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Regionalisation of anterior neuroectoderm and its competence in responding to forebrain and midbrain inducing activities depend on mutual antagonism between OTX2 and GBX2

¹ MRC Centre for Developmental Neurobiology, King’s College London, Guy’s Campus, New Hunt’s House, London SE1 1UL, UK
² International Institute of Genetics and Biophysics, CNR, Via G. Marconi 12, 80125, Naples, Italy

View larger version (90K): [in a new window]   Fig. 1. Morphology of a hOtx12/hOtx12; Gbx2–/– embryo at 9.7 d.p.c. (A-D) Compared with wild-type (A) and Gbx2–/– (B) embryos, hOtx12/hOtx12; Gbx2–/– (D) mutants exhibit severe morphological abnormalities affecting the anterior neuroectoderm and the heart (arrow); however, compared with hOtx12/hOtx12 embryos (C), it is evident that the distance between the otic vesicle (arrowhead in C,D) and the rostral tip of the embryo is increased in hOtx12/hOtx12; Gbx2–/– mutants. fb, forebrain; mb, midbrain; hb, hindbrain; ov and arrowheads, otic vesicle.

View larger version (100K): [in a new window]   Fig. 2. Neural patterning in hOtx12/hOtx12; Gbx2–/– embryos. Whole-mount in situ hybridisation of 8.7 d.p.c. wild-type (A-G), hOtx12/hOtx12 (H-N), Gbx2–/– (O-U) and hOtx12/hOtx12; Gbx2–/– (V-ß') embryos with Otx2 (A,O), hOtx1 (H,V,V'), Gbx2 (B,I,P,W,W'), Fgf8 (C,J,Q,X,X'), Pax2 (D,K,R,Y,Y'), En1 (E,L,S,Z,Z'), Wnt1 (F,M,T,,') and Bf1 (G,N,U,ß,ß') probes shows that hOtx12/hOtx12; Gbx2–/– embryos exhibit widespread expression of hOtx1, Gbx2, Fgf8, Pax2, En1 and Wnt1 genes whilst the forebrain specific gene Bf1 is not transcribed in the anterior neuroectoderm. A-ß are sagittal and V'-ß' are dorsolateral views. Abbreviations as in previous figure plus, MHB, midbrain-hindbrain boundary. The arrow and the arrowhead in ß-ß' indicate the corresponding regions in non-neural tissue.

View larger version (93K): [in a new window]   Fig. 3. Failure in regionalisation of the anterior neural plate in hOtx12/hOtx12; Gbx2–/– embryos. (A-X) In situ hybridisation of sagittal sections of 9.7 d.p.c. wild-type (A-F), hOtx12/hOtx12 (G-L), Gbx2–/– (M-R) and hOtx12/hOtx12; Gbx2–/– (S-X) embryos, with Otx2 (A,M), hOtx1 (G,S), Fgf8 (B,H,N,T), Pax2 (C,I,O,U), En1 (D,J,P,V), Wnt1 (E,K,Q,W) and Atx (F,L,R,X) probes reveals that in hOtx12/hOtx12; Gbx2–/– embryos widespread co-expression of hOtx1, Fgf8, Pax2, En1 and Wnt1 is maintained and that the midbrain-specific gene Atx is not transcribed. Abbreviations as in previous figures. A',G',M',S' are bright-field images of the sections I A,G,M and S.

View larger version (71K): [in a new window]   Fig. 4. Fgf8 is unable to repress Otx2 in the dorsal midbrain of Otx1+/–; Otx2+/– embryos. (A-J) Whole-mount in situ hybridisation of wild-type (A-E) and Otx1+/–; Otx2+/– (F-J) embryos with Otx2 (A,F), Gbx2 (B,G), Fgf8 (C,H), Pax2 (D,I) and En1 (E,J) probes shows that in double heterozygous embryos Fgf8 (arrow in H), Pax2 (arrow in I) and En1 (arrow in J) are ectopically co-expressed in the dorsal midbrain where Otx2 is also transcribed (arrow in F), whilst the Gbx2 stripe of expression (G) is only slightly thickened. Abbreviations as in previous figures.

View larger version (102K): [in a new window]   Fig. 5. Onset of the Otx/Gbx2 abnormal expression occurs at the late headfold-presomite stages and precedes Fgf8 activation. (A-M) Whole-mount in situ hybridisation of wild-type (A-K) hOtx12/hOtx12 (A'-K'), Gbx2–/– (A''-K'') and hOtx12/hOtx12; Gbx2–/– (L-O) embryos with Otx2/Hoxb1 (A,C,E,A'',C'',E''), Otx2 (G,I,G'',I''), hOtx1/Hoxb1 (A',C',E',L,), hOtx1 (G',I',N), Gbx2 (B-B'',D-D'',F-F'',H-H'',J-J'' and M,O) and Fgf8 (K-K'') probes at the stages indicated on the right side of the panel. The arrows indicate the anterior border of Hoxb1 expression in wild-type (A,C,E) and hOtx12/hOtx12 (A',C',E'). In Gbx2–/– embryos at late headfold and presomitic stages this border could not be identified, being fused to the posterior expansion of Otx2 expression (C'',E'').

View larger version (90K): [in a new window]   Fig. 6. Morphology of an Otx2/-; Gbx2–/– embryo at 10.5 d.p.c. Compared with wild-type (A) and Gbx2–/– (B) embryos, Otx2/-; Gbx2–/– embryos (D) show compromised development and severe abnormalities of the anterior CNS; while, compared with the head-less phenotype of Otx2/- embryos (C) it is evident that the anterior neural tube of Otx2/-; Gbx2–/– embryos (D) develop morphological similarities having midbrain and forebrain. Moreover, the heart (D) reveals normal morphology when compared with hOtx12/hOtx12; Gbx2–/– embryos (Fig. 1D). Abbreviations as in previous figures plus, he, heart.

View larger version (56K): [in a new window]   Fig. 7. A minimal amount of OTX2 protein suffices to displace Fgf8 and Gbx2 expression posteriorly and to induce forebrain- and midbrain-specific gene expression. (A-T) Whole-mount in situ hybridisation of wild-type (A-E), Otx2/- (F-J), Gbx2–/– (K-O) and Otx2/-; Gbx2–/– (P-T) embryos with Otx2 (A,F,K,P,), Gbx2 (B,G,L,Q), Fgf8 (C,H,M,R), Bf1 (D,I,N,S,) and Atx (E,J,O,T) probes shows that Otx2/-; Gbx2–/– double mutants recover a wide-spread expression of Otx2 (P), a posterior displacement of Gbx2 (Q) and Fgf8 (R) transcripts and the neuroectodermal expression of both the forebrain gene Bf1 (compare arrow in I and S) and the midbrain gene Atx (T). The arrow in R indicates Fgf8 expression in a region corresponding to the presumptive ANR; wild-type and Gbx2–/– embryos in A-C and K-M are the same as shown in Fig. 2. Abbreviations as in previous figures.