Hypothalamic deiodinase type-3 establishes the period of circannual interval timing in mammals
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eLife Assessment
This study provides potentially important findings on the understanding of circannual timing in mammals, for which iodothyronine deiodinases (DIOs) have been suggested to be of critical importance, yet functional genetic evidence has been missing. The authors aim to implicate dio3, the major inactivator of the biologically active thyroid hormone T3, in circannual timing in Djungarian hamsters, using a combination of correlative and gene knock-out experiments. Currently, several questions have been raised concerning either the methodological description and/or the design of the experiments, and so the experimental evidence is considered incomplete.
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Abstract
Abstract
Animals respond to environmental cues to time phenological events, but the intrinsic mechanism of circannual timing remains elusive. We used transcriptomic sequencing and frequent sampling, during three distinct phases (induction, maintenance and recovery) of circannual interval timing for Djungarian hamster energy balance, to investigate the molecular architecture of a neuroendocrine seasonal clock. Our study identified three distinct phases of transcript changes, with deiodinase type-3 (Dio3) expression activated during the early induction phase. Subsequent work demonstrated that targeted mutation of Dio3 resulted in a shorter period for circannual interval timing. Hamsters that exhibit naturally disrupted Dio3 expression do not show any change in body mass or pelage. Our work demonstrates that changes in Dio3 induction is essential for setting the period of circannual interval timing.
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eLife Assessment
This study provides potentially important findings on the understanding of circannual timing in mammals, for which iodothyronine deiodinases (DIOs) have been suggested to be of critical importance, yet functional genetic evidence has been missing. The authors aim to implicate dio3, the major inactivator of the biologically active thyroid hormone T3, in circannual timing in Djungarian hamsters, using a combination of correlative and gene knock-out experiments. Currently, several questions have been raised concerning either the methodological description and/or the design of the experiments, and so the experimental evidence is considered incomplete.
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Reviewer #1 (Public review):
Circannual timing is a phylogenetically widespread phenomenon in long-lived organisms and is central to the seasonal regulation of reproduction, hibernation, migration, fur color changes, body weight, and fat deposition in response to photoperiodic changes. Photoperiodic control of thyroid hormone T3 levels in the hypothalamus dictates this timing. However, the mechanisms that regulate these changes are not fully understood. The study by Stewart et al. reports that hypothalamic iodothyronine deiodinase 3 (Dio3), the major inactivator of the biologically active thyroid hormone T3, plays a critical role in circannual timing in the Djungarian hamster. Overall, the study yields important results for the field and is well-conducted, with the exception of the CRISPR/Cas9 manipulation.
Figure 1 lays the foundation …
Reviewer #1 (Public review):
Circannual timing is a phylogenetically widespread phenomenon in long-lived organisms and is central to the seasonal regulation of reproduction, hibernation, migration, fur color changes, body weight, and fat deposition in response to photoperiodic changes. Photoperiodic control of thyroid hormone T3 levels in the hypothalamus dictates this timing. However, the mechanisms that regulate these changes are not fully understood. The study by Stewart et al. reports that hypothalamic iodothyronine deiodinase 3 (Dio3), the major inactivator of the biologically active thyroid hormone T3, plays a critical role in circannual timing in the Djungarian hamster. Overall, the study yields important results for the field and is well-conducted, with the exception of the CRISPR/Cas9 manipulation.
Figure 1 lays the foundation for examining circannual timing by establishing the timing of induction, maintenance, and recovery phases of the circannual timer upon exposure of hamsters to short photoperiod (SP) by monitoring morphological and physiological markers. Measures of pelage color, torpor, body mass, plasma glucose, etc, established that the initiation phase occurred by weeks 4-8 in SP, the maintenance by weeks 12-20, and the recovery after week 20, where all morphological and physiological changes started to reverse back to long photoperiod phenotypes. The statistical analyses look fine, and the results are unambiguous. Their representation could, however, be improved. In Figures 1d and 1e, two different measures are plotted on each graph and differentiated by dots and upward or downward arrowheads. The plots are so small, though, that distinguishing between the direction of the arrows is difficult. Some color coding would make it more reader-friendly. The same comment applies to Figure S4. The authors went on to profile the transcriptome of the mediobasal and dorsomedial hypothalamus, paraventricular nucleus, and pituitary gland (all known to be involved in seasonal timing) every 4 weeks over the different phases of the circannual interval timer. A number of transcripts displaying seasonal rhythms in expression levels in each of the investigated structures were identified, including transcripts whose expression peaks during each phase. This included two genes of particular interest due to their known modulation of expression in response to photoperiod, Dio3 and Sst, found among the transcripts upregulated during the induction and maintenance phases, respectively. The experiments are technically sound and properly analyzed, revealing interesting candidates. Again, my main issues lie with the representation in the figure. In particular, the authors should clarify what the heatmaps on the right of Figures 1f and 1g represent. I suspect they are simply heatmaps of averaged expression of all genes within a defined category, but a description is missing in the legend, as well as a scale for color coding near the figure.
