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Emx2 directs the development of diencephalon in cooperation with Otx2

¹ Department of Morphogenesis, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University, Japan
² CREST, JST, Japan
³ Vertebrate Body Plan Group, RIKEN Center for Developmental Biology, 2-2-1 Honjo, Kumamoto-860-0811, Japan
⁴ Department of Anatomy, Nara Medical University, 840 Saijo-machi, Kashihara, Nara-634-8521, Japan*These authors contributed equally to this work.

View larger version (116K): [in a new window]   Fig. 1. Morphological features of Emx2-/-Otx2+/- double mutant brains. (A,E,I,M) Wild-type, (B,F,J,N) Otx2+/-, (C,G,K,O) Emx2-/- and (D,H,L,P) Emx2-/-Otx2+/- embryos. (A-D) Sagittal sections stained with Cresyl Violet, (E-H) frontal sections stained with Cresyl Violet and (M-P) sagittal sections stained with Hematoxylin and Eosin. (I-L) Dorsal views of embryos. (A-H) 15.5 dpc, (I-P) 12.5 dpc. Arrows in A-D indicate the position of posterior commissure; circles in I-L indicate cerebral hemispheres. cb, cerebellum; cp, choroid plexus; Di, diencephalon; dt, dorsal thalamus; ept, epithalamus; hip, hippocampus; hp, hypophysis; ht, hypothalamus; ic, inferior coliculus; lge, lateral ganglionic eminence; ma, mammillary region; Me, mesencephalon; met, metencephalon; mge, medial ganglionic eminence; ncx, neocortex; ob, olfactory bulb; opc, optic chiasma; pt, pretectum; s, septum; spc, superior coliculus; Te, telencephalon; tg, tegmentum; vt, ventral thalamus; 3v, the third ventricle. Scale bars: 500 µm.

View larger version (101K): [in a new window]   Fig. 2. Characterization of diencephalic defects with molecular markers. (A,C,E,G,I,K,M,O,Q) Wild-type and (B,D,F,H,J,L,N,P,R) Emx2-/-Otx2+/- embryos. (A,B) Tcf4, (C,D) Lim1, (E,F) Dlx1, (G,H) Gbx2, (I,J) Ebf1, (K,L) Pax6, (M,N) Otx1, (O,P) Mek4 and (Q,R) ephrin-A2 expressions. (C,D) Whole-mount hybridization of 10.5 dpc embryos; the rest are in situ hybridization of sagittal sections of 11.5 dpc embryos. apt, anterior pretectum; at, anterior tectum; dt, dorsal thalamus; ge, ganglionic eminence; ht, hypothalamus; ppt, posterior pretectum; pt, pretectum; tg, tegmentum; vt, ventral thalamus; zl, zona limitans. Scale bars: 125 µm.

View larger version (96K): [in a new window]   Fig. 3. Characterization of double mutant defects with molecular markers at 9.5 dpc. (A,E,G,I,K,M,O) Wild-type, (C) Otx2+/- and (B,D,F,H,J,L,N,P) Emx2-/-Otx2+/- embryos. (A,B) BF1, (C,D) Otx2, (E,F) Pax6, (G,H) Wnt1, (I,J) En2, (K,L) Pax5, (M,N) Fgf8 and (O,P) Gbx2 expression. Arrows and arrowheads indicate the positions of the telodiencephalicus and isthmus, respectively. Double arrows in G,H indicate the anterior terminus (r3) of Wnt1 expression in the roof of spinal cord, and asterisks in E,F denote eyes. ov, otic vesicle. Scale bars: 250 µm.

View larger version (79K): [in a new window]   Fig. 4. Characterization of double mutant defects with molecular markers at six-somite stage. (A) Otx2+/-, (C,E,G,I,K,M) wild-type and (B,D,F,H,J,L,N) Emx2-/-Otx2+/- embryos. (A,B) Otx2, (C,D) Pax6, (E,F) Wnt1, (G,H) En1, (I,J) Pax5, (K,L) Fgf8 and (M,N) Six3 expression. The Wnt1-negative r1/2 is indicated by arrowheads in E,F. Arrow in K indicates the concentration of the Fgf8 expression into future isthmus at this stage. Asterisks indicate the optic cup. Scale bars: 150 µm.

View larger version (136K): [in a new window]   Fig. 5. Onset of double mutant defects. (A-D,M-P) Otx2, (E-H) Emx2, (I-L,Q-V) Pax2 and Gbx2 (W,X) expression at the three- (A,B,E,F,I,J,M,N,Q,R) and four- (C,D,G,H,K,L,O,P,S-X) somite stages. (A-L,U,W) Wild-type embryos and (M-T,V,X) Emx2-/-Otx2+/- embryos. (A,C,E,G,I,K,M,O,Q,S,W,X) Lateral views; (B,D,F,H,J,L,N,P,R,T) dorsal views; and (U,V) frontal views. Arrows denote the posterior limit and arrowheads the anterior limit of expressions. In S,T, arrows and double arrows indicate the posterior limits of expression in the most medial (future ventral) and lateral (future dorsal) points, respectively; the double arrows coincide with preotic sulcus. Broken lines in U,V indicate the anterior end of the neural plate. In W,X, arrows indicate the anterior limit of the expression, single arrowheads preotic sulcus and double arrowheads otic sulcus. Scale bars: 150 µm.

