XB-ART-7392Development April 1, 2002; 129 (7): 1609-21.
The homeoprotein Xiro1 is required for midbrain-hindbrain boundary formation.
The isthmic organizer, which patterns the anterior hindbrain and midbrain, is one of the most studied secondary organizers. In recent years, new insights have been reported on the molecular nature of its morphogenetic activity. Studies in chick, mouse and zebrafish have converged to show that mutually repressive interactions between the homeoproteins encoded by Otx and Gbx genes position this organizer in the neural primordia. We present evidence that equivalent, in addition to novel, interactions between these and other genes operate in Xenopus embryos to position the isthmic organizer. We made use of fusion proteins in which we combined Otx2 or Gbx2 homeodomains with the E1A activation domain or the EnR repressor element which were then injected into embryos. Our results show that Otx2 and Gbx2 are likely to be transcriptional repressors, and that these two proteins repress each other transcription. Our experiments show that the interaction between these two proteins is required for the positioning of the isthmic organizer genes Fgf8, Pax2 and En2. In this study we also developed a novel in vitro assay for the study of the formation of this organizer. We show that conjugating animal caps previously injected with Otx2 and Gbx2 mRNAs recreate the interactions required for the induction of the isthmic organizer. We have used this assay to determine which cells produce and which cells receive the Fgf signal. Finally, we have added a novel genetic element to this process, Xiro1, which encode another homeoprotein. We show that the Xiro1 expression domain overlaps with territories expressing Otx2, Gbx2 and Fgf8. By expressing wild-type or dominant negative forms of Xiro1, we show that this gene activates the expression of Gbx2 in the hindbrain. In addition, Xiro1 is required in the Otx2 territory to allow cells within this region to respond to the signals produced by adjacent Gbx2 cells. Moreover, Xiro1 is absolutely required for Fgf8 expression at the isthmic organizer. We discuss a model where Xiro1 plays different roles in regulating the genetic cascade of interactions between Otx2 and Gbx2 that are necessary for the specification of the isthmic organizer.
PubMed ID: 11923198
Article link: Development
Genes referenced: en2 fgf8 gal.2 gbx2.1 gbx2.2 irx1 mst1 myc otx2 pax2 wnt1
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|Fig. 1. Comparison between Otx2, Gbx2 and Xiro1 expression. Embryos were fixed at late gastrula (stage 12-12.5) (A,A’,D,G), early neurula (stage 13-14) (B,B’,E,H,J,K) and mid neurula (stage 17-18) (C,C’,F,I,L), and double in situ hybridization and sectioning were carried out for each pair of genes. The whole mounts are dorsal views oriented with anterior to the top and the sections and inset are oriented with anterior to the left. (A-C) Otx2 (green) and Gbx2 (purple) are expressed in complementary domains that overlap in the isthmus region. (A’) Higher magnification of the square shown in A. Notice the overlapping expression of both genes. (B’,C’) Upper panels show a sagittal section of an embryo after the first chromogenic reaction for Otx2 detection (green). Lower panels show the same embryo after the second chromogenic reaction for Gbx2 detection (purple). Notice the overlap in the expression of both genes at the early neurula stage (bracket in B’), which disappears at the mid neurula stage (bracket in C’), to generate a sharp boundary of Otx2/Gbx2 expression. (D-F) Otx2 (purple) and Xiro1 (light blue) overlap at the presumptive midbrain domain. (G-I) Gbx2 expression (purple) is almost completely included in Xiro1 (light blue)-expressing territory. (J) Position of Fgf8 expression. The initial isthmus expression of Fgf8 appears at early neurula stage in the region where Otx2 and Gbx2 are co-expressed (brackets). This early expression precedes the establishment of the sharp border described for Otx2 and Gbx2. Images were taken from the same embryos after the first gene detection (right panels, green for