hwa mRNA into a single blastomere resulted in secondary axes; the induced axis is darker because the ventral side is more pigmented. (F) Hwa secondary axes are blocked by EIPA injection.[49] Scale bars 0.5 mm.
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FIGURE 5
Model of how activation of lysosomes/MVBs might explain the initial dorsal β-catenin signal. The unfertilized egg contains cytoplasmic determinants in the vegetal pole. After fertilization, the centrosome contributed by the sperm nucleates microtubules that direct cortical rotation of the cytoplasmic determinants and formation of the gray crescent. Lysosomes are activated on the dorsal side at the 64-cell stage, and we propose that β-catenin stabilization could result from sequestration of GSK3, Axin1 or other proteins in MVBs in the region in which animal hemisphere cytoplasm mixes with more ventral components.
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FIGURE 6
Multivesicular bodies of special characteristics are enriched specifically on the dorsal side of the Xenopus 64-cell embryo. (A) Large MVB surrounded by tightly packed smaller vesicles with electron dense membranes and contents. Three intraluminal vesicles are present, and a coated pit typical of the process of involution of membranes by the ESCRT machinery is seen near them; the lumen of the limiting membrane has a non-membranous covering. These components are lettered in the graphical abstract of this paper. (B, C) Two additional examples from other embryos are shown; note dorsal MVBs surrounded by clouds of smaller, darker cytoplasmic membranes that we propose may represent cytoplasmic determinants. These previously unpublished electron micrographs were taken by our collaborator Prof. David D. Sabatini at New York University. Regularly cleaving embryos with strong dorsal-ventral polarity at 64-cell stage were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4, and 1 h later dissected with a scalpel into dorsal to ventral fragments of the marginal zone. Embryo fragments were post fixed with 2% Osmium tetroxide and processed for embedding in epoxy resin LX 112, and sections stained with Uranyl acetate and Lead citrate and examined by TEM in a JEOL EX 1200 electron microscope, as described.[59] Scale bars, 500 nm.
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In the unfertilized egg the animal (top) hemisphere is pigmented, showing a clear spot where the large oocyte GV nucleus breaks down during meiotic maturation.
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A Zygote, or fertilized egg, close to the end of the first cell cycle. The sperm entry point on the bottom side of this photo is darker due to accumulation of pigment and the white spot of GV breakdown is erased by cortical movements. The cortical rotation generates a less pigmented side, or gray crescent, opposite the sperm entry point.
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NF stage 2 (2-cell), 90 min after fertilization, showing clear Dorsal-Ventral differences in pigmentation due to cortical rotation.
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NF stage 3 (4-cell) showing the two dorsal blastomeres on the top of the photo. At this stage the dorsal blastomeres are larger in many, but not all, batches of eggs. Cleavage divisions are rapid and occur every 30 min until midblastula when zygotic transcription starts.
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NF stage 4 (8-cell).
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NF stage 5 (16-cell) 6 cell stage. The Dorsal-Ventral contrast in pigmentation increases in subsequent divisions due to cytoplasmic ingression associated with cleavage furrowing.
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A Xenopus fertilized egg, in cross section, at the end of the first cell cycle showing an unlabeled swirl of vegetal cytoplasm on the dorsal side separating the recently endocytosed yolk platelets of the animal pole.
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Xenopus embryo in cross section, at NF stage 7 (64-cell); the arrowhead indicates a ring of recently deposited yolk flanked by unlabeled cytoplasm specifically in the dorsal marginal zone.
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