Lane MC and Keller R (1997), Microtubule disruption reveals that Spemann's o...

XB-ART-16972

Development 1997 Feb 01;1244:895-906. doi: 10.1242/dev.124.4.895.

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Microtubule disruption reveals that Spemann's organizer is subdivided into two domains by the vegetal alignment zone.

Lane MC , Keller R .

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Mediolateral cell intercalation is proposed to drive morphogenesis of the primary embryonic axis in Xenopus. Mediolateral intercalation begins in a group of cells called the vegetal alignment zone, a subpopulation of cells in Spemann's organizer, and spreads through much of the marginal zone. To understand the functions of the vegetal alignment zone during gastrulation and axis formation, we have inhibited its formation by disrupting microtubules with nocodazole in early gastrula embryos. In such embryos, mediolateral intercalation, involution and convergent extension of the marginal zone do not occur. Although cell motility continues, and the anterior notochordal and somitic mesoderm differentiate in the pre-involution marginal zone, posterior notochordal and somitic mesoderm do not differentiate. In contrast, microtubule depolymerization in midgastrula embryos, after the vegetal alignment zone has formed, does not inhibit mediolateral cell intercalation, involution and convergent extension, or differentiation of posterior notochord and somites. We conclude that microtubules are required only for orienting and polarizing at stage 101/2 the first cells that undergo mediolateral intercalation and form the vegetal alignment zone, and not for subsequent morphogenesis. These results demonstrate that microtubules are required to form the vegetal alignment zone, and that both microtubules and the vegetal alignment zone play critical roles in the inductive and morphogenetic activities of Spemann's organizer. In addition, our results suggest that Spemann's organizer contains multiple organizers, which act in succession and change their location and function during gastrulation to generate the anterior/posterior axis in Xenopus.

