XB-ART-16556Development May 1, 1997; 124 (9): 1733-43.
Xotx1 and Xotx2 are two Xenopus homologues of the Drosophila orthodenticle gene that are specifically expressed in presumptive head regions that do not undergo convergent extension movements during gastrulation. We studied the function of Xotx1 and compared it with that of Xotx2. Ectopic expression of each of the two genes has similar effects in impairing trunk and tail development. Experimental evidence suggests that posterior deficiencies observed in microinjected embryos are due to negative interference with convergent extension movements. Transplantations of putative tail-forming regions showed that, while Xotx1 overexpression inhibits tail organizer activity, Xotx2 overexpression is able to turn a tail organizer into a head organizer. Finally, Xotx1 and Xotx2 are activated by factors involved in head formation and repressed by a posteriorizing signal like retinoic acid. Taken together, these data suggest that Xotx genes are involved in head-organizing activity. They also suggest that the head organizer may act not only stimulating the formation of anterior regions, but also repressing the formation of posterior structures.
PubMed ID: 9165121
Article link: Development
Genes referenced: ag1 nog not otx1 otx2 sia1 tbxt wnt8a
Article Images: [+] show captions
|Fig. 1. Phenotypes of embryos injected with Xotx1. The embryos shown were injected with 1.5 ng of Xotx1 RNA at 1-cell stage, except for the top embryo, which was injected with 2.5 ng of DXotx1 RNA and shows with no phenotypic abnormalities. Embryos in the middle row display severe posterior defects, while embryos in the bottom row show a normal trunk but reduced tail structures. Injected embryos were allowed to develop at room temperature until uninjected control embryos reached stage 38. Frequencies of occurrence of the two phenotypes are shown in Table 1.|
|Fig. 2. Xotx1 does not induce ectopic cement glands and, in coinjection experiments, inhibits Xotx2 cement-gland-inducing ability. Embryos at 2-cell stage were injected with 0.8 ng of DXotx1 (A), Xotx1 (B) or Xotx2 RNA (C), or co-injected with 0.8 ng of Xotx1 and 0.8 ng of Xotx2 RNA (D) and the resulting stage 32 embryos were subjected to whole-mount in situ hybridization with XAG-1, a probe specific for the cement gland. Both DXotx1 (A) and Xotx1 (B)- injected embryos display wild-type pattern of XAG-1 expression. Embryos injected with Xotx2 RNA show ectopic expression of XAG- 1 (C, arrows) which is strongly repressed (D, leftmost embryo; arrowhead point to residual ectopic XAG-1 expression) or completely abolished (D, rightmost embryo) in embryos co-injected with both Xotx1 and Xotx2.|
|Fig. 3. Xotx1 microinjection inhibits molecular markers expressed in cells that undergo convergent extension. 0.8 ng of Xotx1 RNA were injected into one blastomere at 2-cell stage and, when control embryos reached stage 14, injected embryos were subjected to whole-mount in situ hybridization with Xbra (A, B) and Xnot2 (C, D) probes. In all cases, anterior is to the top and posterior to the bottom. The black dots demarcate the dorsal midline and ‘inj’ indicates the injected side as determined by coinjection of a fluorescent tracer (fluorescein dextran amine).|
|Fig. 4. Analysis of convergent extension in exogastrulae and dorsal marginal zone explants. Stage 24 exogastrulae obtained from embryos microinjected at 4-cell-stage into both dorsal blastomeres with DXotx1 (A) or Xotx1 (B) RNA and subsequently transferred in a hypertonic solution. Whereas DXotx1 exogastrulae were indistinguishable from uninjected exogastrulae (A), Xotx1 injection yielded exogastrulae that show a reduced elongation; in extreme cases (B, top row, leftmost embryo), strong inhibition of convergent extension gave rise to radially symmetrical exogastrulae. Dorsal marginal zone explants isolated from stage 10 embryos microinjected at 4-cell-stage into both dorsal blastomeres with DXotx1 (C) or Xotx1 (D) RNA and analyzed when control embryos reached stage 23. Morphogenetic movements of the dorsal marginal zone appear to be inhibited by Xotx1 overexpression.|
|Fig. 5. Transplantation of organizers from Xotx1- and Xotx2-injected embryos. Transplantation of putative tail organizers from Xotx2-injected embryos gives rise to anterior structures (A, arrowhead points to an ectopic cement gland) and secondary heads induction (B). Transplantation of putative tail organizers from Xotx1- injected embryos results in ectopic ‘bulging’ structures (C) and ectopic reduced tail structures (D), whereas when putative tail organizers from DXotx1-injected embryos are tranplanted, complete ectopic tails, similar to those induced by uninjected organizers, are obtained (E).|
|Fig. 6. Histological analysis of structures induced by transplantation of putative tail organizers. (A,A¢) Sections at a proximal (A) and distal (A¢) level of a secondary tail induced by putative tail organizer of a DXotx1-injected embryo. (B,B¢) Sections at a proximal (B) and distal (B¢) level of a ‘bulging’ structure induced by putative tail organizer of a Xotx1-injected embryo. (e), endoderm; (m), muscles; (me), mesenchyme; (n), notochord; (nt), neural tube.|
|Fig.7. Xotx1 and Xotx2 transcription regulation by posteriorizing and anteriorizing signals. RNase protection analysis of Xotx1 and Xotx2 expression in embryos that were either treated with retinoic acid (RA, 1 mM continous treatment starting at the 2-cell stage) or microinjected with noggin or Xwnt-8 RNA. Numbers above the lanes refer to embryo stages (Nieuwkoop and Faber, 1967) used for RNase protection analysis. N, normal untreated embryos. The Xenopus laevis gene for ribosomal protein S8 (rp S8) is analyzed as an internal standard.|
|Fig. 8. Whole-mount in situ hybridization performed with Xotx1 (A,B) and Xotx2 (C,D) probes on stage 10.5 / 11 normal embryos (A,C) or embryos of the same stage that were injected with Siamois (B,D).|