Structural characterization of the zebrafish ALP subfamily. (A) Gene structures of zebrafish elfin, mystique, alp, alp-like and ril are shown. Overall alp family genes demonstrate relative conserved intron/exon structures. For all five genes the isolated, largest splice variant are depicted, for Elfin in addition a verified shorter spliceform is shown (CN505168). Other splice forms have been described in different organisms for Alp, Mystique and Ril but not for Elfin (Bashirova et al., 1998, Andersen et al., 2004 and Loughran et al., 2005). Numbers indicated correspond to the amino acid (Aa) sequences. (B) Alignment of Aa sequences of the PDZ- (shaded in blue) and LIM-domains (shaded in yellow) for the zebrafish ALP family. Fully conserved residues are marked in black and partial conservation is indicated in grey. (C) Phylogenetic analysis of zebrafish alp genes. Shown is a phylogenetic tree of the zebrafish ALP subfamily including human and mouse sequences. The unrooted tree was constructed by neighbour-joining analysis based on the amino acid sequences.

Expression pattern of the zebrafish alp gene by whole mount in situ hybridization. Zebrafish embryos at eight different developmental stages are shown and stages were indicated. Expression domains are marked by arrows and expression was found in the following structures: adductor mandibulae (am), branchial arches (ba), cerebellum (ce), diencephalon (di), duct of Cuvier (dc), hindbrain (hb), interhyoideus (ih), intermandibularis posterior (imp), intestine (in), lens (ls), liver (lv), midbrain-hindbrain boundary (mhb), myotomes (myt), otic vesicle (ov), retina (ret), somites (som), tectum (tct), telencephalon (tel). Black arrowheads point to the developing heart.

Expression pattern of the zebrafish-specific alp-like gene. Similar to the alp gene alp-like expression was detected by in situ hybridization at the eight different developmental stages indicated. Specific expression was found in: arterial arches (aa), axial vasculature (av), branchial arches (ba), diencephalic ventricle (dv), epiphysis (epi), intestine (in), lens (ls), midbrain-hindbrain boundary (mhb), midcerebral vein (mcev), myotomes (myt), otic vesicle (ov), retina (ret) and the telencephalic ventricle (tv).

Expression pattern of the zebrafish elfin gene by in situ hybridization. Picture of the in situ experiments with zebrafish elfin specific probes show staining patterns in the regions of the central nervous system (telencephalon (tel), olfactory placode (olf), cerebellum (ce) and hindbrain (hb)), the pectoral fins (pf), branchial arches (ba) and the heart (black arrowhead). Further expression was observed in the axial vasculature (av), epiphysis (epi), midbrain-hindbrain boundary (mhb), myotomes (myt) and the otic vesicle (ov).

Expression pattern of the zebrafish mystique gene by in situ hybridization. mystique expression in eight developmental stages of zebrafish embryogenesis is shown. mystique expression was observed in the CNS, in branchial arches (ba) and the lens (ls). In figure G a frontal view of a 48 h old embryos is shown to illustrate the mystique expression in the intermandibularis posterior (imp) and medial rectus (mr). Further expression was found in the arterial arches (aa), hindbrain (hb), intestine (in), liver (lv), medial rectus (mr), myotomes (myt), otic vesicle (ov), tectum (tct) and the tegmentum (tgm).

Expression pattern of the zebrafish ril gene by in situ hybridization during zebrafish embryonic development. ril expression was found in the branchial arches (ba), caudal presomitic mesoderm (cpsm), hindbrain (hb), intestine (in), lateral line ganglia (llg), lens (ls), liver (lv), myotomes (myt), pigmented epithelium (pe), pectoral fin (pf), spinal cord (sc), somites (som), tectum (tct), telencephalon (tel) and finally the tegmentum (tgm).

Detection of specific expression domains for alp family genes in sections of 2 dpf embryos. Shown are whole mount in situ hybridization pictures of 2 dpf embryos probed for the indicated genes. Abbreviations used are: anterior mandibularis (am), (di): diencephalon, exocrine pancreas progenitor (epp), ganglion cell layer (gcl), inner nuclear layer (inl), inner plexus layer (ipl), mandibular cartiledge (mc), midbrain (mb), neural tube (nt), optic nerve (on), outer nuclear layer (onl), outer plexus layer (opl), pancreatic islet (pi), posterior chiasm (poc), pronephric duct (pd), retinal ganglion cells (rgc) and tectum (tct). (A–E) show transversal brain sections and (F and G) transversal sections through more posterior regions of the embryo. Staining in the gut is marked with an asterisk in F and G.