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The zebrafish spiel-ohne-grenzen (spg) gene encodes the POU domain protein Pou2 related to mammalian Oct4 and is essential for formation of the midbrain and hindbrain, and for pre-gastrula morphogenesis

Shawn Burgess^1,*,, Gerlinde Reim^2,, Wenbiao Chen^1,, Nancy Hopkins¹ and Michael Brand^2,

¹ Massachusetts Institute of Technology, Center for Cancer Research, E17-340, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
² Max Planck Institute for Molecular, Cell Biology and Genetics, Dresden, Pfotenhauer Str. 108, 01307 Dresden, Germany
^* Present address: Genome Technology Branch, National Human Genome Research, National Institutes of Health, Bethesda, MD 20892, USA
Present address: Vollum Institute, Oregon Health Sciences University, Portland, OR 97201, USA
These authors contributed equally to this work

View larger version (92K): [in a new window]   Fig. 1. Morphology of spg mutants. (A-D) Lateral views. (E-H) Dorsal views. (A) Wild-type embryo at 26 hours post fertilization. c, cerebellum; h, hindbrain; o, otic placode; t, tectum. (B) spghi349 homozygous mutant embryo at 26 hpf. There is significant variability of the phenotype. The hindbrain displays disorganization (left arrowhead) and the otic placode is reduced in size, containing only one otolith (right arrowhead); note the proximity of the otolith to anterior brain structures when compared with wild type. (C) spge68 homozygous embryos are less affected at the hindbrain than the insertional mutant (B). (B,C) In spg mutant embryos, no proper MHB structures are visible (red and white arrowheads, respectively). (D) The spghi349 mutation causes a variable shortening and altered morphology of the tail. The embryo at the top is wild type, and the two beneath are both spghi349/spghi349. The effect varies from a very minor kink to major shortening and structural defects (see arrowheads). (E,F) -acetylated tubulin staining recognizing the axonal scaffold in the developing brain at 28 hpf. (E) Wild type. Six bilateral transverse axon bundles mark the borders between single rhombomeres. (F) spg mutant embryo shows strong disorganization of the axonal scaffold within the hindbrain. (G,H) Staining with the monoclonal antibody 3A10 at 30 hpf. (G) The Mauthner neurons in the wild-type embryo are marked with a red arrowhead. (H) spghi349/spghi349 mutant embryo showing complete absence of the Mauthner neurons.

View larger version (31K): [in a new window]   Fig. 2. spg mutations affect the pou2 gene. (A) Part of the Pou2 protein containing the POU-specific and the POU-homeodomain. The insertional mutation spghi349 and the point mutation spge713 are indicated. The point mutation is based on a transition from T to C, leading to an amino acid exchange from leucine to proline. (B) spg is genetically linked to the SSR marker z13467 mapping on chromosome 21 (indicated by an purple arrow in C). z13467 was used as a diagnostic marker in PCR-based mapping of wild type and spge713, resulting in an amplification product of 280 bp for the mutant (purple arrow) and 190 bp for the wild type (black arrow). (C) Detail from chromosome 21. The red and the purple arrow indicate the position of the mapping marker z13467 and pou2/spg, respectively. (D) Syntenic relationship between the zebrafish linkage group 21, human chromosome 6 and mouse chromosome 17. Zebrafish pou2, clic1, efna5a and bf and their mammalian orthologs show conserved synteny (sources, OMIM and ZFIN). The different order of efna5a and bf may be due to smaller inversions that frequently occur in an overall syntenic area (Woods et al., 2000). (E) Zebrafish Pou2 (DrPou2, red arrowhead) and its mouse (MmOct3/4) and human (HsPou5F1) ortholog cluster within the ClassV POU domain protein subfamily in an unrooted phylogenetic tree, the closest mammalian members being in the Oct3/4/Pou5f1 subgroup. (F) PCR genotyping of eight normally expressing pax2.1 embryos and eight embryos with impaired pax2.1 expression showing all pax2.1-deficient embryos are also spghi349/spghi349. PCR product sizes for wild-type genomic DNA (WT) and spghi349 are marked. Among eight embryos normally expressing pax2.1 are pax6 embryos that are heterozygous for spg, as indicated by the occurrence of both the wild-type- and the mutant-specific PCR products.