Figure 2 reveals that SP-programmed body mass loss is correlated to increased Dio3-dependent somatostatin (Sst) expression. First, to distinguish whether the body mass loss was controlled by rheostatic mechanisms and not just acute homeostatic changes in energy balance, experiments from hamsters fed ad lib or experiencing an acute food restriction in both LP and SP were tested. Unlike plasma insulin, food restriction had no additional effect on SP-driven epididymal fat mass loss (Figure S7). This clearly establishes a rheostatic control of body mass loss across weeks in SP conditions. Importantly, Sst expression in the mediobasal hypothalamus increased in both ad lib fed or restriction fed SP hamsters and this increase in expression could be reduced by a single subcutaneous injection of active T3, clearly suggesting that increase in Sst expression in SP is due to a decrease of active T3 likely via Dio3 increase in expression in the hypothalamus. The results are unambiguous.
Figure 3 provides a functional test of Dio3's role in the circannual timer. Mediobasal hypothalamic injections of CRISPR-Cas9 lentiviral vectors expressing two guide RNAs targeting the hamster Dio3 led to a significant reduction in the interval between induction and recovery phases seen in SP as measured by body mass, and diminished the extent of pelage color change by weeks 15-20. In addition, hamsters that failed to respond to SP exposure by decreasing their body mass also had undetectable Dio3 expression in the mediobasal hypothalamus. Together, these data provide strong evidence that Dio3 functions in the circannual timer. I noted, however, a few problems in the way the CRISPR modification of Dio3 in the mediobasal hypothalamus was reported in Figure S8. One is in Figure S8b, where the PAM sites are reported to be 9bp and 11bp downstream of sgRNA1 and sgRNA2, respectively. Is this really the case? If so, I would have expected the experiment to fail to show any effect as PAM sites need to immediately follow the target genomic sequence recognized by the sgRNA for Cas9 to induce a DNA double-stranded break. It seems that each guide contains a 3' NGG sequence that is currently underlined as part of sgRNAs in both Fig S8b and in the method section. If this is not a mistake in reporting the experimental design, I believe that the design is less than optimal and the efficiencies of sgRNAs are rather low, if at all functional. The authors report efficiencies around 60% (line 325), but how these were obtained is not specified. Another unclear point is the degree to which the mediobasal hypothalamus was actually mutated. Only one mutated (truncated) sequence in Figure S8c is reported, but I would have expected a range of mutations in different cells of the tissue of interest. Although the authors clearly find a phenotypic effect with their CRISPR manipulation, I suspect that they may have uncovered greater effects with better sgRNA design. These points need some clarification. I would also argue that repeating this experiment with properly designed sgRNAs would provide much stronger support for causally linking Dio3 in circannual timing.
A proposed schematic model for mechanisms of circannual interval timing is presented in Figure S9. I think this represents a nice summary of the findings put in a broader context and should be presented as a main figure in the manuscript itself rather than being relayed in supplementary materials.
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Reviewer #2 (Public review):
Summary:
Several animals and plants adjust their physiology and behavior to seasons. These changes are timed to precede the seasonal transitions, maximizing chances of survival and reproduction. The molecular mechanisms used for this process are still unclear. Studies in mammals and birds have shown that the expression of deiodinase type-1, 2, and 3 (Dio1, 2, 3) in the hypothalamus spikes right before the transition to winter phenotypes. Yet, whether this change is required or an unrelated product of the seasonal changes has not been shown, particularly because of the genetic intractability of the animal models used to study seasonality. Here, the authors show for the first time a direct link between Dio3 expression and the modulation of circannual rhythms.
