Figures and data
Molecular basis of circannual interval timing for morphology and physiology.
Experimental design in which Djungarian hamsters were kept in long photoperiod (LP) or transferred to short photoperiod (SP) (a). SP induced pelage color change from LP agouti to winter white after 12-weeks which reversed to agouti by 28-weeks (F8, 91 = 50.77; P < 0.001). Created with BioRender.com. (b). Torpor was identified in hamster between 12- and 20-weeks SP exposure indicate by red line (b). SP exposure induced significant reduction in body mass (F8, 91 = 13.428; P < 0.001) (c), food intake (F8, 91 = 10.860; P < 0.001; denoted as downward triangles) (d) and adipose mass (F8, 91 = 27.929; P < 0.001; denoted as upward triangles) (d). Plasma GLP-1 did not vary in response to SP manipulation; denoted as upward triangles (e). SP exposure resulted in significant changes in plasma glucose around the onset of torpor; denoted as downward triangles (F8, 91 = 3.117; P < 0.05) (e). BioDare2.0 heatmaps of mediobasal hypothalamus (f) and pituitary gland (g) transcripts from Djungarian hamster collected at 4-week SP intervals. Transcripts identified as highly rhythmic (FDR < 0.1) showed three distinct phases of expression that coincide with the induction, maintenance and recovery of circannual interval timing. Deiodinase type-3 (Dio3) was upregulated during the induction phase, whereas transcripts associated with energy stability (e.g., somatostatin [Sst and Sstr5]) were upregulated during the maintenance phase. All rhythmic transcripts reverted to the LP condition by 28-weeks SP exposure. Polar scatter chart of significant transcripts from mediobasal hypothalamus and pituitary gland provide a comprehensive seasonal clock for mammalian circannual interval timing across neuroendocrine tissues (h). Green line indicates the induction phase, red line indicates the maintenance phase, and the blue line represents the recovery phase. Letters denote significant differences between treatment groups (P < 0.05) (b-e).
Somatostatin expression reflects programmed circannual interval timing that is dependent on Dio3 regulation of local triiodothyronine signaling.
The experimental design in which SP exposure induced rheostatic reduction in body mass after 12-weeks, and then hamsters experienced either an overnight food restriction (FR) or maintained food ad libitum (AL) (a). SP induced a significant reduction in body mass (F1, 22 = 3.85; P < 0.01). Created with BioRender.com (b). Food restriction further reduced body mass (F1, 22 = 25.46; P < 0.001) (c). Sst expression in the mediobasal hypothalamus was significantly increased in SP (F1, 15 = 5.59; P < 0.05) but did not change after manipulations in nutritional availability (d). Npy expression was increased after food restriction (F1, 15 = 6.12; P < 0.05) but did not change with SP exposure (e). Prolactin (Prl) expression in the pituitary gland was downregulated in response to SP (F1, 20 = 28.53; P < 0.001), and insensitive to food restriction. 12-weeks of SP were found to increase Sst expression in the hamster mediobasal hypothalamus and levels were significantly reduced in response to a single triiodothyronine (T3) injection (g). *P < 0.05 and **P < 0.01 denote significant difference between SP and LP conditions (b). Letters denote significant difference between treatment groups (P < 0.05) (c-g).
Dio3 functions to time circannual interval duration in hamsters.
Hamsters received intracerebroventricular injections to target Dio3 expressing cells in the tanycytes localized to the mediobasal hypothalamus (a). Crispr-Cas9 constructs were packaged into lentiviral vectors to generate blank control hamsters (Dio3wt) or contain gRNAs that mutated the Dio3 gene (Dio3cc). Hamsters were then exposed to SP conditions and the circannual interval timer was assessed by monitoring body mass. Created with BioRender.com (b). Dio3cc hamsters delayed interval timing as evidenced by higher body mass at 10-weeks SP exposure (P < 0.001) and recovered body mass quicker at 26 to 32 (P < 0.05) weeks. Lomb-Scargle analyses identified a significant reduction in the period of the circannual interval timer (t6 = 6.975; P < 0.05) (c,d). Example Dio3cc (hamster #903) and Dio3wt (hamster #825) are presented in (c). A subpopulation of hamsters did not decrease body mass in response to SP exposure and termed non-responsive (NR) (t12 = 7.12; P < 0.001) (e). Dio3 expression in the mediobasal hypothalamus of NR hamster was nearly nondetectable after exposure to 8-12 weeks of SP exposure compared to SP control hamsters (t12 = −3.78; P < 0.005) (f). *P < 0.05, **P < 0.01, and ***P < 0.001.
Plasma insulin reflects changes in transcript expression.
(A) Plasma insulin of Siberian hamsters across 32-weeks of short photoperiod exposure (H (8) = 20.401, P = 0.009). (B) Expression of Dio3 from the MBH of Siberian hamsters, assessed by qPCR, across 32-weeks of short photoperiod exposure (H (8) = 39.771, P < 0.001). (C) Expression of Sst from the MBH of Siberian hamsters, assessed by qPCR, across 32-weeks of short photoperiod exposure (H (8) =16.002, P = 0.042). Letters denote significance between groups.
