QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Boyl, P. P. Articles by Simeone, A. Search for Related Content PubMed PubMed Citation Articles by Boyl, P. P. Articles by Simeone, A.

Forebrain and midbrain development requires epiblast-restricted Otx2 translational control mediated by its 3' UTR

Pietro Pilo Boyl^1,*, Massimo Signore^1,2,*, Dario Acampora^1,2,*, Juan Pedro Martinez-Barbera¹, Cristina Ilengo³, Alessandro Annino², Giorgio Corte^3,4 and Antonio Simeone^1,2,

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
³ IST-National Institute for Cancer Research, Università di Genova, Largo Benzi, 16132 Genova, Italy
⁴ Dipartimento di Oncologia Clinica e Sperimentale, Università di Genova, Largo Benzi, 16132 Genova, Italy
^* These authors contributed equally to this work

View larger version (30K): [in a new window]   Fig. 1. Targeted mutagenesis of the 3' UTR of the Otx2 gene. (A) The targeting vector is shown in third line; fourth line illustrates recombined locus; first and last lines show EcoRI fragments (11 and 7.5 kb) detected by Southern blot using probes (a-e, white boxes) external to the targeting vector or within neomycin gene. Kp, KpnI; Ap ApaI; E, EcoRI; met, methionine; stop, stop codon; pA: polyadenylation signal. (B) Southern blot analysis of one targeted cell line (Otx2/+) and wild-type HM-1 ES cells hybridised with probe b in A. (C) PCR genotyping of a litter from two heterozygotes using the primers indicated as filled and open arrowheads in A. (D) In situ hybridisation of an Otx2/Otx2 embryo at 10 d.p.c. with the Otx2-specific probe (probe d in A).

View larger version (50K): [in a new window]   Fig. 2. Morphology of Otx2/Otx2 and Otx2/- embryos. (A-J) When compared with wild-type (A,F), Otx2/Otx2 embryos exhibited brain abnormalities, the majority of which were exencephaly, lack of closure of neural tube (B,G) or reduction of forebrain-midbrain (C,H); 90% of Otx2/- embryos revealed a severe reduction of anterior CNS with an almost headless phenotype (D,I,J) and 10% of them a phenotype (E) similar to that of Otx2/Otx2 mutants. Fb, forebrain; Hb, hindbrain; Mb, midbrain.

View larger version (101K): [in a new window]   Fig. 3. Distribution of Otx2 mRNA and protein during gastrulation. (A-L) Sagittal sections of 6.5 and 7.75 d.p.c. wild-type (A,A',E,E',I), Otx2+/- (B,B',F,F',J), Otx2/Otx2 (C,C',G,G',K) and Otx2/- (D,D',H,H',L) embryos hybridised with an Otx2- (A,B,E,F) and an Otx2 (C,D,G,H) -specific probe. There are no differences between Otx2 (A,B,E,F) and Otx2 (C,D,G,H) expression patterns. OTX2 protein levels are gradually reduced in the epiblast, anterior neuroectoderm and axial mesendoderm but not in the VE of Otx2/Otx2 (C’,G’,K) and Otx2/- (D’,H’,L) mutants (when compared with wild-type (A’,E’,I) and Otx2+/- (B’,F’,J) embryos. ame, axial mesendoderm; ane, anterior neuroectoderm; AVE, anterior visceral endoderm; epi, epiblast. (I-L) Magnifications of embryos in (E'-H').

View larger version (28K): [in a new window]   Fig. 4. OTX2 protein level in Otx2 mutant embryos. (A) Western blot analysis of 7.5 d.p.c. wild-type, Otx2+/-, Otx2/Otx2 and Otx2/- embryos, showing that in Otx2 mutants the OTX2 protein is remarkably reduced. (B) Coomassie Blue staining of the same gel in A. (C) Graphic representation of densitometric scanning indicating a reduction of 60% of OTX2 protein in Otx2/Otx2 and Otx2/- mutants when compared with wild-type and Otx2+/- embryos, respectively.

View larger version (39K): [in a new window]   Fig. 5. Quantitative analysis and ribosome affinity profiles of Otx2 mRNA. (A) Total, cytoplasmic and nuclear Otx2 mRNA exhibits a reduction of 20% when compared with the wild-type Otx2 mRNA. The Otx2 mRNA percentages are the mean of three independent experiments. (B) Ribosome affinity profiles of Otx2, ß-actin mRNAs and in vitro synthesised Otx2 RNA, showing the distribution of the untranslated Otx2 RNA (see also Materials and Methods). (C,D) Ribosome affinity profiles comparing Otx2, Otx2 and ß-actin mRNAs in Otx2/Otx2 embryos at 7.75 (C) and 10.5 d.p.c. (D) show that while more than 50% of the ß-actin and Otx2 mRNAs is detected in the first two fractions, only 25% of the Otx2 mRNA is concentrated in the same fractions. Note that mRNA profiles are not influenced by any quantitative variation of the mRNAs analysed.

View larger version (123K): [in a new window]   Fig. 6. Gastrulation and early patterning of rostral neuroectoderm are unaffected in Otx2/- embryos. (A-F) Sagittal sections of 6.5 d.p.c. wild-type (A,C,E) and Otx2/- (B,D,F) embryos hybridised with Otx2 (A), Otx2 (B), cer-l (C,D) and Lim1 (E-F). (G-P) Sagittal sections of 7.5 d.p.c. wild-type (G,I,K,M,O) and Otx2/- (H,J,L,N,P) embryos are hybridised with Otx2 (G), Otx2 (H), Gbx2 (I,J), Six3 (K,L) Chd (M,N) and Lim1 (O,P).

View larger version (82K): [in a new window]   Fig. 7. Abnormal patterning of forebrain and midbrain territory in Otx2/Otx2 and Otx2/- mutant embryos. (A-O) Whole-mount in situ hybridisation of 8.5 d.p.c. wild-type (A,D,G,J,M), Otx2/Otx2 (B,E,H,K,N) and Otx2/- (C,F,I,L,O) embryos with probes for Otx2 (A-C), Fgf8 (D-F), Gbx2 (G-I), Wnt1 (J-L) and BF1 (M-O).

View larger version (103K): [in a new window]   Fig. 8. Head abnormalities in Otx2/Otx2 and Otx2/- embryos at 10.5 d.p.c. (A-L) Sagittal sections of 10.5 d.p.c. wild-type (A,D,G,J), Otx2/Otx2 (B,E,H,K) and Otx2/- (C,F,I,L) embryos hybridised with probes for Otx2 (A-C), Fgf8 (D-F), Gbx2 (G-I) and BF1 (J-L). The arrows point to the corresponding position of the Otx2 posterior border of expression. Fb, forebrain; Hb, hindbrain; Mb, midbrain.

View larger version (38K): [in a new window]   Fig. 9. Vertebrate Otx2 genes contain a conserved element within the 3' UTR. (A) Schematic alignment between the mouse and the human, chicken, Xenopus, zebrafish, lamprey and Amphioxus Otx2 3' UTRs, showing the conserved 140 bp element. Percentages of homology between corresponding regions are reported with different colours. In lamprey, only 26 bp of the element are conserved. (B) Percentages of identity of the 140 bp element among the reported gnathostomata. (C) DNA sequence alignment of the conserved element, also including the 26 bp conserved sequence of the lamprey.