Strengths:
The work is concise and presents the …
Reviewer #2 (Public review):
Summary:
Several animals and plants adjust their physiology and behavior to seasons. These changes are timed to precede the seasonal transitions, maximizing chances of survival and reproduction. The molecular mechanisms used for this process are still unclear. Studies in mammals and birds have shown that the expression of deiodinase type-1, 2, and 3 (Dio1, 2, 3) in the hypothalamus spikes right before the transition to winter phenotypes. Yet, whether this change is required or an unrelated product of the seasonal changes has not been shown, particularly because of the genetic intractability of the animal models used to study seasonality. Here, the authors show for the first time a direct link between Dio3 expression and the modulation of circannual rhythms.
Strengths:
The work is concise and presents the data in a clear manner. The data is, for the most part, solid and supports the author's main claims. The use of CRISPR is a clear advancement in the field. This is, to my knowledge, the first study showing a clear (i.e., causal) role of Dio3 in the circannual rhythms in mammals. Having established a clear component of the circannual timing and a clean approach to address causality, this study could serve as a blueprint to decipher other components of the timing mechanism. It could also help to enlighten the elusive nature of the upstream regulators, in particular, on how the integration of day length takes place, maybe within the components in the Pars tuberalis, and the regulation of tanycytes.
Weaknesses:
Due to the nature of the CRISPR manipulation, the low N number is a clear weakness. This is compensated by the fact that the phenotypes shown here are strong enough. Also, this is the only causal evidence of Dio3's role; thus, additional evidence would have significantly strengthened the author's claims. The use of the non-responsive population of hamsters also helps, but it falls within the realm of correlations. Additionally, the consequences of the mutations generated by CRISPR are not detailed; it is not clear if the mutations affect the expression of Dio3 or generate a truncation or deletion, resulting in a shorter protein.
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Reviewer #3 (Public review):
The authors investigated SP-induced physiological and molecular changes in Djungarian hamsters and the endogenous recovery from it after circa half a year. The study aimed to elucidate the intrinsic mechanism and included nice experiments to distinguish between rheostatic effects on energy state and homeostatic cues driven by an interval timer. It also aimed to elucidate the role of Dio3 by introducing a targeted mutation in the MBH by ICV. The experiments and analyses are sound, and the amount of work is impressive. The impact of this study on the field of seasonal chronobiology is probably high.
Even though the general conclusions are well-founded, I have fundamental criticism concerning 3 points, which I recommend revising:
(1) The authors talk about a circannual interval timer, but this is no circannual …
Reviewer #3 (Public review):
The authors investigated SP-induced physiological and molecular changes in Djungarian hamsters and the endogenous recovery from it after circa half a year. The study aimed to elucidate the intrinsic mechanism and included nice experiments to distinguish between rheostatic effects on energy state and homeostatic cues driven by an interval timer. It also aimed to elucidate the role of Dio3 by introducing a targeted mutation in the MBH by ICV. The experiments and analyses are sound, and the amount of work is impressive. The impact of this study on the field of seasonal chronobiology is probably high.
Even though the general conclusions are well-founded, I have fundamental criticism concerning 3 points, which I recommend revising:
(1) The authors talk about a circannual interval timer, but this is no circannual timer. This is a circa-semiannual timer. It is important that the authors use precise wording throughout the manuscript.
(2) The authors put their results in the context of clocks. For example, line 180/181 seasonal clock. But they have described and investigated an interval timer. A clock must be able to complete a full cycle endogenously (and ideally repeatedly) and not only half of it. In contrast, a timer steers a duration. Thus, it is well possible that a circannual clock mechanism and this circa-semiannual timer of photoperiodic species are 2 completely different mechanisms. The argumentation should be changed accordingly.
(3) The authors chose as animal model the Djungarian hamster, which is a predominantly photoperiodic species and not a circannual species. A photoperiodic species has no circannual clock. That is another reason why it is difficult to draw conclusions from the experiment for circannual clocks. However, the Djungarian hamster is kind of "indifferent" concerning its seasonal timing, since a small fraction of them are indeed able to cycle (Anchordoquy HC, Lynch GR (2000), Evidence of an annual rhythm in a small proportion of Siberian hamsters exposed to chronic short days. J Biol Rhythms 15:122-125.). Nevertheless, the proportion is too small to suggest that the findings in the current study might reflect part of the circannual timing.
Therefore, the authors should make a clear distinction between timers and clocks, as well as between circa-annual and circa-semiannual durations/periods.
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