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Expression pattern of Brachyury in the mollusc Patella vulgata suggests a conserved role in the establishment of the AP axis in Bilateria

Nicolas Lartillot1, Olivier Lespinet2, Michel Vervoort1 and André Adoutte1

1 Centre de Génétique Moléculaire, CNRS batiment 26, 1 Avenue de la Terrasse, F-91198 Gif Sur Yvette, France
2 National Institute of Health, Building 38A, Room B2N13E, 8600 Rockville Pike, Bethesda, MD 20894, USA



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Fig. 1. Sequence alignment and phylogenetic tree. (A) The predicted amino acid sequences of the PvuBra fragment has been aligned with a sample of metazoan Brachyury and other T-box genes. Only the T domain is shown. The primers used for PCR amplification are indicated by arrows. (B) Phylogenetic tree (amino acid) of several Brachyury orthologues, reconstructed by quartet puzzling. A sample of other T-box genes has been taken to root the Brachyury subtree (see Materials and Methods).

 


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Fig. 2. Overview of Patella early development: 3D induction and AP axis specification. Unless mentioned explicitly, all views are taken from the vegetal pole. D quadrant (posterior) is to the bottom, so the left side of the embryo is to the right. Drawings are freely adapted from van den Biggelaar (van den Biggelaar, 1977Go), except A, which is adapted from Wilson (Wilson, 1904), and G. (A) 4-cell stage (30 minutes after first cleavage). The four cells are of equal size, and the progeny of each of them is called a quadrant. (B) 32-cell stage (2 hours after first cleavage (h.p.f.c.)), displaying a four-fold symmetry. (C) Early 32-cell stage (2.5 h.p.f.c.), meridional cross section, before 3D determination. Animal is to the top. Two of the four vegetal cells (macromeres) can be seen (3M). During this period, the four macromeres invade the blastocoel, and compete for contacting the animal micromeres that constitute the blastocoel roof. (D) Mid 32-cell stage (3 h.p.f.c.), same section as in C: one of the four macromeres contacts the blastocoel roof, and as a consequence, takes a 3D fate. (E) 60-cell stage (3.5 to 4 h.p.f.c.). 3D looks smaller, since most of its mass is internalised. 3a, 3b, 3c and 3d are in metaphase. Together with 3D, these latter cells’ cleavage adumbrates bilateral symmetry. (F) 64-cell stage (4 h.p.f.c.): 3c and 3d have yielded smaller vegetal cells (3c2 and 3d2) and larger animal cells (3c1 and 3d1), whereas 3a and 3b have divided in an opposite fashion, budding off a larger cell towards the vegetal pole (3a2 and 3b2. Note that 3a1 and 3b1 are not visible on this drawing). 3D is the last cell to divide before completion of the 6th cleavage, and yields 4d, or M, the mesentoblast, that will contribute to most of the adult mesoderm. (G) 2 hours after the 64-cell stage (6 h.p.f.c.). The segregation of the prospective germ layers is now achieved. The endodermal vegetal plate (yellow) is made from all descendants of 3A, 3B, 3C and 4D. 4d, the mesentoblast divides in a bilateral fashion, yielding Ml and Mr, the paired stem cells giving rise to the mesodermal germ bands (red). 3a and 3b derivatives also contribute to mesoderm (light orange) (Dictus and Damen, 1997Go). Cells coloured in dark blue are of ectodermal fate, and delineate the posterior edge of the blastopore. The pink blastomeres are the three stomodaeum founder cells, and are thus called stomatoblasts, as in Nereis (Wilson, 1892). During gastrulation, the vegetal plate is internalised by epiboly: the rim of ectoderm migrating over and enclosing the vegetal plate is equivalent to a blastopore edge which, in the case of gastropods, gives birth to the mouth. The dark blue cells constitute a useful landmark: they are at the edge of the vegetal plate, and will contribute to the ectoderm at the base of the larval mouth, and thus define the posterior edge of the blastopore.

 


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Fig. 3. PvuBra expression during early development. (A-D) In situ hybridisation using PvuBra antisense probe. (E-H) Schematic pictures indicating lineage of expression. (I-L) In situ hybridisation combined with Hoechst staining of the nuclei. (A,E) Between late 32- and 40-cell stage (3 h.p.f.c.). PvuBra is strongly expressed in 3D. (B,F) 64-cell stage (4 h.p.f.c.), showing expression in 3D daughter cells (4D and 4d), as well as a spread of expression in 3c, 3d and 2d2 daughter cells. Sister cells are linked by a short straight line. (C,G) Beginning of the 88-cell stage (5 h.p.f.c.). 3c2 and 3d2 have not divided yet, and show strong PvuBra expression. 3c1 and 3d1 have divided, with their spindles approximately parallel to the plane of bilateral symmetry. Their daughters express weaker levels of PvuBra transcripts, compared to 3c2 and 3d2. The resulting pattern takes on the shape of two bilateral wings. Expression is also strong in 2d22, fainter in 2d212, and has nearly vanished in 4D and 4d. (D,H) 6 h.p.f.c. The two wings (3c1 and 3d1 derivatives) still express PvuBra at a medium level. 3c2 and 3d2 have divided and, in contrast to their sister cells, they have their spindles parallel to the posterior edge of the vegetal plate, so that, together with 2d22, they are the five darkly stained cells at the posterior edge of the blastopore. Behind, 2d212, the midline stem cell (arrowhead, m.s.c.; see text for details), has budded off a new small cell situated just next to 2d22, and which also strongly expresses PvuBra. The midline stem cell itself shows much weaker expression. Notice its typical asymmetrical position, markedly to the right (for us, to the left). Transcripts are no longer detected in 4D, or in Ml and Mr. Gastrulation is about to start. (I) 63-cell stage. (J) 64-cell stage. (K) 30 minutes after the 88-cell stage, the paired mesoteloblasts can be seen below the focal plane, with their spindles in metaphase. (L) 6 h.p.f.c.. The two wings are apparent.