Otx2 and Gbx2) and at after the second chromogenic reaction (left panels, purple for Fgf8). (K) Double in situ hybridization for Fgf8 (purple) and Xiro1 (green) mRNAs. The Fgf8 isthmus expression is included in the Xiro1-positive cells at this stage (arrow). Arrowhead points the anterior limit of Xiro1. (L) Double staining for En2 (purple) and Gbx2 (green). En2 is expressed mainly in the Otx2 domain with a faint graded co-expression with Gbx2 at stage 17 (arrowhead). (M-O) The expression patterns observed by whole-mount in situ hybridization during the three stages described above. The positions of Fgf8 and En2 expression are also shown. Note the refinement in the Otx2-Gbx2 overlapping region and the co-expression domains of Xiro1, Otx2 and Gbx2.|
|Fig. 2. Otx2 and Gbx2 participate as transcriptional repressors in the positioning of the isthmus organizer. Embryos were injected in one blastomere of two-cell stage embryos with 2 ng of Gbx2 (A,D,G,J), 2 ng of the Gbx2 repressor fusion (Gbx-EnR) (B,E,H,K) or 0.3 ng Gbx2 activator fusion (Gbx2-E1A) (C,F,I,L) mRNAs. The expression of Otx2, Fgf8, En2 and Pax2 were analyzed at stage 17 and the injected sides were detected by X-Gal stain. (A-C) Otx2 expression is inhibited in embryos injected with Gbx2 or Gbx2-EnR mRNAs (A,B, broken lines), while is displaced caudally in those injected with Gbx2-E1A mRNA (C, broken lines). (D-F) A rostral shift of Fgf8 isthmic expression territory is observed upon Gbx2 or Gbx2-EnR overexpression (D,E, broken lines), and inhibition and caudal shift of this expression domain occurs in Gbx2-E1A-injected embryos (F, arrowhead). (G-I) En2 is displaced anteriorly in Gbx2- or Gbx2-EnR-injected embryos (G,H, broken lines), while is repressed and shift caudally in those injected with Gbx2-E1A mRNA (I, broken lines). (J-L) Pax2 expression is displaced rostrally in embryos injected with Gbx2 or Gbx2-EnR mRNAs (J,K, broken lines), while a caudal shift occurs in Gbx2-E1A-injected embryos (L, broken lines). The injection at the two-cell stage of 5 ng of Otx2 (M,P,S,V), 2 ng of Otx2-EnR (N,Q,T,W) or 1 ng of Otx2-E1A (O,R,U,X). The expression of Gbx2, Fgf8, En2 and Pax2 were analyzed at stage 17. (M-O) Overexpression of Otx2 or Otx2-EnR mRNAs produce repression and caudal shift of Gbx2 (broken lines) and injection of Otx2-E1A mRNA caused an anterior shift and diffusion of Gbx2 (O, broken lines). (P-R) Fgf8 is shifted posteriorly in embryos injected with Otx2 or Otx2-EnR mRNAs (P,Q, broken lines) while injection of Otx2-E1A mRNA causes inhibition of the isthmus expression of Fgf8 (R, arrowhead). (S-U) En2 is shifted caudally in Otx2 and Otx2-EnR injected embryos (S,T, broken lines), while there is a decrease in En2 expression with an anterior displacement in embryos injected with Otx2-E1A mRNA (U, broken lines). (V-X) Pax2 is shifted caudally in Otx2- and Otx2-EnR-injected embryos (V,W broken lines), while its expression decrease in the Otx2-E1A-injected embryos (X, arrowhead). Arrowheads point to the injected sides. Each experiment was performed at least twice with a minimum of 30 embryos. The percentage of effect for each experiment was ∼ 70%.|
|Fig. 3. The interaction between Otx2 and Gbx2 induce the isthmus. Embryos were injected with different mRNAs (5 ng of Otx2, 2 ng of Gbx2, 0.3 ng of β-galactosidase, 1 ng of XFD) at the one-cell stage. Animal caps were dissected at stage 10 and cultured as conjugates. (A,B) No Fgf8 expression was detected in conjugates of control uninjected animal caps with Gbx2-injected ones (A, 0%, n=20) or with Otx2-expressing caps (B, 0%, n=23) at stage 17. (C-E) Conjugates of Otx2- with Gbx2-expressing caps performed at stage 10 can induce Fgf8 (C; 69%, n=45), En2 (D; 93%, n=109) and Wnt1 (E; 65%, n=17) (arrowheads) at stage 17. β-Galactosidase (arrow) was co-injected with Otx2 (C) or with Gbx2 (D). Fgf8 was induced in the Gbx2 injected cap and En2 in the Otx2-expressing cap as shown by the X-Gal staining. (F) Conjugate of Otx2+XFD- and Gbx2-expressing caps. En2 induction was blocked when XFD was co-expressed with Otx2 (arrow in F shows X-Gal staining in the Otx2+XFD animal cap, 22% of expression, n=37). (G) Conjugate of Otx2- and Gbx2+XFD-expressing caps. XFD co-injected with Gbx2 did not block the induction of En2 (arrowhead, 95% of expression, n=47). Arrow in G shows X-Gal in the Gbx2+XFD cap.|
