Figure 2. The immunostaining against Bra of Colostethus machalilla embryos. Stages of development on the left, and the age of embryos are according to ávila et al. (unpublished observations). The external morphology of C. machalilla embryos is comparable to X. laevis until stage 13 (Nieuwkoop and Faber, ). The diagrams on the left show the Bra-positive regions in purple. The embryos were immunostained in whole-mount with anti-Bra. The dark purple spots correspond to Bra-positive nuclei, whereas the brown color corresponds to the natural pigment that remained after bleaching. Embryos could not be bleached totally because elimination of the pigment also removed the Bra alkaline phosphatase signal. All embryos were cleared in bb/ba to eliminate the opacity of the yolk, except for the embryos shown in (G) and (J). Embryos were photographed in brightfield (A,B,D,E,G,H,J,K,M-P). Embryo sections were viewed with differential interference contrast (C,F,I,L,Q,R). The sections in C,F,L,R were 100 mu m thick, whereas section I was 50 mu m thick, and P,Q were 7 mu m thick. The animal pole is oriented toward the upper left in A and toward the top in C. Anterior is toward the top in D,E,G,H,J,K and toward the right in M-O. The external surface is toward the right in F,I and toward the top in L,P-R. A-C: the blastula (stage 9; age, 1.1 days). A: Animal view of a cleared blastula that was partially bleached. The animal pole (ap) was Bra-negative. Bra-positive nuclei extend to the marginal zone (arrow). B: Vegetal view of the embryo shown in A. The vegetal pole (vp) did not contain Bra-positive nuclei, whereas the surrounding cells were Bra-positive. The cell outlines and the low density of Bra-positive nuclei in this region showed the large size of vegetal blastomeres. C: Parasagittal section of a blastula. Bra-positive nuclei were detected in surface cells. The animal pole (ap) was Bra-negative. The two faintly positive nuclei (arrow) were located on the embryonic surface but were positioned in a different focal plane. D-F: The early gastrula (stages 10-11). The drawing represents a stage 11 embryo. D: Surface view of the dorsal blastopore lip of a stage 10 embryo (age, 1.4 days). The area of the dorsal blastopore lip (b) contained nuclei that were strongly Bra-positive. The Bra-positive signal of the marginal zone decreased in comparison with the blastula (stage 9). E: The blastopore region of a stage 11 embryo (age, 1.9 days). Brachyury-positive nuclei were observedonly near the blastopore lips. The yolk plug and the marginal zone were Bra-negative. F: Parasagittal section through the blastopore of a late stage 11 embryo, showing the ventral lip. The Bra-positive nuclei were superficial (arrow). Bottle cells (bc) and those of the yolk plug were Bra-negative. G-I: The midgastrula (stage 12; age, 3 days). G: Surface view of the blastopore in an uncleared embryo. Some nuclei of the blastopore lips were Bra-positive. The yolk plug (yp) is Bra-negative. H: The same embryo shown in G after clearing. The Bra-positive nuclei formed an internal ring around the blastopore. I: Section through the blastopore of an advanced stage 12 embryo. Bra-positive nuclei were found in a deep layer (arrow), in contrast to stage 11 (F). The yolk plug (yp) was Bra-negative. J-L: The small blastopore stage (stage 12.5; age, 3.5 days). J: Surface view of the blastopore in an uncleared embryo. The yolk plug has been retracted, but the blastopore was still open. Few Bra-positive nuclei were observed in the surface near the blastopore (arrow), in comparison to stage 12 (G). K: The same embryo shown in J after clearing. The Bra-positive nuclei formed an internal ring around the blastopore as in H. L: Parasagittal section through the blastopore of a slightly earlier embryo. The small yolk plug was not retracted. The internal nuclei of the blastopore lips were Bra-positive. M-O: The slit blastopore stage and the tail bud (stages 13 and 17). M: The slit blastopore stage (early stage 13 embryo; age 4.1 days). After closure of the blastopore, the notochord began to elongate. The nascent notochord (n) and the tail bud (tb) were Bra-positive. N,O: A late stage 13 (age, 4.4 days) embryo in N and a tail bud stage embryo (stage 17; age, 8 days) in O. In both embryos, Bra-positive nuclei were found in the notochord (n) and in the tail bud (tb). P-R: Cross-sections through the notochord of a late stage 13 embryo and a tail bud stage embryo (stage 17). The sections in P,Q were from an embryo equivalent to N, and the section in R was from an embryo equivalent to O. The sections represent the posterior (P) and anterior (Q) regions of the notochord of a late stage 13 embryo. P: Cross-section through the posterior region of the notochord (n) of a late stage 13 embryo. The notochord lined the roof of the original cavity of the gastrula, the gastrocoel (g). Q: Cross-section through the anterior region of the notochord of the same late stage 13 embryo in P. The notochord n was internalized and covered with endodermal cells (e). R: Cross-section through the midregion of the notochord of a tail bud embryo (stage 17). The notochord (n) is located under the neural tube and above the endodermal roof of the archenteron; this cavity was totally covered with endoderm. Scale bars = 200 mu m in G,J,M, 100 mu m in A,B,D,E,H,I,K,L,N-P, 50 mu m in C,F,Q,R.