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Otx2 and Gbx2 are required for refinement and not induction of mid-hindbrain gene expression

¹ Howard Hughes Medical Institute and Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA
² Departments of Cell Biology and Physiology, and Neuroscience, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA

View larger version (50K): [in a new window]   Fig. 1. An interaction between Gbx2 and Otx2 defines the limits of their respective expression domains at the start of somitogenesis. (A) Expression of Gbx2 and Hesx1 in wild-type and Otx2hOtx1/hOtx1 embryos at the EHF stage. The anterior limit of Gbx2 expression in the midline (arrow), relative to the position of the node (arrowhead) in Otx2hOtx1/hOtx1 and wild-type embryos is comparable, although the lateral expression of Gbx2 (asterisk) appears slightly expanded anteriorly in the mutant. (B) Gbx2 expression is significantly expanded anteriorly in Otx2hOtx1/hOtx1 embryos at the LHF stage, compared with wild-type controls. (C,D) Expression of Otx2 in wild-type and Gbx2–/– embryos at the EHF (C) and LHF (D) stage. Relative to the position of the node (arrowhead), the posterior limit of Otx2 (arrow) is not altered in Gbx2–/– embryos at the EHF stage, but shifted caudally by the LFH stage, particularly in the midline. (E) Expression of Gbx2 is normal in Gbx2–/– embryos at the EHF stage. (F) Hesx1 expression (brackets) is not changed in Gbx2–/– embryos, whereas Gbx2 expression is reduced and its anterior limit (arrow) is shifted caudally compared with wild-type controls at the LHF stage. The apparently stronger staining of Hesx1 in the Gbx2–/– embryo is due to a prolonged color reaction in the mutant compared with the wild type in order to visualize Gbx2 staining. (B,F) Dorsal views of flat-mount embryos.

View larger version (78K): [in a new window]   Fig. 2. Gbx2 is required for repression of Otx2 by exogenous RA. (A-D) Expression of Gbx2 in wild-type control (A,B) and RA-treated embryos (C,D). (E-J) Expression of Otx2 in a wild-type control (E,F) and RA-treated wild-type embryo (G,H) and RA-treated Gbx2–/– embryo (I,J). (C,G,I) Embryos 4 hours after RA treatment. (D,H,J) Embryos 24 hours after RA treatment. Note that by 4 hours, Gbx2 is already induced anteriorly by RA (C), whereas Otx2 is only significantly repressed by 24 hours and restricted to the most anterior tip (arrowhead) of the embryo (G,H). The repression of Otx2 by RA is inhibited in Gbx2–/– embryos (I) and 24 hours after RA treatment Otx2 is expanded posteriorly to the presumptive r3/4 border (arrow) (J), similar to that in untreated Gbx2–/– embryos at E8.5 (inset in J). Anterior is towards the left.

View larger version (48K): [in a new window]   Fig. 3. Expression of mes-met genes in single Otx2 or Gbx2 and double homozygous mutant embryos at early somite stages. (A,B) Expression of Fgf8 at the four-somite stage. Insets in the images show expression of Hoxb1 (arrowheads) and Fgf8 (brackets) in six- to seven-somite stage embryos. Note that in the Gbx2–/– embryo (inset in C), broad and weak Fgf8 expression forms a gradient in the anterior hindbrain with highest expression overlapping with Hoxb1 expression in r4. By contrast, Fgf8 is expressed broadly in the anterior neuroectoderm of Gbx2–/–; Otx2hOtx1/hOtx1 embryos (inset in D) and its posterior limit ends a few cell diameters from Hoxb1 expression in r4. (E-H) Expression of Wnt1 in the posterior hindbrain (arrows) remains unchanged in embryos that lack Otx2 (F), Gbx2 (G) or both (H) at the four- to five-somite stage. The transverse band of Wnt1 expression in the mesencephalon (brackets), however, is affected in these embryos. Inset in F shows an Otx2hOtx1/hOtx1 embryos at the eight-somite stage with Wnt1 expression in the lateral edges of the neural plate extending from the posterior hindbrain to the anterior extreme of the embryo. (I-L) and (M-P) Expression of Pax2 at the LHF stage and four-somite stage, respectively, in embryos lacking Otx2 (J,N), Gbx2 (K,O) or both genes (L,P). The mes-met expression of Pax2 is indicated by a bracket. Expression of Pax2 in the pre-otic ectoderm is marked by arrows. (Q-T) En1 expression (brackets) is shifted anteriorly in embryos that lack Otx2 (R,T), whereas En1 expression is expanded posteriorly in embryos that lack Gbx2 (S,T). The En1 expression level in Otx2hOtx1/hOtx1 embryos is also reduced. (U-W) Human OTX1 is expressed from the Otx2 locus in Otx2 heterozygous (U), Otx2hOtx1/hOtx1 (V) and Gbx2–/–; Otx2hOtx1/hOtx1 (W) embryos. Human OTX1 is expressed only in the anteriormost endoderm and ectoderm (arrowhead), but not in the neuroectoderm of Otx2hOtx1/hOtx1 embryos, whereas in Gbx2–/–; Otx2hOtx1/hOtx1 embryos human OTX1 is expressed in a broad anterior domain of the neuroectoderm. Note that all the Otx2hOtx1/hOtx1 and Gbx2–/–; Otx2hOtx1/hOtx1 embryos have a similar anterior truncation (compare embryos in the second column with those in the forth column). The number of somites in the embryos is indicated in the lower right-hand corner of each panel. Anterior is towards the left, except for I-L,Q-T, which are dorsal views of embryos with anterior to the top.

