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OTD/OTX2 functional equivalence depends on 5' and 3' UTR-mediated control of Otx2 mRNA for nucleo-cytoplasmic export and epiblast-restricted translation

Dario Acampora^1,2,, Pietro Pilo Boyl^1,, Massimo Signore¹, Juan Pedro Martinez-Barbera¹, Cristina Ilengo³, Eduardo Puelles¹, Alessandro Annino², Heinrich Reichert⁴, Giorgio Corte^3,5 and Antonio Simeone^1,2,*

¹ MRC Centre for Developmental Neurobiology, King’s College London, Guy’s Campus, New Hunts House, London SE1 9RT, UK
² International Institute of Genetics and Biophysics, CNR, Via G. Marconi 12, 80125 Naples, Italy
³ IST-National Institute for Cancer Research,
⁵ Dipartimento di Oncologia Clinica e Sperimentale, Università di Genova, Largo Benzi, 16132 Genova, Italy
⁴ Institute of Zoology, University of Basel, Rheinsprung 9, CH-4051 Basel, Switzerland
These authors contributed equally to the work

View larger version (39K): [in a new window]   Fig. 1. Genomic organisation of the otd2 and otd2FL targeted loci (A). The targeted loci are shown in the third and fourth line; first and last lines show HindIII fragments (5.3 and 3.0 kb) detected by southern blot analysis using probes (hatched boxes) external to the targeting vector (probe c) or within the neomycin gene (probe f). S, SmaI; H, HindIII; N, NsiI; met, methionine; stop, stop codon; pA, polyadenylation signal. Dark hatched rectangles represent Otx2 5' and 3' UTRs, thinner open rectangles Drosophila otd UTRs. (B) Southern blot analysis of one representative targeted cell line for both molecules and wild-type HM-1 ES cells hybridised with probe c (see A). (C) PCR genotyping of two litters from two heterozygotes using the primers indicated in A as filled arrowheads (wild-type allele), open arrowheads (otd2 allele) and open arrows (otd2FL allele). (D-G) In situ hybridisation of an otd2/+ and an otd2FL/+ embryo at 10.5 d.p.c. with Otx2 (D,E) and otd-specific probes (probe a and e in A; F,G). fb, forebrain; mb, midbrain; hb, hindbrain.

View larger version (65K): [in a new window]   Fig. 2. Morphology of otd2/otd2 and otd2FL/otd2FL embryos. (A-J) Compared to 10.5 and 16 d.p.c. wild-type embryos (A,F), otd2/otd2 mutants exhibits a headless phenotype and normal body axis (B,G); while otd2FL/otd2FL embryos at 10.5 d.p.c. were classified on the basis of head abnormalities as severe (C), moderate (D,H,I) and mild (E,J). Abbreviations as in the previous figure.

View larger version (81K): [in a new window]   Fig. 3. Distribution of otd2, otd2FL and Otx2 mRNAs and proteins during gastrulation. (A-N’) Adjacent sagittal sections of 6.5 and 7.75 d.p.c. wild-type (A,B,A’,B’), otd2/+ (C,D,C’,D’) otd2/otd2 (E,F,E’,F’,K,L,L’), otd2FL/+ (G,H,G’,H’) and otd2FL/otd2FL (I,J,I’,J’,M,N,N’) embryos hybridised with probes specific for Otx2 (A,C,E,G,I,K,M) and otd (B,D,F,H,J,L,N) mRNAs or immunostained with OTX2 (A’,C’,E’,G’,I’) and OTD (B’,D’,F’,H’,J’,L’,N’) antibodies. Abbreviations: AVE, anterior visceral endoderm; epi, epiblast.

View larger version (136K): [in a new window]   Fig. 4. Normal gastrulation and early anterior patterning in otd2/otd2 embryos. (A-R) Sagittal sections of 6.5 d.p.c. (A-H) and 7.5 d.p.c. (I-R) wild-type (A,C,E,G,I,K,M,O,Q) and otd2/otd2 (B,D,F,H,J,L,N,P,R) embryos hybridised with Otx2 (A,I), otd (B,J), Lim1 (C,D), cer-l (E,F), Hesx1 (G,H), Six3 (K,L), Gbx2 (M,N), Nog (O,P) and Chd (Q,R).

