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Development and differentiation of the vertebrate caudal midbrain and anteriorhindbrain are dependent on the isthmic organizer signals at the midbrain/hindbrain boundary (MHB). The future MHB forms at the boundary between the Otx2 and Gbx2 expression domains. Recent studies in mice and chick suggested that the apposition of Otx2- and Gbx2-expressing cells is instrumental for the positioning and early induction of the MHB genetic cascade. We show that Otx2 and Gbx2 perform different roles in this process. We find that ectopically expressed Otx2 on its own can induce a substantial part of the MHB genetic network, namely En2, Wnt1, Pax-2, Fgf8 and Gbx2, in a concentration-dependent manner. This induction does not require protein synthesis and ends during neurulation. In contrast, Gbx2 is a negative regulator of Otx2 and the MHB genes. Based on the temporal patterns of expression of the genes involved, we propose that Otx2 might be the early inducer of the isthmic organizer genetic network while Gbx2 restricts Otx2 expression along the anterior-posterior axis and establishes an Otx2 gradient.
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Fig. 2. Otx2 induces broad ectopic expression of En2, while Xgbx2a inhibits it. Embryos were injected unilaterally with RNA encoding Xgbx2a/GR, Otx2/GR or both, according to the scheme in Fig. 1. In all cases, LacZ RNA was co-injected with one of the RNAs to positively determine the mRNA injected in each side. The hormone-inducible proteins were activated at stage 11. Embryos were harvested at stage 18 and processed for double in situ hybridization with En2 (magenta) and LacZ probes (turquoise). The injection scheme based on Fig. 1 is marked in each panel; Otx2 RNA, red; Xgbx2 mRNA, green; Xgbx2 antisense RNA, yellow; Otx2 mRNA+Xgbx2a antisense RNA, orange. (A) Control embryo. (B) Embryo injected unilaterally with Xgbx2a/GR RNA exhibits inhibition of En2 on the injected side. (C) Embryo injected with Xgbx2a/GR and Otx2/GR RNA in adjacent cells shows En2 inhibition on the side injected with Xgbx2a/GR. On the side injected with Otx2/GR, En2 expression at the MHB is shifted posteriorly and ectopic En2 expression is activated along the anterior-posterior axis (arrowheads). (D) Otx2/GR alone can induce En2 ectopic expression along the anterior-posterior axis (arrowheads). (E) Otx2 injection unilaterally can induce expression of En2 in the lateralepidermis (arrows). (F) The expression of En2 in the antisense Xgbx2a-injected half is slightly enlarged, probably due to the up-regulation of Otx2. (G) Otx2/GR mRNA and antisense Xgbx2a RNA injected in the same cell. The down-regulation of Xgbx2a in the Otx2/GR-expressing half does not affect the ability of Otx2/GR to ectopically activate En2. (H) In embryos where Otx2 mRNA was injected in one side and Xgbx2a antisense RNA was on the other side, En2 was ectopically activated in the Otx2/GR-expressing half, albeit with the down-regulation of Xgbx2a in the opposite side.
Fig. 3. Otx2 induces ectopic expression of Wnt1, Pax2 and Xgbx2a. Embryos were injected unilaterally with Otx2/GR and LacZ mRNA as a lineage tracer. Dex was added at stage 11. The injected side is marked with an asterisk. The lineage tracer was detected by in situ hybridization with a LacZ probe (turquoise) in (A,B,E,F) or with LacZ enzymatic activity (red) in (C,D). (A) In untreated embryos at stage 20, Wnt1 is expressed at the MHB (black arrow) and along the dorsal midline posterior to rhombomere 2 (white arrow). (B) The expression of Wnt1 in the Otx2/GR-injected side is strongly activated, creating an uninterrupted line of expression from the MHB to the posterior region of the embryo. (C) Pax2 normal expression at stage 22. The two main sites of expression are the MHB (black arrow) and the otic vesicles (white arrow). (D) On the non-injected side Pax2 expression remains unchanged. On the Otx2/GR-injected side, the MHB and the otic vesicle domains of expression are combined creating a large Pax2-expressing patch situated posterior to the normal MHB. (E) Embryo injected with Otx2/GR RNA but not treated with dex. In untreated embryos at stage 18, Xgbx2a is expressed in the MHB and the lateralepidermis. (F) Otx2/GR overexpression resulted in strong ectopic expression of Xgbx2a in the dorsolateral region of the embryo at the edges of the lineage tracer staining. (G) Normal Fgf8 expression in stage 23 embryos. The weak Fgf8 expression across the MHB is observed (black arrow). (H) Fgf8 expression in embryos unilaterally expressing Otx2. The change in the Fgf8 pattern of expression as a result of Otx2 activity can be seen (arrowheads). The Otx2-injected sides are marked (*).