View larger version (94K): [in a new window]   Fig. 4. Gbx2–/–; Otx2hOtx1/hOtx1 embryos have an anterior truncation and exencephaly. (A,B) Morphology of wild-type (left in A), Otx2hOtx1/hOtx1 (right in A) and Gbx2–/–; Otx2hOtx1/hOtx1 embryos (B) at E12.5. (C-E) Sagittal sections of wild-type (C), Otx2hOtx1/hOtx1 (D) and Gbx2–/–; Otx2hOtx1/hOtx1 embryos (F) at E12.5. The anterior structures of Otx2hOtx1/hOtx1 and Gbx2–/–; Otx2hOtx1/hOtx1 embryos are largely truncated. There is more anterior tissue, particularly head mesenchyme (asterisk) in Gbx2–/–; Otx2hOtx1/hOtx1 embryos than in Otx2hOtx1/hOtx1 embryos. There is an additional thin epithelium (bracket) extending from the presumptive hindbrain in Gbx2–/–; Otx2hOtx1/hOtx1 embryos. D,E are at a higher magnification than C. The junction between the spinal cord and hindbrain is marked by arrowheads. Cb, cerebellum; Dt, dorsal thalamus; IV, IVth ventricle; Mb, midbrain; Sc, spinal cord; Tel, telecephalon.

View larger version (66K): [in a new window]   Fig. 5. Mes-met genes are maintained and co-expressed in the anterior neuroectoderm of Gbx2–/–; Otx2hOtx1/hOtx1 embryos at E9.5. (A-H) Expression of Fgf8 (A,B), Wnt1 (C,D), En1 (E,F) and human OTX1 (G,H) in wild type (left column) and Gbx2–/–; Otx2hOtx1/hOtx1 embryos (right column) at E9.5. Fgf8 and En1 are strongly expressed in a broad anterior region of the double mutant embryos and their expression appears to co-localize with that of Wnt1 and human OTX1.

View larger version (85K): [in a new window]   Fig. 6. Spatial relationship of the expression domains of mes-met genes in Otx2hOtx1/hOtx1 embryos at E10.5. (A) The normal expression patterns of Wnt1, Fgf8, Pax2, En1 and En2 in the mid/hindbrain region at E10.5 (Joyner et al., 2000). (B-F) In situ hybridization analysis of expression of Fgf8 (B), Wnt1 (C), Pax2 (D), En1 (E) and En2 (F) on near adjacent sagittal sections of a Gbx2–/–; Otx2hOtx1/hOtx1 embryo. Note that the posterior limits (arrows) of the expression domains of Wnt1, Fgf8 and Pax2 are similar, whereas the expression domains of En1 and En2 encompass those of Wnt1, Fgf8 and Pax2 and their posterior limits (arrowheads) are successively extended more caudally, with a decreasing gradient.

View larger version (80K): [in a new window]   Fig. 7. Expression of Krox20 in r3 is restored in Gbx2 mutants by removing Otx2 function. (A-D) Double labeling for Six3 (arrowheads) and Krox20 (arrows) expression in wild-type (A), Gbx2–/– (B), Otx2hOtx1/hOtx1 (C) and Gbx2–/–; Otx2hOtx1/hOtx1 (D) embryos at the eight-somite stage. Insets show Six3 expression in embryos of corresponding genotypes at the 3-somite stage. Two stripes of Krox20 expressing cells (arrows), corresponding to r3 and r5, are found in wild type, Otx2hOtx1/hOtx1 and Gbx2–/–; Otx2hOtx1/hOtx1 embryos, but there is only a single stripe of Krox20 expression in r5 of Gbx2–/– embryos. (E-H) Hoxa2 expression in wild-type (E), Gbx2–/– (F), Otx2hOtx1/hOtx1 (G) and Gbx2–/–; Otx2hOtx1/hOtx1 (H) embryos at E9.5. Hoxa2 expression in rhombomeres is bracketed, whereas the expression in neural crest cells migrating from r4 is indicated by arrows. R3 expression of Hoxa2 is rescued in Gbx2–/–; Otx2hOtx1/hOtx1 mutant embryos, whereas r2 expression of Hoxa2 is missing.

View larger version (21K): [in a new window]   Fig. 8. Establishment of the spatial relationships of mes-met genes at the Otx2-Gbx2 border. (A) Expression of Wnt1, Fgf8 and En1 in wild-type, Otx2hOtx1/hOtx1, Gbx2–/– and Gbx2–/–; Otx2hOtx1/hOtx1 embryos at the three- to four-somite and six- to eight-somite stages. Thickness of the bars represents the level of gene expression. (B) Model of how the mes-met genes are induced and how the normal spatial expression of these genes is established in the neuroectoderm. Opposing interactions between posteriorizing and anteriorizing signals determine the position of the Otx2/Gbx2 border, as well as a mes-met competence domain by E7.5. The entire presumptive mes-met region is competent to respond to a signal that induces expression of Wnt1, Fgf8 and other mes-met genes. Expression of Otx2 and Gbx2 in the neuroectoderm at E7.75 subdivides the presumptive mes-met region into two distinct domains. Negative regulation of Wnt1 by Gbx2, and of Fgf8 by Otx2 results in the initial restriction of Wnt1 and Fgf8 expression specifically to the Gbx2- and Otx2-negative positive regions, respectively, at E8.5. At later stages, expression of Wnt1 and Fgf8 is maintained only in cells adjacent to each other through mutual positive feedback between Fgf8 and Wnt1 (or an unknown secreted factor in the midbrain) and between all mes-met genes. Otx2 and Gbx2 continue to negatively regulate Fgf8 and Wnt1 expression, respectively. MHB, mid-hindbrain boundary.