TAC3 Gene Products Regulate Brain and Digestive System Gene Expression in the Spotted Sea Bass (Lateolabrax maculatus)

Abstract

Neurokinin B (NKB) is a member of the tachykinin (tac) family that plays important roles in mammalian growth by modulating prolactin (PRL) synthesis and secretion and causing contraction of the stomach and intestine. However, its potential role in regulating growth of teleosts is less clear. We aimed to explore the role that NKB plays in regulating fish growth using the spotted sea bass (Lateolabrax maculatus) as a model. In the present study, two tac3 and two tacr3 genes were identified in the spotted sea bass. Sequence analysis showed that two tac3 transcripts, tac3a and tac3b, encode four NKBs: NKBa-13, NKBa-10, NKBb-13, and NKBb-10. Expression analysis in different tissues showed that both genes are highly expressed in the brain, stomach and intestine of the spotted sea bass. In situ hybridization indicated that the tac3a and tac3b mRNAs are both localized in several brain regions, such as the telencephalon and hypothalamus, and that tacr3a and tacr3b are localized in the intestinal villus and gastric gland. To investigate the potential role of NKBs in regulating growth, in vitro experiments were performed to detect the effect of NKBs on growth-related gene expression in the brain and brain-gut peptide (BGP)-related genes in the stomach and intestine. NKBb-13 was the most critical ligand in regulating the expression of growth-related genes in the brain and brain-gut peptide (BGP)-related genes in the stomach. The expression of cholecystokinin (cck) was enhanced by NKBa-13, NKBa-10, and NKBb-10 but not NKBb-13 in the intestine. In general, our results showed that NKBs participate in regulating the growth of spotted sea bass.

Keywords: TAC3/TACR3 system; growth; in situ hybridization; neurokinin B; spotted sea bass.

Figure 1

Syntenic analysis of tac3s (A) in zebrafish and spotted sea bass and tacr3s (B) in tilapia and spotted sea bass. In this map, the spotted sea bass genes are immediately proximal to each other on the chromosome, and the zebrafish tac3s genes and tilapia tacr3s genes are located in the same chromosomal region.

Figure 2

Nucleotide and deduced amino acid sequences of spotted sea bass tac3a (A) and tac3b (B). The putative signal peptides are underlined. The putative NKB peptides are boxed.

Figure 3

Comparison of the amino acid sequences of spotted sea bass tac3a (A) and tac3b (B) precursors from different species. The boxed letters indicate the sequence of mature NKB peptides in the detected species.

Figure 4

Phylogenetic analysis of the tac (A) and tacr (B) gene family in vertebrates. The phylogenetic tree was constructed by MEGA 6 using the neighbor-joining method. Data were resampled with 1,000 bootstrap replicates.

Figure 5

Expression of tac3s (A) and tacr3s (B) mRNAs in various tissues (telencephalon, midbrain, hypothalamus, cerebellum, medulla oblongata, pituitary, gill, liver, intestine, stomach, spleen, heart, kidney, head kidney, muscle, and testis) of male spotted sea bass. The X axis indicates different tissues. The results are normalized to 18S RNA. The data are shown as the mean ± S.E.M (n = 3) and the mRNA levels of tac3a and tac3b were calculated as fold change relative to the mRNA levels of the heart and spleen, respectively, while the mRNA levels of tacr3a and tacr3b were calculated as fold change relative to the mRNA levels of the heart and pituitary, respectively.

Figure 6

Photomicrographs and schematic illustrations of the mRNA levels of tac3s in the brain of the spotted sea bass, as detected by in situ hybridization (ISH). Schematic illustrations showing the positions of the slides within the brain (A). Schematic drawings of spotted sea bass telencephalon (B). Photomicrographs of tac3a and tac3b sense probe results in the telencephalon (C). Positive signal of tac3a and tac3b in the telencephalon (D). VM, ventromedial thalamic nucleus; Vv, area ventralis telencephali pars ventralis; Vs, area ventralis telencephali pars supracommissuralis; Vd, Area ventralis telencephali pars dorsalis; Dl, area dorsalis telencephali pars lateralis; Vl, area ventralis telencephali pars lateralis; Dm, area dorsalis telencephali pars medialis; Dc, area dorsalis telencephali pars centralis; Dd, area dorsalis telencephali pars dorsalis; AC, anterior commissure; Vd, area ventralis telencephali pars dorsalis. Red and gray dots represent the locations of tac3a and tac3b, respectively.