View larger version (98K): [in a new window]   Fig. 5. Lack of forebrain and midbrain identities in the otd2/otd2 mutant is rescued in otd2FL/otd2FL embryos. (A-E’’) Whole-mount in situ hybridisation of 8.5 d.p.c. wild-type (A-E), otd2/otd2 (A’-E’) and otd2FL/otd2FL (A’’-E’’) embryos with Otx2 (A) otd (A’,A’’), Fgf8 (B,B’,B’’), Gbx2 (C,C’,C’’), Wnt1 (D,D’,D’’) and Bf1 (E,E’,E’’). (F-J’’) Adjacent sagittal sections of 10.5 d.p.c. wild-type (F-J), otd2/otd2 (F’-J’) and otd2FL/otd2FL (F’’-J’’) embryos hybridised with Otx2 (F), otd (F’,F’’), Fgf8 (G,G’,G’’), Wnt1 (H,H’,H’’), Bf1 (I,I’,I’’) and Tbr1 (J,J’,J’’). Abbreviations as in previous figures.

View larger version (44K): [in a new window]   Fig. 6. Genomic organisation of the Otx22c targeted locus. (A) The targeted locus is shown in the third line; first and last lines show HindIII fragments (5.3 and 3.0 kb) detected by Southern blot using the same probes (hatched boxes c and f) as in Fig. 1A. Abbreviations are as in legend to Fig. 1. (B) Southern blot analysis of one representative targeted cell line and wild-type HM-1 ES cells hybridised with probe c (see A). (C) PCR genotyping of a litter from two heterozygotes using the primers indicated in (A) as filled arrowheads (wild-type allele) and open arrowheads (Otx22c allele). (D-K) Compared to 10.5 and 16 d.p.c. wild-type embryos (D,H), Otx22c/Otx22c (E-G,I-K) phenotypes were classified on the basis of head abnormalities as severe (E,I), moderate (F,J) and mild (G,K). Note the similarity between Otx22c/Otx22c and otd2FL/otd2FL (Fig. 2) phenotypes. Abbreviations as in previous figures.

View larger version (118K): [in a new window]   Fig. 7. Otx22c/Otx22c embryos exhibit similarities with otd2FL/otd2FL embryos in forebrain and midbrain patterning. (A-D’) Whole-mount in situ hybridisation of 8.5 d.p.c. wild-type (A-D) and Otx22c/Otx22c (A’-D’) embryos with Otx2 (A,A’), Fgf8 (B,B’), Gbx2 (C,C’) and Bf1 (D,D’). (E-H’) Adjacent sagittal sections of 10.5 d.p.c. wild-type (E-H) and Otx22c/Otx22c (E’-H’) embryos, immunostained with OTX2 (E,E’) or hybridised with Fgf8 (F,F’), Gbx2 (G,G’) and Bf1 (H,H’). Abbreviations as in the previous figures.

View larger version (54K): [in a new window]   Fig. 8. Quantitative analysis of OTD and OTX2 proteins, otd2, otd2FL and Otx22c mRNAs and their stability in ES cells. (A) Western blot analysis of extracts from HeLa cells, HeLa cells transfected with an otd-expressing vector, and 7.5 d.p.c. wild-type, otd2/+ and otd2FL/+ embryos. (B) Western blot analysis on extracts from 7.5 d.p.c wild-type, Otx2+/– and Otx22c/Otx22c embryos. (C,D) Coomassie Blue staining of the gels in A and B. (E) otd2, otd2FL and Otx22c cytoplasmic and nuclear mRNA levels were determined in otd2/+, otd2FL/+ and Otx22c/+ embryos and are reported as percentages of the Otx2 mRNA. otd2 mRNA was also analysed at 7.5 d.p.c. Note that while the otd2 cytoplasmic mRNA is considerably reduced, the otd2 nuclear mRNA has accumulated significantly. (F) Stability of otd2, otd2FL and Otx22c mRNA is comparable to that of Otx2 mRNA in actinomycin D experiments performed on heterozygous ES cell lines.

View larger version (33K): [in a new window]   Fig. 9. otd2, otd2FL, Otx22c and hOtx1 mRNAs form polyribosome complexes with different efficiencies. (A) RNAse protection experiments showing the distribution of Otx2, otd2, otd2FL, Otx22c and ß-actin mRNAs along the fractions of polysome sucrose gradients. (B-D) Ribosome affinity profiles comparing the distribution of Otx2 and ß-actin mRNAs with that of otd2 (B), otd2FL (C) and Otx22c (D) mRNAs. (E) Quantitative analysis of hOtx1 cytoplasmic and nuclear mRNA showing a strong accumulation of nuclear RNA. (F) Ribosome affinity profile of the hOtx1 mRNA indicates a severe impairment in forming efficient polyribosome complexes. Note that mRNA profiles are not influenced by any quantitative variation of the mRNA analysed.