Fig. 4. Otx2 is a direct activator of Wnt1, Xgbx2a and Pax2. RNA encoding the hormone-inducible form of Otx2 was injected unilaterally (the injected side is indicated with an asterisk) together with LacZ mRNA. Cycloheximide was added 30 min prior to the activation of Otx2/GR by dexamethasone to inhibit protein synthesis. The embryos were fixed 2.5 h after the addition of dexamethasone and the expression of Wnt1 (A–D), Xgbx2a (E–H) and Pax2 (I–L) was studied. (A) Expression of Wnt1 in the absence of dexamethasone. (B) Activation of Otx2/GR by dexamethasone resulted in Wnt1 expression along the neural tube. (C) Cycloheximide treatment inhibited the normal endogenous expression of Wnt1. (D) Broad ectopic expression of Wnt1 was observed in embryos incubated with dexamethasone and cycloheximide. The endogenous domains of Wnt1 were inhibited by the cycloheximide treatment. (E) Control Xgbx2a expression in the absence of dexamethasone. (F) Activation of Otx2/GR induced broad ectopic activation of Xgbx2a at the edges of the injected region. (G) Inhibition of protein synthesis causes a general increase in Xgbx2a transcription. (H) Xgbx2a is ectopically activated in embryos incubated with dexamethasone in the presence of cycloheximide. (I) In the absence of activator, Otx2/GR does not affect the normal Pax2 expression. (J) Pax2 is broadly activated in embryos treated with dexamethasone. (K) Inhibition of protein synthesis does not affect the MHB-specific expression of Pax2. (L) In the presence of cycloheximide and dexamethasone Pax2 is activated in the injected side (arrow).
Fig. 5. Time course of the ability of Otx2 and Xgbx2a to affect En2 expression. Embryos were injected unilaterally at the two-cell stage either with Xgbx2a/GR or Otx2/GR RNA, or with both in adjacent cells. The hormone-inducible proteins were activated either at stage 10.5 (A,C,E) or at stage 15 (B,D,F). In all instances LacZ mRNA was co-injected as a lineage tracer. (A) Embryo injected unilaterally with Xgbx2a/GR. Dexamethasone was added at stage 10.5. En2 expression was inhibited on the injected side. (B) Embryo injected similarly to (A), but activation by dexamethasone was at stage 15. The ability of Xgbx2a/GR to inhibit En2 is maintained at stage 15. (C) Embryo injected with Otx2/GR. Dexamethasone was added at stage 10.5. On the injected side, En2 expression was ectopically activated in posterior and lateral regions. (D) Embryo injected similarly to (C), but dexamethasone was added at stage 15. The ability of Otx2/GR to induce ectopic En2 expression was considerably diminished as compared to stage 10.5 activation. The ectopic expression (arrowhead) is restricted to a thin stripe along the midline. (E,F) Embryos injected with Otx2/GR into one cell and Xgbx2a/GR along with the lineage tracer into the adjacent cell. (E) Activation of the proteins at stage 10.5 causes inhibition of En2 expression at the MHB in both the Xgbx2a/GR- and Otx2/GR-injected sides and its posterior ectopic activation in the Otx2/GR-injected side. (F) Dexamethasone addition at stage 15. The MHB expression of En2 is inhibited by both constructs. Weak ectopic activation of En2 is seen along the dorsal midline (arrowheads).
Fig. 1. Establishing an artificial boundary between Xgbx2a and Otx2 – experimental design. Xenopus embryos were injected at the two-cell stage with Otx2/GR RNA in one cell and Gbx2/GR mRNA in the other. This injection scheme establishes a novel boundary between Otx2 and Gbx2 expression domains along the dorsal midline of the embryo.