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Species referenced: Xenopus
Genes referenced: mtor tbx2
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	Fig. 1. The marginal zone in Xenopus. Convergence of the marginal zone in the mediolateral direction after stage 10G produces extension, or lengthening of the dorsal mesodermal tissues in the anterior/posterior direction. (A,B) A simplified stage 10 fate map is shown on the embryo on the left, with the head mesoderm (blue), the prospective axial tissue (notochord; anterior is red, posterior is orange) and paraxial mesoderm tissue (somites in brown; anterior is heavily stippled, posterior is lightly stippled) shown centered on the dorsal midline (dashed line). Mediolateral intercalation results in notochordal and somitic tissue involution, which inverts the anterior/posterior directional polarity of the tissues with respect to the animal/vegetal axis of the embryo. Mediolateral intercalation also causes convergence upon the dorsal midline (B; stage 23) and extension of the anterior/posterior axis. (C) Dorsovegetal view of a stage 10G embryo, showing the relationship between the vegetal alignment zone, the prospective anterior notochord and somites, and the prospective posterior notochord and somites. A®P indicates the orientation of the anterior/posterior axis in the notochordal and somitic fields. The gastrulation movements bring these essentially perpendicular axes into parallel alignment. In this view, the epithelium is removed, revealing the deep mesodermal component of the marginal zone. Head mesoderm cells have moved beneath the prospective notochord. The vegetal alignment zone (black cells) begins and ends to either side of the dorsal marginal zone, and traverses the prospective anterior notochordal and somitic tissues. (D) In a sagittal section, the arc (black) is present in the preinvolution mesoderm, beneath the epithelium of the organizer. The head mesoderm is migrating on the deep surface of the prospective notochord. an, anterior; A, animal; Bl, blastocoel; DL, dorsal lip of blastopore; LI, limit of involution; p, posterior; V, vegetal; VAZ, vegetal alignment zone; VL, ventral lip of blastopore.
	Fig. 2. A subpopulation of cells in the dorsal lip/organizer which become bipolar in the mediolateral axis during mid-gastrulation. Cell outlines are revealed by immunostaining with the anti-integrin antibody, 8C8. In all panels, the animal pole is up and the dorsal lip is down. (A,A¢). The epithelium of the organizer is composed of isodiametric, polyhedral cells at stage 10+ (shown) and 10G (not shown). (B,B¢) Cells in the first deep cell layer beneath the organizer epithlium at stage 10+ are likewise polyhedral, and essentially isodiametric. These cells are undergoing radial intercalation (in and out of the focal plane). (C,C¢) Some cells in the first deep cell layer beneath the organizer epithelium at stage 10G are bipolar and elongated (solid circles) in the mediolateral axis of the embryo. Cells in the first deep cell layer situated more animally in the organizer show no mediolateral elongation at stage 10G (stars), while cells vegetal of alignment zone are also not mediolaterally elongated (squares). Scale bar, 25 mm.
	Fig. 3. Nocodazole treatment before stage 10G inhibits closure of the blastopore and formation of bipolar cells. Dorsal is up in A-C and the dorsal lip is down in D-F, respectively. Open squares in A-C indicate the regions shown in D-F, respectively. (A) Control embryos at stage 13. The blastopore is closed and streaks of pigmentation indicate the developing neural plate. (B) Embryos treated with nocodazole from stage 10G, at control stage 13. The blastopores are closing. There is no apparent neural plate at this time. (C) Embryos treated with nocodazole from stage 10S, at control stage 13. Bottle cells form around the circumference of the blastopore, but closure is not occurring. (D) Cell shapes in the dorsal mesoderm in control embryos at stage 12G. Cells are mediolaterally elongated (arrowheads) in both the notochordal and somitic regions and the notochord/somite boundary has formed (black arrow). (E) Cells in the involuted dorsal mesoderm of embryos treated with nocodazole from stages 10G to 12G have become mediolaterally elongated (arrowheads), but the notochord/somite boundary has not yet formed. (F) Cells in the dorsal mesoderm of embryos treated with nocodazole from stages 10+ to 12G do not become mediolaterally elongated, but remain isodiametric (arrowheads). The cells in this field remain in their pre-involution position because gastrulation is blocked. an, anterior; dl, dorsal lip; n, notochord; s, somite. Scale bar (D-F), 50 mm.
	Fig. 4. Convergence extension and notochord/somite boundary formation in open-face explants are inhibited by nocodazole, but not by taxol. The explants were fixed and stained for microtubules at control stage 20. Microtubules outline cell shapes, especially in the notochord. The original animal end of the explant is up in all panels. (A) A control explant that has undergone extensive elongation. The notochord/somite boundary is visible (arrow). Dark shadows (white rings) are caused by the absorption of fluor-emitted light by pigmented epithelial cells lying between the optical section and the collecting pinhole of the confocal microscope. (B) Taxol treatment at stage 10+ does not inhibit convergent extension, nor notochord/boundary formation (arrow). (C) An explant treated with nocodazole from stage 10+ does not converge, extend or form notochord/somite boundaries. Scale bar, 250 mm. All optical settings on the confocal microscope were held constant for A and B. Staining intensity is much greater in taxol-treated than control tissue because microtubules are bundled and assembly is stimulated. For C, the confocal settings used to collect images in A and B yielded a black screen. In order to collect adequate light to reveal the shape of the explant, the pinhole was opened and filtering of the laser was decreased. As this explant contained no microtubules when examined at high magnification, this image does not represent microtubule staining.
	Fig. 5. Nocodazole-blocked embryos express notochordal and somitic differentiation markers. Anterior is to the left in A,C and D. Animal is up in B. (A) Dorso-vegetal view of a control embryo, immunostained with the Tor 70 and 12-101 antibodies at stage 23. (B) Dorsal view of an embryo treated with nocodazole from stage 10S, at stage 23. A button-like notochord (n) with small blocks of somitic tissue (s) on either side sits above the dorsal lip of the blastopore (arrows). (C) Dorsal view of an embryo treated with nocodazole from stage 10G, at stage 23. More notochordal and somitic tissue is present and arranged as an axis. Yolky endoderm (y) protrudes from the blastopore. (D) Lateral view of an embryo treated with nocodazole at stage 11. The dorsal mesoderm consists of an extended notochord with two paraxial somitic files. The archenteron (ar) is present ventral to the axial mesoderm. (E) A transverse section through the axial mesoderm of an embryo similar to the one shown in (B). No archenteron is present and notochordal and somitic tissues are in their pre-involution positions. (F) A cross-section through an embryo similar to the one shown in C. Notochordal and somitic tissue are situated in their post-involution positions and an archenteron is present. (G,H) Gastrulation is rescued when nocodazole is withdrawn. (G) Lateral view of an embryo treated with nocodazole for 2 hours beginning at stage 10+, and subsequently rescued. After removal of the drug, morphogenesis resumes. The notochord, somites and archenteron are visible. (H) A crosssection of an embryo similar to the one shown in G. The dorsal mesodermal structures are in their normal post-involution positions, and an archenteron is present. vp, vegetal pole; an, animal pole.
	Fig. 6. Nocodazole does not inhibit cell motility. Rhodaminedextran- labeled cells were grafted to explants of the dorsal marginal zone, and the explants treated with DMSO or nocodazole. (A) A clump of labeled notochordal cells grafted into the notochordal region, after 15 minutes. (B) At stage 20 in a control explant constructed at stage 10G, the clump of cells is dispersed in an explant that converged and extended. Labeled cells joined with host cells in forming notochordal and somitic tissue. (C) By stage 20 in a nocodazole-treated explant constructed at stage 10+, the labeled cells are dispersed, demonstrating that cells are motile. The explant did not converge and extend, and cells did not assume notochordal nor somitic morphologies (arrow). (D) By stage 20 in a nocodazoletreated explant constructed at stage 10G, labeled cells have assumed notochordal morphology (arrow) in an explant that underwent convergent extension. Dashed lines indicate the edges of explants. Scale bar, 35 mm.
	Fig. 7. Cells remain motile in nocodazole. Grafted, rhodamine-dextran-labeled mesoderm cells are dispersed 5G hours after nocodazole treatment was initiated. (A) Two groups are visible. (B) Approximately 1 hour later, the clumps have changed slightly, and two cells (marked with arrows) have moved into view from deeper in the explant. (C) Approximately 30 minutes later, one cell (straight arrow) has migrated laterally and another cell (small curved arrow) has migrated posteriorly in the explant. A third cell has surfaced (large open arrow). Every cell, including those in clumps, undergoes extensive shape changes between 5G and 7 hours of nocodazole exposure.
	Fig. 8. The original and revised ‘multiple organizer’ hypotheses. (A) The original hypothesis, formulated by Stewart (1990) and Gerhart et al. (1991). (B) The revised hypothesis. See text for details. GO, gastrula organizer; LBO, late blastula organizer; NC, Nieuwkoop Center.