RESULTS:
Embryos injected with different doses of DN-XRhoA mRNA showed varying degrees of inhibition of Vegetal Rotation (figures 1, 2 & 8). The severity of the phenotype correlated directly to the dose of the mRNA injected into the embryo (figure 1). In contrast the embryos injected with β-Gal mRNA alone showed no signs of inhibition of Vegetal Rotation (figure 1 A & B). The embryos injected with 184 pg/blastomere of the DN-XRhoA mRNA showed a weak inhibition (figure 1 C). Tissue separation at the Brachet’s cleft was apparent and there was significant upwelling of vegetal cells on both the dorsal and ventral leading edges. However, both the dorsal and ventral leading edges were significantly less pronounced than those of the control embryos (figure 1 A & B). Moreover, the dorsal lip of blastopore was also very faint and much higher than those of the controls (figure 1 A & B). Archenteron was also absent. The embryos injected with 368 pg/blastomere of the DN-XRhoA mRNA showed a more severe phenotype than the former (figure 1 D). The specimen in the figure was in fact injected on the ventral side as opposed to dorsal, however, it still shows quite severe inhibition of vegetal cell movement on both the dorsal and ventral sides. There is a hint of Brachet’s cleft on the dorsal side, but the dorsal lip of blastopore is almost inexistent. Given the fact that the specimen was injected on the ventral side (as determined by the staining pattern), this level of inhibition is still quite severe. With no dorsal lip of blastopore there is definitely no question of the presence of an archenteron. The blastocoel floor on the injected side is also completely flat with no hint of tissue separation between the BCR and the vegetal cell mass. The embryos injected with 552 pg/blastomere of the DN-XRhoA mRNA (figure 1 E) showed the most severe phenotype with no trace of a Brachet’s cleft on the dorsal side even in embryos injected in the appropriate region. There was no tissue separation between the vegetal cell mass and the BCR on either dorsal or ventral side as is apparent in the specimen in figure 1 E. The blastocoel floor is also completely flat as if in a blastula and the BCR (blastocoel roof) is also very thick as opposed to the controls (figure 1 A&B) and weakly inhibited embryos (Figure 1 C). In addition to the severity of the phenotype, high doses of DN-XRhoA mRNA also caused significant death of cells as well as of embryos. In the 184pg class none of five embryos died, and the surviving embryos also did not show significant cell death and necrosis. However, two out of seven embryos injected with 368 pg/blastomere DN-XRhoA mRNA died; and five out of fourteen embryos that were injected with 552 pg/blastomere DN-XRhoA mRNA died. The embryos injected with 552 pg/blastomere DN-XRhoA mRNA also showed large chunks of necrotic tissue in some cases indicating localized cell death (figure 1 F). Furthermore, the injected sites in almost all embryos had larger cells compared to the surrounding tissue and this effect was more severe at higher doses. Notice the variable size of red spots in the DN-XRhoA mRNA-injected embryos (figure 1 C-E) as opposed to uniform size of spots in the β-Gal control (Figure 1 B). Each spot represents a nucleus (β-Gal used had a nuclear localizing signal), and larger spots represent larger nuclei.
In a follow up experiment using embryos from a different batch, the same phenotypic effect on vegetal rotation was reproduced as described above. Since 184 pg/blastomere yielded too weak an effect and 368 pg/blastomere caused localized necrosis as well as death, this time each embryo was injected with ~250 pg/blastomere of DN-XRhoA mRNA. The results (figures 2 & 8) were somewhat similar to that in figure 1 C; however, the phenotype was quite consistent among different individuals. No embryos died this time. Almost all embryos had a Brachet’s cleft on the dorsal side as well as dorsal and ventral Mesendodermal leading edges (figure 2 A-C). Nonetheless, vegetal cell upwelling was far weaker than in controls (figure 2 D-F). The archenteron was also absent in all the injected specimens while clearly present in the controls.
ROK
Inhibition also prevents vegetal rotation
BCR-less embryos treated with ROK inhibitor showed much less upwelling of vegetal cells compared to the controls (figure 3). The archenterons in the treated BCR-less embryos were also far smaller and shallower than those of the controls. In addition, the treated BCR-less embryos also became very flaccid and fragile after BCR removal, as opposed to the controls that became more firm and compact after BCR removal.
When
injected into the blastocoel cavity, the ROK inhibitor still elicited its
inhibitory effect on vegetal rotation. In embryos that were left in ficoll
solution since after injection until they were fixed, ROK inhibitor
significantly inhibited vegetal cell upwelling. However, the phenotype was a
bit confusing, since ficoll itself elicited some aberrant effect on
gastrulation. Although the control as well as injected embryos externally
appeared as if they were stage 10+ according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1967), internally the control embryos (judging by the
size of archenteron) appeared more similar to stage 11.5 (figure 4 A-C).
Nonetheless, the injected embryos were significantly different from the
controls (figure 4 D-F).
When the embryos were injected much earlier than the above trial and allowed to recover in 0.1X MBS before being fixed (see methods section for details), they developed normally and possible to be staged according to according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). These embryos were fixed at stage 10.5 as previous experiments. The injected and the control embryos were very different with respect to vegetal rotation and tissue separation at Brachet’s cleft and on the ventral side (figures 5& 9). Eleven out of thirty-nine injected embryos showed no tissue separation at all between the vegetal cells and the BCR (figure 5 D-F & figure 9). Two embryos died while three were unaffected (figure 9). Twenty-three out of thirty-nine embryos showed some upwelling of vegetal cells and very faint tissue separation at the dorsal and ventral mesendodermal leading edges. Very faint dorsal blastopore lips could also be identified in these embryos (figure 6 A&B). However in all these embryos, the blastocoels appeared abnormally shrunken suggesting that the injection might have caused the blastocoel fluid to leak out and as the blastocoel collapsed due to loss of fluid, it also lost the injected inhibitor. However, these embryos may be considered as a milder instance of the phenotype elicited by ROK inhibitor.
When compared, both DN-XRhoA as well as ROK inhibitor produced somewhat similar phenotypes (figure 7). However, in this comparison, DN-XRhoA mRNA-injected embryos (figure 7 A-C) appear somewhat more flaccid than the ROK injected embryos (figure 7 D-F). This might have to do with fixation period, since DN-XRhoA mRNA-injected embryos had to be stained with β-Gal-substrate, they could not be fixed for a long time and thus were slightly sheared and deformed during fracturing. The fractures themselves are also not as clean and levelled in case of since DN-XRhoA mRNA-injected embryos as for ROK injected embryos. In addition, ROK inhibitor effect is also very uniform and consistent all across the blastocoel floor as compared to DN-XRhoA that elicited a more localized effect.