Shih LJ et al. (2007), Characterization of the agr2 gene, a homologue ...

XB-ART-35054

Gene Expr Patterns 2007 Feb 01;74:452-60. doi: 10.1016/j.modgep.2006.11.003.

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Characterization of the agr2 gene, a homologue of X. laevis anterior gradient 2, from the zebrafish, Danio rerio.

Shih LJ , Lu YF , Chen YH , Lin CC , Chen JA , Hwang SP .

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We characterized a zebrafish (Danio rerio) anterior gradient 2 homologue (agr2) gene. agr2 contains an open reading frame of 513bp encoding 171 amino acids. Deduced amino acid sequence comparison showed that the zebrafish agr2 protein shares high (80-89%) amino acid sequence similarity with those homologues of anterior gradient 2 (HAGR2, MAgr2, Tagr2, and Sagr2) from the human, mouse, pufferfish, and Atlantic salmon, while sharing less (67-71%) sequence similarity with those anterior gradient 2 genes (XAG-2, XAG-1, XAgr2, MAgr3, and HAGR3) from Xenopus laevis, mouse, and human. Both phylogenetic and syntenic analyses indicate that zebrafish agr2 is the orthologue of human AGR2 and mouse Agr2 genes. Whole-mount in situ hybridization indicated that zebrafish agr2 is expressed in most organs, such as epidermis, olfactory bulbs, otic vesicles, pharynx, esophagus, pneumatic duct, swim bladder, and intestine, which contain mucus-secreting cells. Moreover, semi-quantitative RT-PCR demonstrated agr2 is expressed in the gill, pharynx/esophagus, swim bladder/pneumatic duct, and intestine in the adult fish. In contrast, Xenopus anterior gradient 2 homologues are mainly expressed in ectoderm-derived organs including the cement gland and otic vesicles, while human and mouse anterior gradient 2 orthologues are mainly distributed in endoderm-derived organs including the trachea, lungs, stomach, intestines, and colon.

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Species referenced: Xenopus laevis
Genes referenced: ag1 agr2 ids ins

