Exploring the potential for an evolutionarily conserved role of the taste 1 receptor gene family in gut sensing mechanisms of fish

Abstract

In this study, we investigated the transcriptional spatio-temporal dynamics of the taste 1 receptor (T1R) gene family repertoire in seabream (Sparus aurata [sa]), during larval ontogeny and in adult tissues. In early larval development, saT1R expression arises heterochronously, i.e. the extraoral taste-related perception in the gastrointestinal tract (GIT) anticipates first exogenous feeding (at 9 days post hatching [dph]), followed by the buccal/intraoral perception from 14 dph onwards, supporting the hypothesis that the early onset of the molecular machinery underlying saT1R expression in the GIT is not induced by food but rather genetically hardwired. During adulthood, we characterized the expression patterns of sa T1R within specific tissues (n = 4) distributed in oropharingeal, GIT and brain regions substantiating their functional versatility as chemosensory signaling players to a variety of biological functions beyond oral taste sensation. Further, we provided for the first time direct evidences in fish for mRNA co-expression of a subset of saT1R genes (mostly sa T1R3, i.e. the common subunit of the heterodimeric T1R complexes for the detection of "sweet" and "umami" substances), with the selected gut peptides ghrelin (ghr), cholecystokinin (cck), hormone peptide yy (pyy) and proglucagon (pg). Each peptide defines the enteroendocrine cells (ECCs) identity, and establishes on morphological basis, a direct link for T1R chemosensing in the regulation of fish digestive processes. Finally, we analyzed the spatial gene expression patterns of 2 taste signaling components functionally homologous to the mammalian G(i)α subunit gustducin, namely sa G( i )α1 and sa G( i )α2, and demonstrated their co-localization with the saT1R3 in EECs, thus validating their direct involvement in taste-like transduction mechanisms of the fish GIT. In conclusion, data provide new insights in the evolutionary conservation of gut sensing in fish suggesting a conserved role for nutrient sensors modulating entero-endocrine secretion.

Keywords: Fish larvae; G(i) alpha protein subunits 1 and 2; Gut nutrient sensing; Gut peptides; Taste 1 receptor.

Graphical abstract

Fig. 1

Graphical representation of RT qPCR analyses in whole seabream larvae for seabream (sa) taste 1 receptor (T1R) subunits, saT1R1 (A), saT1R2a (B), saT1R2b (C), saT1R2d (D), saT1R2e (E), saT1R2f (F), saT1R3 (G) at 1, 3, 5, 7, 10, 12 d post hatching (dph). saT1R2b mRNAs expressed roughly 300-fold higher levels of saT1R2f and 60, 30, 10 and 5-folds of saT1R3, saT1R2e, saT1R2a and saT1R1, respectively (H). Data are represented as the fold change differences of target gene expression to the reference gene-elongation factor 2, per 100 ng of input RNA/sample. All experiments were performed in triplicates (n = 3), each containing approximately 15 pooled whole-body larvae; data are expressed as means ± standard error of the mean (SEM). Different letters indicate significant differences between experimental groups. Asterisks indicate significance levels (*P < 0.05, **P < 0.01, ***P < 0.001) after one-way ANOVA followed by Tukey's multiple comparisons tests (GraphPad Prism version 8.0).

Fig. 2

Graphical representation of RT qPCR analyses of seabream (sa) taste 1 receptor (T1R) subunits, saT1R1, saT1R2a-f and saT1R3 in adult seabream tissues of oropharyngeal (A), gastrointestinal tract (B) and brain tissues (C). Data are represented as the fold change differences of target gene expression to the reference gene-elongation factor 2, per 100 ng of input RNA/tissue. L = lips; G = gill filaments; T = tongue; Oc = oral cavity epithelium; FG = foregut; MG = midgut; HG = hindgut; FB = forebrain; MB = midbrain; HB = hindbrain. All experiments were performed in quadruplicates (n = 4); data are expressed as means ± standard error of the mean (SEM). Different letters indicate significant differences after one-way ANOVA followed by Tukey's multiple comparisons tests (see section of results for significance levels).

