spiel ohne grenzen/pou2 is required during establishment of the zebrafish midbrain-hindbrain boundary organizer
Heinz-Georg Belting1,*,
Giselbert Hauptmann1,*,
Dirk Meyer1,
Salim Abdelilah-Seyfried2,
Ajay Chitnis3,
Cathrin Eschbach1,
Iris Söll1,
Christine Thisse4,
Bernard Thisse4,
Kristin B. Artinger5,
Karen Lunde1 and
Wolfgang Driever1,
1 Albert-Ludwigs-Universität Freiburg, Institut für Biologie I, Hauptstrasse 1, D-79104 Freiburg, Germany
2 Department of Physiology, UCSF, Third and Parnassus Avenue, San Francisco, CA 94143-0725, USA
3 Unit on Vertebrate Neural Development, NIH/NICHD Lab of Molecular Genetics, Building 6B, Room 3B-315, 9000 Rockville Pike, Bethesda MD 20892, USA
4 IGBMC, 1, rue Laurent Fries, BP163, 67404 Illkirch Cedex, France
5 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
* These authors contributed equally to this work
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Fig. 1. Mutations in spiel ohne grenzen (spg) affect MHB formation. (A-E) Wild-type, (F-J) spgm216 and (K-O) spgm793 mutant live (A,F,K) or whole-mount embryos at 24 hours post fertilization (hpf). (B,G,L) otx2 expression in the pretectum and tectum; (C,H,M) fgf8 expression at the midbrain-hindbrain boundary; and (D,I,N) strong zp50 expression in the cerebellum. zp50 is expressed in a complex pattern in all major subdivisions of the brain. (E,J,O) zash1a expression in the tegmentum and ventral hindbrain. Arrows indicate the position of MHB. Arrowheads indicate the limits of zash1a expression in the tegmentum and ventral rhombomere 1. In all embryos, anterior is towards the left and dorsal is upwards. cb, cerebellum; ey, eye; hb, hindbrain; mhb, midbrain-hindbrain boundary; ov, otic vesicle; tc, tectum; te, telencephalon; tg, tegmentum.
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Fig. 2. The spiel ohne grenzen mutations are linked to point mutations in pou2. (A) Genomic structure of the transcribed region of the pou2 locus. The coding region is interrupted by four introns. Translational start site and stop codon are indicated. The POU-specific domain is shown in blue, and the POU-homeodomain is shown in green. (B) Mapping of the spg locus, and demonstration of tight linkage to the pou2 gene. Molecular markers used for meiotic mapping are indicated in blue. PACs isolated in the genomic walk are designated A, B and C (see Materials and Methods). Recombination frequencies of the polymorphic markers used are indicated. The map positions of the markers are from the Stanford HS panel (Kelly et al., 2000), except for Z13467 (six recombinants/168 meioses), Z21718 (2/556), Z12068 (2/1666) and Z7224 (21/556), which are from the MGH panel (Shimoda et al., 1999). Z21718 maps on the HS panel at 33.1 cM. (C) Putative Pou2 protein products generated by spg mutant alleles. Wild-type and mutant proteins are shown at top and bottom, respectively. Foreign sequences, generated by frameshift, are indicated in red. The asterisk shows the position of the mis-sense mutation in helix 1 of the homeodomain in spgm216. The splice acceptor sites in spgm793 and spgm308 are highlighted in gray. (D) Top: sequence comparison of pou2 genomic DNA from wild-type embryos and mutants. Mutated positions are underlined. Bottom: genotype analysis of single embryos. Each spg mutation eliminates a restriction site. As expected, these restriction site polymorphisms segregate with the mutant phenotypes. Genotypes are indicated by ‘+’ (wild type) and ‘-’ (mutant). wt, wild type; un, undigested PCR product.
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Fig. 3. pou2 expression in wild-type, spg and noi mutant embryos. Expression of pou2 in wild-type (A-E), spgm793 (F-H), spgm216 (I), and noitu29a (J) mutant embryos. MHB expression of pax2.1 (red; D,I) or eng2 (orange; E,J) relative to that of pou2 (blue). Dorsal (B-E,G-J) or lateral (A,F) views of whole-mount embryos with animal pole/anterior at the top; (D,E,I,J) dorsal views of the neural plate at higher magnification. Embryonic genotypes are indicated in the top right-hand corner of each image; developmental stages are indicated in the bottom right-hand corner. hb, hindbrain; mb, midbrain; %, % epiboly; tb, tailbud stage; som, somites (stage).
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Fig. 4. Expression of MHB genes is reduced in spgm793 mutants. Expression of wnt1 (A-H), pax2.1 (I-P) and eng2 (Q-X) in wild-type and spgm793 mutant embryos. Embryonic genotypes are indicated at left, developmental stages, above. 90-100% epiboly and the two-somite stage embryos are shown in dorsal views, anterior/animal pole is at top. 90-100% epiboly stage embryos were genotyped by allele-specific PCR (see Materials and Methods). Five-somite stage and 1 dpf embryos are depicted in lateral views of the head region, anterior towards the left and dorsal upwards; images are focused at a mid-sagittal plane. 90%-100%, 90%-100% epiboly; som, somite (stage); hb, hindbrain; os, optic stalk; op, otic placode; rp, roof plate.
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Fig. 5. Rescue of the spg mutant phenotype by pou2 mRNA injection. Wild-type and spgm793 mutant embryos were injected at the one-cell stage with pou2 mRNA, fixed at the five- to six-somite stage (A-C) or at 24 hpf (D-I), and assayed for the expression of pax2.1 (A-C), pax2.1 and krx20 (D-F), or wnt1 (G-I). All embryos shown were genotyped by allele-specific PCR. Arrows indicate the position of the MHB. Orientation: (A-C) dorsal views, anterior at the top; (D-I) lateral views, anterior towards the left.
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Fig. 6. Expression of pax2.2, pax5 and pax8 is absent from the MHB region of spgm793 mutant embryos. Expression of pax2.2 (A-D), pax5 (E-H) and pax8 (I-L) at the five-somite stage (A,B,E,F), the nine-somite stage (I,J) and at 26 hpf (C,D,G,H,K,L) is shown. pax8 expression is first detected in wild-type embryos at the nine-somite stage (Pfeffer et al., 1998). Embryonic genotypes are indicated at top. Embryos are shown in lateral view, anterior towards the left and dorsal upwards. hb, hindbrain; os, optic stalk; ov, otic vesicle; s, somite (stage). Arrows indicate position of MHB.
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Fig. 7. Expression of otx2 and gbx1 and initial expression of fgf8, appear normal in spgm793 mutant embryos. Expression of otx2, gbx2, fgf8 (dark blue), and pax2.1 (red) in wild-type and spgm793 mutant embryos. Embryonic genotypes are indicated at the top; the genes for which expression is shown are on the left and right; and embryonic stages are indicated below. The genotype of embryos shown in A,B,E,F,I,J was determined by allele-specific PCR. Embryos are viewed from the dorsal side, anterior towards the top.
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Fig. 8. Function of spg/pou2 during MHB establishment. Model showing spg/pou2 activity positioned within the cascade of known MHB patterning genes in zebrafish. Three parallel pathways are involved in the activation of wnt1, pax2.1 and fgf8 in the mid-/hindbrain (Lun and Brand, 1998) during MHB establishment. Our data suggests that spg/pou2 functions upstream of both pax2.1 and wnt1. fgf8 is initiated normally in the anterior hindbrain during late gastrulation, but requires pou2 for its continued expression during MHB establishment. Arrows do not necessarily indicate direct interactions.
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© The Company of Biologists Ltd 2001