 


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Fig. 4. PvuBra in situ hybridisation, later stages. (A) Posterior view of a 8 h.p.f.c. embryo. The vegetal plate (v.p.) is just visible above the five stained cells marking its posterior edge. The apical pole (a.p.) is below. Staining is now barely visible in the wings, and not apparent in the midline stem cell. (B) 10 h.p.f.c. embryo, ventral view. The posterior edge of the blastopore takes a V-shape (arrowhead). Posteriorly, the ventral midline is being formed, and also expresses PvuBra. A spot of expression appears terminally, consistently on the right side. It probably corresponds to the midline stem cell (m.s.c.). The prototroch (pt.) is visible in the background. (C) Young trochophore (13 h.p.f.c.), three quarter view, from the right-ventral side. Gastrulation has proceeded to its end. Stomodaeum (st.) is to the right, ventral midline to the bottom, and terminal cells (t.c.) to the left. (D) Older trochophore (24 h.p.f.c.). ventral-posterior view. The ectodermal cells underlying the stomodaeum (st.) still show transcripts. Midline and terminal expression has fainted away. More dorsally, right at the mantle edge (m.e.), four new spots of expression can now be seen. sb., stomatoblast; pt., prototroch; m.e., mantle edge.

 


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Fig. 5. Summary of PvuBra expression, from 3D induction to the end of gastrulation. Color shading corresponds to observed PvuBra RNA expression. (A) 32-cell stage (3 h.p.f.c.). (B) 64-cell stage (4 h.p.f.c.). (C) 88-cell stage (5 h.p.f.c.). Division of 3c1, 3d1 and 2d2 (see text for details). (D) 6 h.p.f.c.. Division of 3c2, 3d2 and the midline stem cell, so that the midline is now made of two cells. 3D derivatives are now omitted. (E) 10 h.p.f.c.. The posterior edge of the blastopore migrates anteriorly, together with the two lateral stomatoblasts. Terminal cells have lost PvuBra expression. A third small cell is added to the midline by the stem cell. (F,G) Late gastrulae, showing the growing midline, and the closing blastopore. (H) 13 h.p.f.c. young trochophore expression pattern. v.p., vegetal plate; t.c. teminal cell; m.s.c. midline stem cell; sb. stomatoblast; st. stomodaeum.

 


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Fig. 6. Localisation of activated ERK in a 32-cell stage embryo. (A) Vegetal view. The labelled cell is one of the four macromeres. (B) Side view of the same embryo. The labelled cell is contacting the blastocoel roof, and thus can be identified as 3D. Labelling is strong in the nucleus (arrow) and along the zone of contact with the cap of micromeres (arrowhead).

 


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Fig. 7. Mitotic pattern and PvuBra expression in ERK inhibited embryos (medium dose). Unless specified otherwise, all views are from the vegetal pole. Presumed D quadrant to the bottom. (A) In situ hybridisation, combined with Hoechst staining of the nuclei, on a 64-cell stage embryo, showing an equalised cleavage pattern (compare with Fig. 3I,J). Sister cells are indicated by a line. (B) 64-cell stage, side views, showing PvuBra expression in 3c1, 3c2, 3d1, 3d2, and at a weaker level, in 2d22 and 2d21. No staining is seen in 4D and 4d (the cleavage is equal: compare with Fig. 3B). (C) Late 64-cell stage: some PvuBra expression is now seen in 4D and 4d. (D) Early trochophore: PvuBra expression is detected in cells scattered on the ventral side of the trochophore. pt., prototroche; ft., foot lobe; st., stomodaeum.

 


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Fig. 8. Amphistomy, AP axis formation, and Brachyury expression (blue) in the common ancestor of the Bilateria. (A) Following AP axis specification, the vegetal region of the embryo becomes subdivided into an anterior, Brachyury negative, and posterior, Brachyury positive, domains. (B) The blastopore closes in an amphistomous fashion. (C) The posteriorly located growth zone is responsible for the AP axis elongation (arrow). As a result of the whole process, Brachyury is expressed posteriorly and on the ventral midline.

 





© The Company of Biologists Ltd 2002