Fig 1.
Overview of the brain transcriptome in Atlantic salmon before (720 dd) and after (920 dd) the onset of exogenous feeding.
(A) Hierarchically clustered heatmap of the whole brain transcriptome (36 734 genes) at 720 dd and 920 dd, scaled by row, shows the average normalized counts for each gene per sampling time point (n = 13 per stage) using the merged normalized count file. (B) Venn diagram showing the presence of appetite-related genes (see S2 Table for more details) in the three normalized count files (for 720 dd, 920 dd, and merged 720 dd and 920 dd). Figure made in: https://bioinfogp.cnb.csic.es/tools/venny/ (C) Hierarchically clustered heatmap of grouped genes related to appetite and metabolism at 720 dd and 920 dd (358 genes), scaled by row, shows the average normalized counts for each group per sampling point (n = 13 per stage) using the merged normalized count file. The lower bar at the x-axis indicates the light period (light/dark) and feeding time twice a day (brown) for 920 dd. For the expression pattern of individual genes related to appetite, see S1 Fig. Abbreviation: dd, day-degree..
Table 1.
Significant cycling appetite-related genes at 720 dd and 920 dd in Atlantic salmon.
Table 2.
Significantly differentially expressed appetite-related genes between time points at 920 dd Atlantic salmon (p < 0.05).
Fig 2.
Appetite-related genes that have a significant change in their normalized count levels at 920 dd in Atlantic salmon brain.
A-G: Normalized counts for appetite-related genes at 920 dd with significant changes in expression levels from DESeq2 analysis (p-value < 0.05). Boxplot color coding indicates daytime (blue) or nighttime (red). (A) cnr1 (ENSSSAG00000091836); (B) insrs2b (ENSSSAG00000031099); (C) insrs2b (ENSSSAG00000065410). (D) nppc (ENSSSAG00000074569); (E) cart4 (ENSSSAG00000069679); (F) npp-r1-like (ENSSSAG00000048802); (G) mrap.3 (ENSSSAG00000103286). The grey background indicates nighttime (22:00-08:00). The yellow background indicates the feeding times (09:00-09:30 and 16:00-20:00). Asterix (*) indicates significant (DESeq2 analysis p-value < 0.05) differences between time points. (H) Acrophase distribution of significantly (MetaCycle analysis p-value < 0.05) appetite-related genes and clock genes at 920 dd in Atlantic salmon. Color coding bars indicate whether the acrophase occurs during the daytime (blue) or nighttime (red) (14:12 LD). Appetite-related genes (black) and clock genes (grey) are listed under the respective acrophases. The orange background indicates feeding times. Each radius of the rose plot represents one gene. Data for the clock genes were obtained from Fig 3 in Eilertsen et al., [12].
Fig 3.
Expression of melanocortin system genes in Atlantic salmon brain.
(A) npya mRNA expression (indicated by black arrow) in the anterior tuberal nucleus at 740 dd. (B) cart2b mRNA expression in the anterior tuberal nucleus at 740 dd. (C) agrp1 mRNA expression in the lateral tuberal nucleus at 740 dd. (D) pomca mRNA expression in lateral tuberal nucleus and adenohypophysis at 740 dd. (E) Schematic illustrating key neuropeptides of the melanocortin system (indicated by color dots) expressed in the hypothalamus in endogenous feeding Atlantic salmon. (F) Conceptual model of melanocortin signaling: agrp/npy and cart/pomc provide opposing neuropeptide input on downstream effector neuron expressing mc4r and mrap. (G) Gene expression analyses of neuropeptides, receptors, and accessory proteins of the melanocortin system between 720 dd and 920 dd in Atlantic salmon. Asterisks (*) show significant levels (DESeq2 analyses) (* indicates p-value < 0.001). Abbreviations: NAT; nucleus anterior tuberis, NLT; nucleus lateralis tuberis, NPT; nucleus posterior tuberis, pit; pituitary. Scalebar A-D: 114 µm.
Fig 4.
Schematic overview of factors involved in the regulation of energy expenditure and nutrient supply in hypothalamic and extrahypothalamic brain regions during endogenous and exogenous feeding in Atlantic salmon.
The brain responds to hormonal and nutrient-sensing signals that modulate downstream signaling cascades and regulate the expression of key neuropeptides in the melanocortin system. The daily cycle is controlled by oscillations of signal factors. (A) During endogenous feeding at 720 dd only a few appetite-related genes cycled. (B) During the first-feeding period at 920 dd, several genes had started to cycle and were upregulated compared to 720 dd. Color codes are based on the average normalized counts in the heatmap of grouped genes related to appetite and metabolism at 720 dd and 920 dd (Fig 1C) and the heatmap for the expression pattern of individual appetite-related genes (S2 Fig). The cycling symbol indicates if there was a significant cycling (MetaCycle analyses, p-value < 0.05) for a gene or a gene within a group (i.e., genes linked to nutrient sensing) (Table 1). The gene agrp1 was absent in the normalized count file for 720 dd and 920 dd and was given no color code at 720 dd, while at 920 dd the color code is based on the 920 dd normalized count file. Abbreviations: agrp, agouti-related peptide; akt, protein kinase B; ampkα, AMP-activated protein kinase; bsx, brain homeobox transcription factor; cart, cocaine- and amphetamine-related transcript; cck, cholecystokinin; creb, cAMP response-element binding protein; foxo1, forkhead box protein O1; mtor, mechanistic target of rapamycin; NLTv, lateral tuber nucleus pars ventralis; npy, neuropeptide Y; pomca, pro-opiomelanocortin a. The schematic drawing is modified from Soengas [32].