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spiel ohne grenzen/pou2 is required for zebrafish hindbrain segmentation

¹ Albert-Ludwigs-Universität Freiburg, Institut für Biologie I, Hauptstrasse 1, D-79104 Freiburg i. Br., Germany
² Max Delbrueck Center for Molecular Medicine, Robert-Roessle Strasse 10, D-13125 Berlin, Germany
³ Department of Organismal Biology and Anatomy, University of Chicago, 1027 E. 57th St, Chicago, IL 60637, USA

View larger version (104K): [in a new window]   Fig. 1. Inter-rhombomeric boundaries and segmental neuronal differentiation are affected in spg mutants. (A-C) Lateral views of the hindbrain of live wild-type (A), spgm216 (B) and spgm793 (C) embryos at the 22-somite stage. Red arrows indicate the positions of rhombomere boundaries and black arrowheads mark the size of the otic vesicle (ov). (D-F) Dorsal views of the hindbrain of wild-type (D), spgm216 (E), and spgm793 (F) embryos at 1 dpf that were assayed for pax6.1 expression by WISH. (G-I) Lateral views of wild-type (G), spgm216 (H), and spgm793 (I) embryos at 1 dpf that were assayed for lim1 expression by WISH. (J-L) Confocal images of wild-type (J), spgm216 (K) and spgm793 (L) embryos at 5 dpf in which hindbrain reticulospinal neurons were back-filled with lysinated rhodamine-dextran. Stacks of confocal images were merged to produce a composite image in which the signal from each layer was depth-coded in color. A color-coded depth scale (in µm) is shown below each figure (dorsal-most blue to ventral-most red). In the spgm216 mutant embryo shown (K), reticulospinal neurons in r1 and r6 are not seen while the large Mauthner neurons in r4 are duplicated. In spgm793 mutant embryos (L), fully-differentiated primary reticulospinal neurons are not observed. Anterior is to the left in all panels. 1-7, rhombomeres 1-7; g, ganglia of the posterior lateral line neurons; m, nuclei of the medial longitudinal fascicles; tg, tegmentum; v, vestibular nuclei.

View larger version (84K): [in a new window]   Fig. 2. Abnormal hox gene expression in spg mutants at the 10-somite stage. Dorsal (A-C,G-I,M-W) and lateral (D-F,J-L) views of the hindbrain of wild-type (left column), spgm216 (middle column) and spgm793 (right column) embryos at the 10-somite stage. Embryos have been assayed for expression of hox genes (blue) by WISH as indicated at left. krx20 expression (red) in r3 and r5 was also detected. Schematic summary of hox, krx20, ephA4 and val expression in wild-type (X), spgm216 (Y) and spgm793 (Z) embryos at the 10-somite stage. The diagrams do not exactly represent the proportional sizes of rhombomeres. The shape of r1 in (Z) is deduced from ephA4 expression at 1 dpf (Fig. 5G,I). In all photographs, anterior is to the left. In D-F,J-L, dorsal is up. 1-7, rhombomeres 1-7.

View larger version (85K): [in a new window]   Fig. 3. Early expression of krx20 and val is severely reduced in spgm793 mutants. Dorsal views of wild-type embryos (A,C,E,G,I,K) and spgm793 mutants (B,D,F,H,J,L) at 100% epiboly (A-D), 2-somite (E-H), and 5-somite (I-L) stages showing detection of krx20 (A,B,E,F,I,J) or val (C,D,G,H,K,L) expression by WISH. Anterior is to the top. 3, 5, 6; rhombomeres 3, 5 and 6.

View larger version (95K): [in a new window]   Fig. 4. Injection of pou2 mRNA in spgm793 mutants can rescue segmental expression of krx20 and val. (A-F) Dorsal views of pou2 expression (purple/blue) during late gastrula to early somitogenesis stages in comparison with that of val in r5/r6 (red; D,E) and krx20 in r3/r5 (red; F). (A-C) High-level pou2 expression in the hindbrain primordium is marked by a red arrow. (G-J) Restriction of pou2 expression to the epiblast. Sagittal (H) and transverse (G,I,J) sections through the neural plate and notochord of wild-type embryos stained for pou2 (blue) and ntl (red). (I) Arrows and arrowheads mark strong lateral and medial pou2 expression in the hindbrain primordium, respectively. Embryonic stages are indicated. (K-R) Dorsal views of the hindbrain primordia of uninjected and pou2 mRNA-injected embryos at the 5- to 6-somite stage. The genotypes of the embryos are wild-type or spgm793, as indicated at the top. Genotypes were determined by an allele-specific restriction polymorphism detected in genomic DNA amplified from the embryos shown. Expression of krx20 (K-N) and val (O-R) was detected by WISH. pax2.1 expression was also assayed (K-N) to compare rescue of hindbrain gene expression in spgm793 mutants with that of the MHB. While pax2.1 expression in the MHB is not shown, pax2.1 in the otic placodes is visible on the images (K-N; lateral edges between hindbrain and otic placodes highlighted by dotted white lines). Embryos in O and Q were determined to be +/+ for spg. Anterior is up in all whole-mount embryos shown. 2-6, rhombomeres 2-6; ep, epiblast; h, hindbrain; hy, hypoblast; m, midbrain; no, notochord; tb, tailbud stage.

View larger version (121K): [in a new window]   Fig. 5. Hindbrain expression of ephA4 is altered in spg mutants. Dorsal (A-C,G-I) and lateral (D-F) hindbrain views of wild-type (A,D,G), spgm216 (B,E,H) and spgm793 (C,F,I) mutant embryos at the 10-somite stage (A-C) and at 1 dpf (D-I). Expression of ephA4 is visualized by WISH. In all panels, anterior is to the left. In D-F, dorsal at top. 1, 3, 5, rhombomeres 1, 3and 5; op, otic placode.

View larger version (74K): [in a new window]   Fig. 6. Early segmental expression of hoxa2, hoxb2 and hoxb3 is altered in spgm793 mutants. Wild-type (A,C,E,G,I,K,M,O,Q,S) and spgm793 mutant embryos (B,D,F,H,J,L,N,P,R,T) at the 100% epiboly (M,N,Q,R), 2-somite (A,B,E,F,I,J,O,P,S,T) and 5-somite (C,D,G,H,K,L) stages showing expression of hoxa2 (A-D), hoxb2 (E-H), hoxb3 (I-L), hoxb1b (M-P), and hoxb1a (Q-T) (WISH; purple/blue). To identify spg mutants, pax2.1 (A,B,E,F,I,J,M,N,Q,R) or krx20 (O,P,S,T) expression (red) was also assayed. All panels show dorsal views, anterior is to the left (A-L) or up (M-T). 2-6, rhombomeres 2-6.