Fig. 3

Representative seabream larvae images of the localization of expression for seabream (sa) taste 1 receptor (T1R) subunits, saT1R1 (A/O), saT1R2a (B/R), saT1R2b (C/S) and saT1R2d (D/T) genes, at (A–D) 5, (E–H) 11, (I–L) 14, (M−P) 17, and (Q–T) 21 d post hatching determined by the whole-mount mRNA in situ hybridization. Ov = otic vesicle; St = stomach; P = exocrine pancreas; Fg = foregut; Mg = midgut; Hg = hindgut; T = tongue; Oc = oral cavity; Phy = pharynx. Scale bar = 100 μm (Q), 150 μm (B), 200 μm (A, I), 300 μm (C–H, J–P, R–T).

Fig. 4

Representative seabream larvae images of the localization of expression for seabream (sa) taste 1 receptor (T1R) subunits, saT1R2e (A/M), saT1R2f (B/N) and saT1R3 (C/O) genes, at (A–C) 5, (D–F) 11, (G–I) 14, (J–L) 17, and (M−O) 21 d post hatching determined by the whole-mount mRNA in situ hybridization analyses. Ov = otic vesicle (Ov); St = stomach (St); P = exocrine pancreas (P); Fg = foregut (Fg); Mg = midgut (Mg); Hg = hindgut (Hg); T = tongue (T); Oc = oral cavity (Oc); Phy = pharynx (Phy). Scale bar = 300 μm (A–O).

Fig. 5

Representative seabream larvae images as determined by the whole-mount mRNA in situ hybridization analyses of negative sense controls for seabream (sa) taste 1 receptor (T1R) subunits, (A) saT1R1, (B) saT1R2b, and (C) saT1R3 at 11 d post hatching (dph), and positive antisense controls using the pituitary/hypothalamic gene marker proopiomelanocortin β at (D) 5 and (E, F) 11 dph. Ov = otic vesicle; bHy = basal hypothalamus; Pit = pituitary gland. Scale bar = 200 μm (A-F).

Fig. 6

Representative microphotographs of single chromogenic (A–G), single and dual fluorescent (H–J; and K–N3, respectively) in situ hybridization analyses of nd1, saT1R1, saT1R2b and saT1R3 genes in pyloric caeca (Pyl), foregut (Fg), midgut (Mg), and hindgut (Hg) segments of the seabream gastrointestinal tract. Gene names and probe combinations are indicated in the upper left-hand corner of each panel. Signal color corresponds to probe name; Hoechst 33342 (blue) fluorescent dye was used for nuclear DNA counterstain. nd1 = neurogenic differentiation 1; gl = goblet cells; L = intestinal lumen; lp = lamina propia. Scale bar = 10 μm (A–C, E–K2), 20 μm (D; L–N3).

Fig. 7

Representative microphotographs of chromogenic and dual fluorescent in situ hybridization analyses of saG(i)α1 (A–C and D–D3, respectively) and saG(i)α2 (E–G and H–H3, respectively) genes in pyloric caeca (Pyl), foregut (Fg) and midgut (Mg) segments of the seabream gastrointestinal tract. Gene names and probe combinations are indicated in the upper left-hand corner of each panel. Signal color corresponds to probe name. gl = goblet cells (gl); L = intestinal lumen (L); lp = lamina propia (lp) Scale bar = 10 μm (A–H3).

Fig. 8

Representative microphotographs of chromogenic (B, F–H), single and dual fluorescent in situ hybridization (A and C–E3/I–J3, respectively) of ghre and cck genes in stomach (St), pyloric caeca (Pyl), foregut (Fg), midgut (Mg), and hindgut (Hg) segments of the seabream gastrointestinal tract. Gene names and probe combinations are indicated in the upper left-hand corner of each panel. Signal color corresponds to probe name. ghre = ghrelin; cck = cholecystokinin; gl = goblet cells (gl); L = intestinal lumen; lp = lamina propia (lp). Scale bar = 10 μm (B, E–E3; J–J3), 20 μm (G–I), 30 μm (A, C–D; F).

Fig. 9

Representative microphotographs of chromogenic (A, C, F, and H), single (B–B2) and dual fluorescent in situ hybridization (D–D2; E–E2; G–G2) of pyy and pg genes in foregut (Fg), midgut (Mg), and hindgut (Hg) segments of the seabream gastrointestinal tract. Gene names and probe combinations are indicated in the upper left-hand corner of each panel. Signal color corresponds to probe name. pyy = peptide YY; pg = proglucagon; gl = goblet cells; L = intestinal lumen; lp = lamina propia. Scale bar = 10 μm (B–H), 20 μm (A).