View larger version (67K): [in a new window]   Fig. 6. Targeted knock-in of Emx2 cDNA into the Otx2 locus. (A) Diagrammatic representation of knock-in strategy. Thick lines represent Otx2 genomic sequences; thin lines, pBluescript sequences; black boxes, Otx2 coding exons; black triangles, loxP sequences. White boxes denoted as Em, neor and DT-A indicate Emx2 cDNA lacking 3'UTR, neomycin-resistant gene lacking polyadenylation signal driven by Pgk1 promoter and diphtheria toxin-A fragment gene with MCI promoter, respectively. S is the probe for Southern blotting used to identify homologous recombinant ES cells shown in B; p1 and p2 are PCR primers used in the detection of the homologous recombinants in ES cells, and p3 and p4 are PCR primers used in the detection of the deletion of the neor cassette in F1 heterozygotes. H, HindIII; N, NotI; Nl, NlaIII; S, SalI; Sm, SmaI; Sp, SphI. (B) The top panel displays an example of Southern blotting used to identify homologous recombinants in ES cells; the bottom panel displays an example of the PCR genotyping for the deletion of neor cassette in F1 offspring obtained by mating between male chimera and Cre females. (C,D) RNA expressions from both endogenous and knocked-in Emx2 at 10.5 dpc in a wild-type embryo (C) and a knock-in mutant (D). Emx2 is expressed in the caudal diencephalon and mesencephalon where endogenous Emx2 is absent. (E,F) EMX2 protein expressions at 10.5 dpc in a wild-type (E) and a knock-in embryo (F). Consistent with the ectopic mRNA expression, EMX2 protein is detected in the caudal diencephalon and mesencephalon. The endogenous Otx2 expression and thus the knocked-in Emx2 expression is somewhat weaker in the midbrain than in the forebrain at this stage. (G,H) Enlarged views of E,F (respectively) at the telodiencephalicus level indicated by arrows. Arrowheads indicate the isthmus. Scale bars: 125 µm.

View larger version (89K): [in a new window]   Fig. 7. Forebrain phenotype in knock-in mutants; loss of commissural region of pretectum. (A,B) Parasagittal sections of 12.5 dpc wild-type (A) and knock-in embryos (B) stained with Hematoxylin and Eosin. 1, ventral thalamus; 2, dorsal thalamus; 3, anterior pretectum; and 4, posterior pretectum. The area of low cell density indicated as 4 in A is not apparent in the knock-in mutant (B). (C-R) Analyses with forebrain markers in wild type (C,E,G,I,K,M,O,Q) and in knock-in mutants (D,F,H,J,L,N,P,R). Endogenous Emx2 (C,D), Emx1 (E,F), Wnt7b (G, H), Tcf4 (I, J), Gbx2 (K,L), Pax6 (M-P), Lim1 (Q,R) expression in whole-mount (C-J,M,N,Q,R) and in midsagittal (K,L,O,P) views at 9.5 dpc (G,H), 10.5 dpc (C-F,I,J,M,N,Q,R) and 12.5 dpc (K,L,O,P). (S,T) Midsagittal views of anti-GAP43 staining of 12.5 dpc wild-type and knock-in embryos, respectively. Arrows in G-J,M,N indicate the caudal limits of the expressions, arrowheads in G,H indicate the anterior end of the midbrain, arrowheads in O,R indicate expression in the posterior pretectum and in the tegmentum, respectively. Asterisks in S indicate the anti-GAP43-positive posterior commissure. Scale bars: 250 µm.

View larger version (53K): [in a new window]   Fig. 8. Midbrain phenotype in knock-in mutants. (A,C,E,G,I,K) Wild-type and (B,D,F,H,J,L) knock-in mutant embryos. (A,B) Wnt1, (C,D) Ebf3, (E,F) COUP-TFI, (G,H) En1, (I,J) Pax5 and (K,L) Fgf8 expression at 9.5 dpc (K,L) and 10.5 dpc (A-J). Scale bars: 125 µm.

View larger version (31K): [in a new window]   Fig. 9. An interpretation of Emx2-/-Otx2+/- double and Otx2+/Emx2 knock-in mutant defects. (A) The onset of double mutant defects at the four-somite stage. Emx2-positive domain shown in green horizontal lines was lost in the double mutants. Consequently Otx2-positive region shown in red oblique lines was reduced both rostrocaudally and lateromedially. Moreover, the Pax2-positive area shown in blue oblique lines shifted rostrally, resulting in the reduction of anterior Otx2-positive and Pax2-negative area. Concomitantly Gbx2-positive area rostral to preotic sulcus (POS), painted in yellow, expanded. (B) A prosomeric view of affected region in double and knock-in mutants. P3 ventral thalamus, P2 dorsal thalamus and rostral P1 anterior pretectum (shown in red) were lost by the defects at the three- to four-somite stage in double mutants. P4 archipallium shown in purple was probably also lost by the defects at the same stage, though no detailed analysis was conducted in the present study. The r1 and r2 shown in yellow expand to compensate for the loss of the anterior structures in the double mutants. Posterior pretectum shown in blue develops in double mutants, but is specifically lost in knock-in mutants. Emx2, Otx2, Otx1, Pax6, Tcf4, Lim1, Dlx1, Gbx2, Ebf1 and GAP43 give their dorsal expression at 11.5dpc. Intense and weak expression are shown by thick and thin lines, respectively, in Otx1, Pax6 and Tcf4. Fgf8, Gbx2, Wnt1, En2 and Pax5 give the expression at 9.5 dpc. ACX, archicortex; APT, anterior pretectum; DT, dorsal thalamus; EMT, eminentia thalami; ET, epithalamus; HPT, habenulopeduncular tract; HT, hypothalamus; Is, isthmus; M, midbrain; MA, mammillary area; ov, otic vesicle; PC, posterior commissure; POS, preotic sulcus; PPT, posterior pretectum; RM, retro mammillary area; SPV, suprapreoptic paraventricular area; TG, tegmentum; TU, tuberal hypothalamus; VT, ventral thalamus; Zlth, zonalimitans interthalamica. Red lines indicate the boundary between alar and basal plates.