View larger version (65K): [in a new window]   Fig. 3. Abnormal development of the hindbrain- and the MHB primordium in spg mutants. (A-D) krox20 is not properly initiated in r3 (B,D) and r5 (D); in spghi349 mutant embryos, krox20 staining often fails to fuse at the midline in mutant embryos (D). (E) pax6 is expressed within the forebrain and the hindbrain, but spared at the MHB in wild-type embryos. (F) The forebrain expression domain of pax6 invades into the prospective MHB region (arrow). (G) pax2.1 expression is initiated between 80 and 90% of epiboly in wild-type embryos at the MHB. (H) In spghi349 mutant embryos, pax2.1 is downregulated from its onset of expression at the MHB.

View larger version (101K): [in a new window]   Fig. 4. pou2 expression during embryogenesis. Shown are wild-type (A-D,F-G) and spghi349/spghi349 mutant embryos (E). (A) Animal pole upwards. (B,C,F,G) Dorsal views, anterior is upwards; (D,E) Lateral view, anterior towards the left. (A) pou2 is ubiquitously expressed before gastrulation. (B) pou2 expression refines within the neuroectoderm at the end of gastrulation. (C) During early somitogenesis, pou2 expression becomes restricted to the hindbrain within r2 and r4, and to a ventromedial patch at the MHB. (D) Wild-type embryo at the tailbud stage. Expression in rhombomeres 2 and 4 is indicated by asterisks. Beside the expression within the brain, pou2 is also expressed within the posterior spinal cord. pou2 vanishes during gastrulation in spghi349 embryos (not shown) and at tailbud stage (E), no pou2 expression can be detected anymore throughout the embryo. (F,G) Double in situ hybridization with pou2 (blue, long brackets) and pax2.1 (red, short brackets). At 90% epiboly, MHB expression of pax2.1 is completely contained within the anterior pou2 expression domain (F) but slightly later, at tb stage, expression domains of pax2.1 and pou2 start to separate from each other (G).

View larger version (74K): [in a new window]   Fig. 5. pou2 mRNA injection rescues spg mutants. (A-H) Injections of pou2 mRNA and lacZ mRNA. The dorsal midline of the injected embryos is indicated by a white arrowhead, to show the unilateral injection of lacZ/pou2. (A-D) pax2.1 staining. Wild-type embryo showing laterally expanded pax2.1 expression on the injected side. (B-D) spghi349/spghi349 mutant embryos injected into one cell of a two-cell stage embryo with pou2 and lacZ mRNA and fixed at the three-somite stage. pax2.1 staining is purple, lacZ staining in brown. (B,C) pax2.1 expression at the MHB is clearly rescued on the injected side of the embryo. (D) Dorsoposterior view of a mutant embryo, showing pax2.1 staining in the intermediate mesoderm. pax2.1 expression is not affected in this tissue. (E,F) Lateral expansion of wnt1 after pou2 mRNA injection in the wild-type embryo (E) and rescue of expression in the mutant embryo (F). (G,H) Rescue of krox20 expression after pou2 mRNA injection.

View larger version (92K): [in a new window]   Fig. 6. Morpholinos phenocopy the spg mutant phenotype. (A-L) Results of morpholino experiments where pou2 antisense morpholinos (B,D,E,F,H,J,L) or control morpholinos (A,C,G,I,K) were injected at the one-cell stage. (A) Control embryo 26 hours after injection. (B) Embryos injected with lower dose of morpholinos phenocopy the phenotype of spg 26 hours after injection. Loss of the MHB and reduced size of otic placode are indicated by red arrowheads. (C) Control embryos at 80% of epiboly. (D) Morpholinos were injected at high concentration at the one-cell stage. Cells arrest development at the sphere-dome stage. Embryos were photographed at the same timepoint after injection as Control embryos in C. (E) Embryos continue development after co-injecting morpholino 1 (E) or morpholino 2 (F) with a non-inhibitable pou2 mRNA(-5'UTR), showing that the observed blastula arrest after morpholino injection is specific. (G-L) Molecular defects of morpholino-injected embryos. pax2.1, wnt1 and krox20 expression at the end of gastrulation is strongly reduced in morpholino-injected embryos, as seen in spg mutants (H,J,K).

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