|Fig. 4. Xiro1 participates in the positioning of the isthmus organizer. Embryos were injected in one blastomere at the two-cell stage with 2 ng of Xiro1 mRNA (A,D,G,J), 0.5 ng of HD-GR-EnR (B,E,H,K) or HD-GR-E1A (C,F,I,L); the inducible constructs were induced around stage 12.5. The injected side is marked by X-Gal stain in the Xiro1-injected embryos and by Myc staining in the case of the inducible constructs. (A) Xiro1 overexpression promotes an expansion and caudal shift of Gbx2. (B) HD-GR-EnR mRNA injection causes expansion and anterior shift of Gbx2 expression. (C) Gbx2 is repressed in embryos injected with HD-GR-E1A mRNA. (D) In embryos injected with Xiro1 mRNA Otx2 midbrain expression domain is expanded caudally. (E) However, injection of HD-GR-EnR mRNA caused an anterior shift of the Otx2 expression domain. (F) A caudal expansion of Otx2 when HD-GR-E1A mRNA is overexpressed. (G) Fgf8 expression is displaced posteriorly in embryos injected with Xiro1 mRNA. (H) Overexpression of HD-GR-EnR promotes an expansion and anterior shift of the isthmus domain of Fgf8. (I) This domain is repressed in HD-GR-E1A-injected embryos. (J) In embryos injected with Xiro1 mRNA, Pax2 is expanded. (K) HD-GR-EnR mRNA injection causes an anterior shift of Pax2 expression. (L) Pax2 is repressed and shifted caudally in embryos injected with HD-GR-E1A mRNA. Broken lines show the described effects. Arrowheads indicate the injected sides. Each experiment was performed at least twice with a minimum of 45 embryos. The percentage of effect for each experiment was ∼70%.|
|Fig. 5. Xiro1 controls the expression of Otx2 and Gbx2 at different developmental stages. Embryos were injected in one blastomere at two-cell stage with 2 ng of Xiro1 mRNA (A-C), 0.5 ng of HD-GR-EnR (D,G,J,M,P,S), 0.5 ng of HD-GR-E1A (E,H,K,N,Q,T) or 0.5 ng of HD-GR (F,I,L,O,R,U) and the expression of Otx2, Gbx2 and Pax2 were analyzed at early neurula stage (stage 14). Activation of the inducible constructs was achieved by adding dexamethasone at stage 9.5-10 (D-L) or at stage 12-12.5 (M-U). Embryos injected with Xiro1 mRNA show a caudal expansion of Otx2 (A, broken lines), expansion and caudal shift of Gbx2 (B, broken lines), and Pax2 is displaced caudally (C, broken lines). (D-I) Otx2 (green) and Gbx2 (purple) were expanded and shifted caudally in embryos injected with HD-GR-EnR mRNA (D,G, broken lines). HD-GR-E1A and HD-GR repressed Otx2 and Gbx2 expression when activated at stage 9.5-10 (E,H and F,I, arrowheads). A caudal shift of Pax2 expression is observed in embryos injected with HD-GR-EnR when activated at stage 9.5-10 (J, broken lines). The injection of both HD-GR-E1A and HD-GR repress Pax2 midbrain expression domain (K,L, arrowheads). (M-O) Otx2 midbrain territory is inhibited and shifted rostrally in embryos injected with HD-GR-EnR mRNA (M, broken lines). A caudal expansion in Otx2 expression is produced by HD-GR-E1A and HD-GR overexpression and activation at stage 12-12.5 (N,O, broken lines). (P-R) Gbx2 expression is expanded anteriorly in embryos injected with HD-GR-EnR mRNA and activated at stage 12-12.5 (P, broken lines), while the injection of HD-GR-E1A and HD-GR mRNAs promote repression of Gbx2 (Q,R, arrowheads). (S-U) Embryos injected with HD-GR-EnR and activated at stage 12-12.5 causes an anterior shift of Pax2 expression (S, broken lines), while HD-GR-E1A and HD-GR produce repression and caudal displacement of Pax2 expression when activated at stage 12-12.5 (T,U, broken lines). Arrowheads indicate the injected sides. Each experiment was performed at least twice with a minimum of 20 embryos. The percentage of effect for each experiment was ∼70%.|