Figure 7

Photomicrographs and schematic illustrations of the mRNA levels of tac3s in the midbrain of the spotted sea bass, as detected by in situ hybridization (ISH). Schematic drawings of spotted sea bass midbrain (A). Photomicrographs of tac3a and tac3b sense probe results in the midbrain (B). Positive signal of tac3a and tac3b in the midbrain (C). Abbreviations: TeO, tectum opticum; Val, lateral division of the valvula cerebella. Red and gray dots represent the locations of tac3a and tac3b, respectively.

Figure 8

Photomicrographs and schematic illustrations of the mRNA levels of tac3s in the hypothalamus of the spotted sea bass, as detected by in situ hybridization (ISH). Photomicrographs of tac3a and tac3b sense probe results in the hypothalamus (B,D). Positive signal of tac3a and tac3b in the hypothalamus (A,C). NDLl, diffuse nucleus of the inferior lobe; NLT, lateral tuberal nucleus; NAT, Nucleus anterior tuberis; NGp, posterior nucleus glomerulosus; TeO, tectum opticum; Val, lateral division of the valvula cerebella; Red and gray dots represent the locations of tac3a and tac3b, respectively.

Figure 9

Photomicrographs and schematic illustrations of the mRNA levels of tacr3s in the intestine of the spotted sea bass, as detected by in situ hybridization (ISH). Positive signal of tacr3a and tacr3b in the intestine (A). Photomicrographs of tacr3a and tacr3b sense probe results in the intestine (B). Schematic diagram for intestine histology (C). GS, gastric serosa; ML, muscular layer; IG, intestinal gland; IV, intestinal villus; LP, lamina propria; EC, epithelial cell.

Figure 10

Photomicrographs and schematic illustrations of the mRNA levels of tacr3s in the stomach of the spotted sea bass, as detected by in situ hybridization (ISH). Positive signal of tacr3a and tacr3b in the stomach (A). Photomicrographs of tacr3a and tacr3b sense probe results in the stomach (B). Schematic diagram for stomach histology (C). ML, muscular layer; EC, epithelial cell; GM, gastric mucosa; Gsu, gastric submucosa; GS, gastric serosa; GG, gastric gland.

Figure 11

Effect of NKBs on the expression of growth-related genes in the brain. The X axis indicates hours after NKB treatment. (A,B) Effect of NKBb-13 and NKBb-10 on the mRNA levels of ghrh in the brain. (C–E) Effect of NKBa-13, NKBa-10, and NKBb-13 on the mRNA levels of prlh in the brain. (F) Effect of NKBb-13 on the mRNA levels of igf1 in the brain. The results were represented as the mean ± SEM and expressed as fold of the expression level in controls. Significant differences are noted by different letters for each concentration (P < 0.05).

Figure 12

Effect of NKBs on the expression of brain-gut peptide (BGP)-related genes in the stomach. The X axis indicates hours after NKB treatment. (A,B) Effect of NKBa-13 and NKBb-13 on the mRNA levels of gas in the stomach; (C,D) Effect of NKBb-13 on the mRNA levels of gas and ghrl in the stomach; the results are represented as the mean ± SEM and expressed as fold of the expression level in controls. Significant differences are noted by different letters for each concentration (P < 0.05).

Figure 13

Effect of NKBs on the expression of brain-gut peptide (BGP)-related genes in the intestine. (A–C) Effect of NKBa-13, NKBa-10, and NKBb-10 on the mRNA levels of cck in the intestine. The X axis indicates hours after NKB treatment. (D) Effect of NKBb-10 on the mRNA levels of gas in the intestine. (E) Effect of NKBa-10 on the mRNA levels of ghrl in the intestine. The results are represented as the mean ± SEM and expressed as fold of the expression level in controls. Significant differences are noted by different letters for each concentration (P < 0.05).