Gene ontology analysis of MBH sequencing reveals well known seasonal pathways.
(A) Gene ontology enrichment analysis of significant transcripts from sequencing of Djungarian hamster MBH across 32-weeks of short photoperiod exposure. (B) Interaction network of enriched gene ontology terms. Size of individual points represent the number of transcripts in each term. Thickness of interactions represents shared transcripts between terms.
Gene ontology analysis of pituitary gland unveils potential mechanisms for seasonal changes in protein processing and release.
(A) Gene ontology enrichment analysis of significant transcripts from sequencing of Djungarian hamster pituitary gland across 32-weeks of short photoperiod exposure. (B) Interaction network of enriched gene ontology terms. Size of individual points represent the number of transcripts in each term. Thickness of interactions represents shared transcripts between terms.
Paraventricular and dorsomedial hypothalamus sequencing unveils novel transcripts and widespread seasonal interval timing within the hypothalamus.
(A-B) Heatmaps of significant (FDR < 0.1) Djungarian hamster transcripts displaying sine or cosine rhythmicity were selected from (A) paraventricular hypothalamus and (B) dorsomedial hypothalamus. (C) Polar scatter chart of significant transcripts from Djungarian hamster paraventricular and dorsomedial hypothalamus, displaying peak of expression and −log(FDR).
Gene ontology analysis of paraventricular hypothalamic sequencing.
(A) Gene ontology enrichment analysis of significant transcripts from sequencing of Djungarian hamster paraventricular hypothalamus across 32-weeks of short photoperiod exposure.
Gene ontology analysis of dorsomedial hypothalamus suggests widespread immune involvement and cellular differentiation.
(A) Gene ontology enrichment analysis of significant transcripts from sequencing of Djungarian hamster dorsomedial hypothalamus across 32-weeks of short photoperiod exposure. (B) Interaction network of enriched gene ontology terms. Size of individual points represent the number of transcripts in each term. Thickness of interactions represents shared transcripts between terms.
Rheostatic mechanism controlling body mass change in Djungarian hamsters.
(A) Epidydimal fat mass in long- and short-photoperiod exposed Djungarian hamsters (F1, 20 = 59.136; P < 0.001) after an overnight food restriction, or ad libitum, feeding protocol (F1, 22 = 2.738; P = 0.114). (B) Pituitary gland expression of Gh from in long- and short-photoperiod exposed Djungarian hamsters (F1, 20 = 2.738; P = 0.114) after an overnight food restriction, or ad libitum, feeding protocol, assessed by qPCR (F1, 20 = 0.518; P = 0.480). (C) Plasma insulin in long- and short-photoperiod exposed Djungarian hamsters (F1, 31 = 29.865; P < 0.001) after an overnight food restriction, or ad libitum, feeding protocol (F1, 31 = 6.863; P = 0.014). Letters denote significant main effect of photoperiod (a). Asterisk indicates significant effect of photoperiod (P < 0.005), and hashtag denotes significant main effect of food restriction (P < 0.05).
Evidence of CRISPR modification in Dio3cc and Dio3wt Djungarian hamsters.
(A) Binding positions of gRNA1 and gRNA2, injected into Djungarian hamster hypothalamus (B) Sequence of gRNA1 and gRNA2 binding and PAM sites. (C) Representative Dio3 sequence alignment with gRNA1 and PAM sites indicated for a representative Dio3wt (control) and Dio3cc (CRISPR) hamster. (D) Decomposition error difference for Dio3 sequence alignment to determine genomic modification (t5 = 2.66, p < 0.05) (E) Pelage change in Dio3wt and Dio3cc Djungarian hamsters across 32 weeks of SP exposure, Dio3wt showed significantly increased pelage score at 13 (P < 0.001) and 14 (p < 0.001).
Schematic model for the mechanisms of circannual interval timing.
(a) Hamsters held in long photoperiod (LP) have high body mass, thyrotropin-stimulating hormone beta (Tshβ) and low somatostatin (Sst) and deiodinase type-3 (Dio3). Transfer to short photoperiod (SP) results in the induction of circannual interval timing characterized by a reduction in body mass, low Tshβ in the pars tuberalis and increased Dio3 expression in tanycytes. The maintenance phase is associated with increased Sst expression which serves to inhibit growth. The onset of the recovery phase is associated with a complete reversal in whole organismal physiology to the LP phenotype. (b) tanycytes along the third ventricle in the mediobasal hypothalamus integrate photic cues derived from Tshβ and are sensitive to nutritional cues. The triiodothyronine (T3) output signal from tanycytes regulates Sst expression leading to long-term programmed rheostatic changes in body mass that serve to control circannual interval timing in multiple physiological systems. Conversely, homeostatic stability is maintained despite large scale seasonal rhythms in body mass. Homeostatic energy balance is established by well characterized circuits that include neuropeptide Y (Npy), agouti-related peptide (Agrp) and the melanocortin receptor 4 (Mc4r). Created with BioRender.com.
