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.