|Fig. 6. Rescue experiments. Embryos were injected in one blastomere at the two-cell stage with Xiro1 (1 ng) and HD-GR-EnR (0.5 ng) (A,D,G,J), or HD-GR-E1A (0.5 ng) (B,E,H,K), or HD-GR (0.5 ng) (C,F,I,L). The inducible constructs were activated around stage 10 (A-F) or 12.5 (G-L) and the expression of Otx2 and Gbx2 were analyzed at early neurula stage. Embryos injected with a mixture of Xiro1 and HD-GR-EnR and activated around stage 10 show an expansion and caudal shift of Gbx2 (A, broken lines, 90%, n=27) and a caudal expansion of Otx2 midbrain expression domain (D, broken lines, 70%, n=23). The overexpression of Xiro1 with HD-GR-E1A or with HD-GR and activation at stage 10 rescue the expression of both Otx2 and Gbx2 (B,E and C,F, respectively). The expression of Otx2 and Gbx2 is rescued in the embryos injected with mixtures of Xiro1 with HD-GR-EnR (G,J), with HD-GR-E1A (H,K) or with HD-GR (I,L) activated at stage 12-12.5. Broken lines show the displacements of gene expression. Arrowheads indicate the injected sides. The percentage of rescue of normal expression for each experiment was ∼75%.|
|Fig. 7. Gbx2 rescue Otx2 but not Fgf8 expression. Embryos were injected in one blastomere at the two cells stage with 0.5 ng of HD-GR (A,C), or with 0.5ng of HD-GR and 1 ng of Gbx2 (B,D). The inducible constructs were induced around stage 12.5 and the injected side was detected by the Myc immunostaining. (A) Caudal expansion of the Otx2 midbrain domain (black lines) in embryos injected with HD-GR mRNA. (B) A nearly normal Otx2 expression is restored with the co-expression of Gbx2 and HD-GR (black lines). (C) Injection of HD-GR produced a complete inhibition of Gbx2. (D) The co-injection of HD-GR and Gbx2 did not rescue the expression of Fgf8, even though it produced a nearly normal Otx2 expression. Arrowheads show the injected sides and point the effects described above. Each experiment was carried out at least twice with a minimum of 54 embryos. The percentage of effect (or rescue) for each experiment was ∼70%.|
|Fig. 8. Role of Xiro1 on isthmic organizer in vitro. Embryos were injected at one-cell stage with the mRNAs described, the animal caps were explanted and conjugated at stage 10 and cultured until the equivalent of stage 17. At this stage the gene expression was assayed. (A) Injection of 2 ng of Gbx2 mRNA do not induce Xiro1 expression (0%, n=36). (B) In caps injected with 5 ng of Otx2 mRNA, Xiro1 expression is induced (arrowheads, 65%, n=23; inset shows uninjected animal caps). (C) Caps injected with 2 ng Xiro1-EnR mRNA express Gbx2 (arrowheads, 57%, n=46; inset shows uninjected animal caps). (D) Otx2(5 ng)//Xiro1(2 ng) conjugates express En2 (arrowheads, 90%, n=30) in the Otx2 territory (arrow indicates the X-Gal stain in the Xiro1-expressing caps). (E) Fgf8 also is induced in these conjugates (arrowhead, 71%, n=34, arrow shows the X-Gal stain in the Xiro1 caps). (F) Interference with Xiro1 function with HD-GR-E1A (0.5 ng) at stage 12 suppressed En2 expression in the Otx2 expressing cap (40%, n=33).|
|Fig. 9. A model for the induction and positioning of the isthmus organizer. (A) Gastrula. Xiro1 encompasses Gbx2 expressing domain and the presumptive midbrain territory of Otx2 and participate in the activation of both genes (arrows). In addition, Otx2 also activates Xiro1 expression in the midbrain. At this stage Otx2 and Gbx2 expression domains overlap in the prospective isthmus and the mutual repressive activities between the corresponding proteins begin (red lines) (B) Early neurula. The expression domains of Otx2 and Gbx2 start to separate although a faint overlapping is still detected. At this stage, Xiro1 is no longer able to activate Otx2. In addition, Fgf8 expression, and therefore the establishment of the isthmus, begins as a result of the overlapping domain created by Otx2 and Gbx2 (broken arrows) and the activity of Xiro1 in this region (arrow). (C) Mid neurula. A sharp boundary between Otx2 and Gbx2 arises, which is probably due to an equilibrium reached by their cross-inhibitory activities (red lines). The interaction between Otx2 and Gbx2 maintains Fgf8, which reinforces the expression of Gbx2 in the caudal face of the isthmus (arrow). In addition, Fgf8 induces En2 expression in the competent territory defined by the co-expression of Otx2 and Xiro1. a, anterior; p, posterior.|