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	Fig. 1. Nucleic acid and the deduced amino acid sequences of zebrafish agr2. The putative signal peptide of zebrafish agr2 is shown in gray boxes. Numbers on the right indicate respective positions of nucleic acid and amino acid sequences.
	Fig. 2. Alignment of the amino acid sequence of zebrafish agr2 with other anterior gradient 2 homologues from the pufferfish, Atlantic salmon, Xenopus, human, and mouse. Identical amino acid sequences which are greater than or equal to seven are shown in black boxes. Gaps are introduced as represented by dots to optimize the alignment. An arrow indicates the residue used as the first N-terminal amino acid for phylogenetic analyses. The percentage of amino acid similarity between zebrafish agr2 and each homolog from other vertebrate organisms is shown. Sequences are pufferfish Tagr2 (Tetraodon nigroviridis; CAF94244); Atlantic salmon Sagr2 (Salmo salar; ABB96968); human HAGR2 (NM_006408) and HAGR3 (AY069977); mouse MAgr2 (BC013334) and MAgr3 (Q8R3W7); zebrafish Zagr2 (AY796218; this study); and Xenopus XAG-1 (U76752), XAG-2 (AF025474), and XAgr2 (AF314056).
	Fig. 3. Phylogenetic tree analyses of zebrafish agr2 and homologues from the pufferfish, Atlantic salmon, Xenopus, mouse, and human. A Neighbor-joining (NJ) tree is presented. The bar represents the number of estimated differences for a unit branch length. Bootstrap values are shown. Sequences used are pufferfish Tagr2 (Tetraodon nigroviridis; CAF94244); Atlantic salmon Sagr2 (Salmo salar; ABB96968); human HAGR2 (NM_006408) and HAGR3 (AY069977); mouse MAgr2 (BC013334) and MAgr3 (Q8R3W7); zebrafish Zagr2 (AY796218; this study); and Xenopus XAG-1 (U76752), XAG-2 (AF025474), and XAgr2 (AF314056).
	Fig. 4. Spatial mRNA expression patterns of agr2 during 24 to 72 h of development. agr2 was expressed in the otic vesicles of 24- (A), 36- (B and C), 48- (D and E), and 72-h post-fertilization (hpf) embryos (F, G). Expression of agr2 in mucous cells was observed in 24- (A), 36- (B), 48- (D), and 72-hpf embryos (F). agr2 was expressed in the hatching gland of 24- (A) and 36-hpf embryos (B). Expression of agr2 was detected in the olfactory bulbs of 48- (D) and 72-hpf embryos (K). agr2 was expressed in the anterior endoderm in 36- (B and C) and 48-hpf embryos (D and E). Expression of agr2 in the pharynx, esophagus, pneumatic duct, and swim bladder was detected in 72-hpf embryos (F and G). Images from two color double in situ hybridization using insulin (blue)/agr2 (red) (H) and LFABP (blue)/agr2 (red) (I) as probes are shown. Longitudinal paraffin sections of 72-hpf embryos revealed detailed expression of agr2 in the pharynx, pneumatic duct, esophagus, swim bladder (J), olfactory bulb (K), and semicircular canal of the inner ear (L). No expression of agr2 can be found in the notochord of 72-hpf embryos (J). All embryos are shown with the anterior to the left. Embryos in A, B, D, and F are shown in lateral view, while embryos in C, E, G, H, and I are shown in ventral view. Ae, anterior endoderm; an, anus; es, esophagus; hg, hatching gland; nt, notochord; ob, olfactory bulb; ov, otic vesicle; pd, pneumatic duct; ph, pharynx; sb, swim bladder; sc, semicircular canal. Bars represent 100 μm.
	Fig. 5. Spatial mRNA expression patterns of agr2 during 96–120 h of development. agr2 was expressed in the otic vesicles of 96- (A), 98- (D, F), and 120-h post-fertilization (hpf) embryos (K, M). Expression of agr2 in mucous cells and anus was observed in 96- (A), 98- (D), and 120-hpf embryos (K). Expression of agr2 was detected in the olfactory bulbs of 96- (A), 98- embryos (E), and 120-hpf embryos (K). Expression of agr2 in the pharynx was observed from 96 to 120 h of development (A–D, F, H, I, K, L, and N). Expression of agr2 in the esophagus, and pneumatic duct was detected in 96-h (B and C) and 98- hpf embryos (D, H, and I). Expression of agr2 in the swim bladder can only be detected in 96-hpf embryos (B and C). Trace amount of agr2 expression in the esophagus can be observed in paraffin sections of 120-hpf embryos (N). agr2 was expressed in the goblet cells located in the mid-intestine region of 98- (D, G, H, and J) and 120-hpf embryos (K and O). Higher magnification view of agr2-expressing goblet cells was shown as insets in J and O, respectively. No expression of agr2 can be observed in the notochord of 96- (C) and 98-hpf embryos (H and I). Longitudinal paraffin sections of 96- (C), 98- (H–J), and 120-hpf embryos (M–O) revealed agr2 expression in the pharynx, esophagus, pneumatic duct, swim bladder, goblet cells, semicircular canal, and anus. All embryos are shown with the anterior to the left. Embryos in A, D, and K are shown in lateral view, embryos in B, G, and L are shown in ventral view, and embryos in E and F are shown in their dorsal view. Ae, anterior endoderm; an, anus; es, esophagus; hg, hatching gland; nt, notochord; ob, olfactory bulb; ov, otic vesicle; pd, pneumatic duct; ph, pharynx; sb, swim bladder; sc, semicircular canal. Bars represent 100 μm.
	Fig. 6. Temporal expression pattern of agr2 during different developmental stages analyzed by RT-PCR. DNase I-treated total RNA isolated from 24-, 48-, 72-, and 96-h post-fertilization, 6-, 7-, 14-, 30-day post-fertilization, and adult fish (Ad) was used to conduct RT reaction. (A) An agr2-specific primer pair was used in PCR and resultant 510 bp PCR product was analyzed. (B) An α-actin-specific primer pair was used in PCR and amplified 350 bp DNA fragments were examined by agarose electrophoresis. No template controls (C) in respective PCRs were shown.
	Fig. 7. Distribution of agr2 in different tissues or organs isolated from adult fish. DNase I-treated total RNA isolated from the eye (E), brain (B), gill (G), heart (H), pharynx/esophagus (P), swim bladder/pneumatic duct (S), liver (L), intestine (I), and muscle (M) was used to perform RT reaction. An agr2-specific primer pair was used in PCR and 510 bp amplified DNA fragments were analyzed and shown in A. An α-actin-specific primer pair was used in PCR and amplified 350 bp DNA fragments were examined and shown in B. No template controls (C) in respective PCRs were shown.
	Fig. 8. Maps showing gene order with conserved syntenic groups between human chromosome 7 and respective zebrafish linkage group 7 and 19. Genes sharing equal or greater than 60% amino acid sequence identity and their start positions (million base pair) on respective zebrafish linkage group 7 (LG 7), 19 (LG 19), and human chromosome 7 (Hsa 7) are shown. The entire chromosome (grey box) is shown, but the scale is different in different chromosomes. Names of the zebrafish external gene ID are shown next to both zebrafish linkage group 7 and 19. Some zebrafish external gene IDs are italicized and they represent genes showing syntenic conservation with those in mouse chromosome 12 where Agr2 lies.