Embryos had been injected at the one-cell stage with a Lissamine-labeled control MO.
(A–C) Bright field views. (D–F) Fluorescent views. D' shows a high-power view of cells within the animal hemisphere of an embryo at the early gastrula stage.
(A and D) Early gastrula stage 10–11. (B and E) Tailbud stage 28. (C and F) Tadpole stage 41.

(A–C) MOs directed against 14-3-3η. (A) Control MO; embryos develop normally.
(B) Embryos injected with MO1, directed against the translation start site of 14-3-3η, develop with a shortened antero-posterior axis.
(C) Embryos injected with MO2, directed against sequence 5′ of the translation start site of 14-3-3η, resemble those injected with MO1.
(D–F) MOs directed against Xnr3. (D) Control MO; embryos develop normally. (E) Embryos injected with MO1, directed against the translation start site of Xnr3, exhibit an upturned tail. (F) Embryos injected with MO2, directed against sequence 5′ of the translation start site of Xnr3, also have an upturned tail, but they differ slightly from those injected with MO1 because their antero-posterior axes are slightly shortened.
(G–J) MOs directed against Tbx3. (G and I) Embryos injected with control MOs develop normally. (H) Embryos injected with MO1, directed against the translation start site of Tbx3, have a normal body axis but their tails are slightly wavy. (J) Embryos injected with MO2, directed against sequence 5′ of the translation start site of Tbx3, have a more severe phenotype than those injected with MO1, in which the antero-posterior axis of the embryo is shortened. MO1, primary MOs; MO2, second site MOs.

Note that the blastopore in control embryos is closing normally, but is either absent or severely delayed in embryos in which the functions of the indicated genes are inhibited. All embryos shown are at gastrula stage 10.5 to 11.5. In this figure and in Figures 5–13, the number in the top left hand side of each panel represents the synphenotype group to which the embryos belong, and the name of the gene in question is shown bottom left.

This class can be subdivided into six synphenotype groups, as indicated in Figure 3 and Table 4. Members of the first three synphenotype groups (involution defects, gastrula or neurula defects, and short axis surviving to tailbud) are shown at tailbud stage (stage 24–28), while the member of the second synphenotype group shown here (short axis surviving to tadpole) is shown at tadpole stage 35–41. Lines in this and subsequent figures demarcate the different synexpression groups.

This class can be subdivided into six synphenotype groups, as indicated in Figure 3 and Table 4. The figure shows examples of the fourth synphenotype group (short axis surviving to tadpole) at tadpole stages 35–41.

This class can be subdivided into six synphenotype groups, as indicated in Figure 3 and Table 4. The figure shows examples of the second three synphenotype groups (short axis surviving to tadpole, normal body short tail, and proportionately small) at tadpole stages 35–41.

Embryos injected with MOs targeting this gene appear perfectly normal to early tailbud stage 30 but then rapidly disintegrate. Embryos are shown at the tailbud stage (stage 24–28).

Embryos are shown at the tadpole stage (stage 35–41).

This class can be subdivided into two synphenotype groups, as indicated in Figure 3 and Table 4. All embryos are shown at the tadpole stage (stage 35–41).

This class can be subdivided into five synphenotype groups, as indicated in Figure 3 and Table 4. All embryos are shown at the tadpole stage (stage 35–41).

This class can be subdivided into five synphenotype groups, as indicated in Figure 3 and Table 4. All embryos are shown at the tadpole stage (stage 35–41).

This class can be subdivided into three synphenotype groups, as indicated in Figure 3 and Table 4. All embryos are shown at the tadpole stage (stage 35–41).

Embryos were injected at the one-cell stage with 15 ng of the indicated MO and allowed to develop to the equivalent of the tailbud stage, when they were examined by TUNEL staining. Note that only the Tbx3.2, Xnr3.2, and Xbra.2 MOs caused a level of apoptosis that exceeded the level observed in control embryos (injected with 30 ng of the Gene Tools control MO).

The specificities of the MOs used to define this phenotypic class were investigated by injecting 10–15 ng of the Gene Tools standard control MO (Column 1); the original antisense MO (Column 2); MO1 together with 1 ng of a form of the target RNA that lacks the MO target sequence (Column 3); MO1 (or, in the case of Dp71, MO2) with five mismatched bases (Column 4); MO2 (Column 5); MO2 together with 1 ng of a form of the target RNA that lacks the MO target sequence (Column 6).
The results of these experiments are summarized in Table 5. In Column 1 (control MO) embryos are shown at the mid-gastrula stage. Embryos in Column 2 (MO1) are at the same stage as those in Column 1, but (with the exception of Dp71) gastrulation is delayed or inhibited. In the case of Dp71, MO1 does not inhibit gastrulation but does cause embryos to develop with a shortened axis. Column 3 indicates that for five of the nine MOs studied, complete or partial rescue of the phenotype was obtained by injection of the cognate RNA. In these experiments, embryos were allowed to develop beyond gastrula stages to tailbud or tadpole stages. In the case of D1LIC, rescue was more complete at tailbud stages (upper panel) than tadpole stages (lower panel). Column 4 shows that for each of the nine MOs, changing five bases caused them to lose the ability to disrupt development.
Use of a second site MO usually causes a milder phenotype than is observed with MO1 (Column 5), but the phenotype is usually specific, in the sense that it can frequently be rescued by injection of the cognate RNA (Column 6).
MO1, original antisense oligonucleotide; MO2, second site MO.

The six examples shown here are all from the motility defects class. (A–C) Expression patterns of the three members of the swimming in circles synphenotype group. (D–F) Expression patterns of three members of the normal appearance but paralyzed synphenotype group. Members of each group are not expressed in the same patterns and so do not belong to the same synexpression group (see text).