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 M_l and M_r. 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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