Two dorsal blastomeres of 4-cell stage embryos were injected with 4 ng of Xom
RNA (Ladher et al., 1996) or 1 (or 2) ng of P V .l RNA (Ault et al., 1996), respectively, and the resulting phenotypes were analysed at stage 35. Each individual injection experiment was always performed with eggs from the same female. The results from 5 independent experiments are compiled in Table 5.1.
T able 5.1:
Morphological phenotypes observed after five independent dorsal injections of Xom or P V .l in Xenopus embryo.RNA
Phenotype (%), Stage 35
n Normal Severe ® Mild'’ Very Mild*’ Gastrulation
defects'*
Non injected 60 0 0 3 14(0) 30
Xom (4 ng) 12 32 22 16 18(12) 50
P V .l (1 or 2 ng) 3 37 17 0 43(31) 35
eyes. Mild phenotypes correspond to embryos with small head, small cement gland and no eyes or eye defects. ‘ Very mild corresponds to embryos with eye defects only, or sometimes also with a small cement gland but always a normal sized head. Eye defects include: small eyes (on one side or both sides), one eye absent (on the right or the left side) and combinations of these phenotypes. ‘‘Gastrulation defects include embryos with different extents o f non-closure of the blastopore lip; the num ber betw een brackets corresponds to the percentage of embryos with gastrulation defects lacking cement glands, n, total number of embryos.
Analysis of Table 5.1 indicates that Xom injections generated 32% of embryos that were severely ventralised, with no head or small heads, lacking cement gland and eyes. This phenotype, however, was not observed in all experiments performed. Mild and very m ild ventralisation was observed in 22% and 16%, respectively, o f X om
injected embryos. Figure 5.1 shows examples of very mild ventralisation as a result of mis-expression of Xom, whereas uninjected embryos are not ventralised (Fig. 5.1 A, B and inset). Occasionally, very mild phenotypes were accom panied by shortening and bending of the tail, which may indicate defects in convergence-extension movements, characteristic of gastrula stage dorsal involuting tissue. By contrast, P V .l injected embryos generated more embryos with a severe ventralised phenotype (37%), which were present in all experiments performed (Table 5.1 and Fig. 5.1 C). Embryos with a very mild ventralisation phenotype were never observed (Table 5.1). I note that in these experiments P V .l RNA was always used in lower doses than Xom RNA. In a substantial proportion o f embryos presenting gastrulation defects, the cem ent gland was also missing in injected embryos (Table 5.1; Fig. 5.1 B and C), while control non-injected embryos still form ed cement gland. This further indicates that patterning did not occurred normally in injected embryos. Taken together, these results indicate that in the range of concentrations used, Xom has a milder ventralising phenotype than P V .l.
Xom and P V .l injected embryos from one of the experiments described in Table 5.1 were further analysed by whole mount antibody staining using the antibody MZ15, which recognises the notochord, and the results are presented in Table 5.2.
Chapter 5, Function o f Xom in the Xenopus embryo
Table 5.2
Whole mount antibody staining using MZ15 antibody.RNA
Notochord staining (%), Stage 35
n
Normal Fragmented* Absent
Uninjected 100 0 0 10
Xom (4ng) 41 53 6 17
PV .l (1 ng) 40 60 0 10
* Corresponds to MZ15 staining in fragments of notochord spread along the axis of the tadpole, which can cover from almost 100% to almost 0% of the length of the axis.
M is-expression of X om and P V .l RNA causes frequent disruption of the notochord, and in one case complete absence (Table 5.2 and 5.1 B, D and E). These results confirm that the ventralisation phenotypes observed in X om and f y. 7-injected embryos correlates with a disruption in dorsal structures, such as the notochord.
In summary, in my hands and in the range o f concentrations used, X om has a variable and, generally, milder ventralising phenotype than P V .l. However, neither Xom
nor P V .l generated completely ventralised embryos, as has previously been reported (Ault et al., 1996; Onichtchouk et al., 1996; Rastegar et al., 1999; Schmidt et al., 1996). Similar phenotypes for P V .l (Xvent-1) injections as the ones presented here have been observed by Gawantka et al. (1995). It remains possible that higher doses of RNA will generate completely ventralised embryos, although increased concentrations of Xom
T
- 1
Chapter 5, Function o f Xom in the Xenopus em bryo
Fig. 5.1
Over-expression of Xom and P V .l causes ventralisation of X enopusembryos and affects expression of dorsally-expressed genes. Phenotypes of uninjected embryos at tadpole stage (A) or of embryos injected dorsally at the 4-cell stage with 4 ng of X om RNA (B) or 2 ng of P V .l RNA (C). In this experim ent, P V .l injections generated severe ventralised embryos with no head or small heads and a shortened body, whereas Xom injections generated milder phenotypes, defective in the formation of the eyes and/or cement gland but with normal head and body size. Embryos with severely impaired gastrulation are also shown (B, C). The inset in (B) shows an embryo injected with Xom lacking one eye while the inset in (A) shows the normal head of an uninjected embryo. (D-F) Notochord staining of uninjected em bryos (D) and dorsally injected embryos with 4 ng of X o m (E) or 2 ng of P V .l (F). N ote discontinuous notochord staining in injected embryos. (G) FRK H l expression in control embryos at early gastrula stage 10.5 injected with the fluorescein dextran lineage m arker and analysed by whole mount in situ hybridisation with a FRKHl probe and by whole mount antibody staining to show fluorescein dextran staining. Vegetal view of three embryos. (H, I) F R K H l
expression in stage 10.5 embryos co-injected with fluorescein dextran and 4 ng of Xom
(H) or 2 ng of P V .l (1). Dorso-vegetal view. Note up-regulation of F R K H l expression in Zorn-injected em bryos and FR K H l dow n-regulation in PV. 7-injected em bryos.
Goosecoid expression in uninjected embryos (J) or in em bryos injected in both blastomeres of 2-cell stage embryo with 4 ng Xom RNA (K) analysed by whole mount in situ hybridisation at stage 10.5. Vegetal views; dorsal to the top. Note down-regulation of goosecoid expression in injected embryos.