Uncovering thermoregulation in laboratory mice

Gene expression , mice , thermoregulation , evolution , endothermy  

2021.04866.BD FCT grant

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From an evolutionary point of view, the endothermic homeothermic thermoregulation in small altricial mammals, like rodents, develops at around one third to a half of the animal's adult size ⁸. By having a small hairless body, pups are constantly losing heat, having to manage a higher and unbalanced energetic challenge, relying on adult parents or nests to keep a high and stable body temperature. To overcome this challenge, they usually resort to heterothermic physiology. This way, during their development, pups can have a controlled reduction of their metabolism and body temperature, saving energy that is crucial for them to grow ⁸.

Although this heterothermic physiology is very likely to be employed by many species, until the present date, it has only been reported in 4 bird orders and 1 marsupial order. In these two cases, the hatchlings are poikilothermic at birth following a heterothermic thermoregulation afterwards, during cold exposure. By this, it is possible to infer that daily heterothermy is phylogenetically old and likely plesiomorphic. Besides birds and marsupials, daily heterothermy in placental mammals has only been described in two orders, Insectivora and Rodentia. However, juveniles from other orders, like bats, are known to enter a torpor state ⁹. Yet, no studies were published on detailed developmental sequences, identifying the transition between poikilothermy and heterothermy or endothermy. The main difference between the studied marsupials and the placentals is that, in the latter, poikilothermy at birth is followed by a homeothermic phase after which the endothermic thermoregulation is established, meaning that the ability to employ daily heterothermy appears later in life (i.e. poikilothermy–endothermy–heterothermy) ⁸ This suggests that in placentals, daily heterothermy is a trait that evolved secondarily after a homeothermic phase, in response to energetic challenges, probably due to climate change ⁵. As mammals and birds arose from different reptilian lineages, it is likely that endothermy emerged from two separate classes, and daily heterothermy seems to have evolved at least three times, given the differences in developmental sequence between marsupials and placentals ⁸.

 

On an ontogenetic point of view, a recent study compared pups (0-4 days) from four different rodent species, ranging in different heterothermy levels as adults ¹⁰. They tested the adaptation to cold during hypoxia conditions, concluding that heterotherms retain traits that are common to all newborn mammals ¹¹, including high tolerance to body temperature decreases. Even as newborns, the heterotherms species were more tolerant of cold temperatures and O2 limitation than homeotherms^12,13. Additionally, from the tested species, the rat was the one showing the greatest thermogenic response, followed by the facultative heterotherm species, and lastly by the hibernator (squirrel) with almost no response. These differences were coherent with the ones seen on the same adult species¹⁴, indicating that the adult phenotype characteristics are already present at birth. This was the first comparative study that attempted to test a link between pup and adult stages in different species¹⁰, concerning the heterothermy and endothermy physiologies, studying some environmental parameters (temperature and O2 levels). Nonetheless, the question of when (ontogenetically) is the homeothermic stage achieved and how (which genes trigger it), still remains unanswered. For this project we will test whether the endothermic regulation starts around weaning age. Specifically, we aim to answer the following questions: 1) is endothermy developed gradually, rather than abruptly?; 2) is endothermy a pleiotropic trait, where previously studied metabolic control and temperature-related genes take part?; and 3) is a high level of gene expression after weaning indicative of emergence of endothermy?. We will use an inbred Mus musculus strain (C57BL/6J) in a laboratory setting as a model for a facultative heterotherm species¹⁵ as adults. To test our main hypothesis and answer our additional experimental questions, we will reproduce the laboratory mice in-house. Firstly, we will measure and study the relation between acute and short-term surface (Ts) and internal (Tb) body temperature loss in hairless pups temporarily within the litter (0 and 3min – pup mass) and separated (6min) from and, during their active (night) and inactive (day) periods; and monitor Ts and Tb body temperature in juveniles. To account for the effect of nesting material in insulation and thermoregulation, we will remove it for 60 min prior to measurements, in half of the cages (assigned randomly), from P15 to P40. To identify the ontogenic onset of endothermy, two tissue samples will be taken before weaning and two afterwards, in spaced time-points (at P4, P15, P23 and P40), namely brain (hippocampus and amygdala), and liver, which are markedly related with thermoregulation control and thermogenesis ^16–19, that will be posteriorly sequenced (RNAseq) in order to assess their gene expression.

Confirmatory

Main hypothesis being tested: Endothermic regulation starts around weaning age Secondary hypotheses: 1. Endothermy develops gradually, rather than abruptly 2. Endothermy is a pleiotropic trait, where previously studied metabolic control and temperature-related genes take part. 3. High level of gene expression after weaning indicates the emergence of endothermy

Experimental unit: litter

Timeline:

P0 - pups birth

 

from P0 to P40 - Daily TS and TB measurements of the pups (0, 3 and 6 min)

 

P4 - experimental endpoint - random euthanasia of one pup/litter; dissection and sampling; P15 - experimental endpoint - random euthanasia of one pup/litter; dissection and sampling; P21 - separation of the males and females in two cages by sex; pups weaning

P21-P40 (every 2 days: P22, P24, P28, P30, P32, P34, P36, P38) - random assign of "control" and " experimental" groups to either the male or female cage for the nesting inhibition experiment (30min-1h)

P23 - experimental endpoint - random euthanasia of one pup/litter; dissection and sampling;

P40 - experimental endpoint - random euthanasia of one pup/litter; dissection and sampling;

The researcher analyzing the data will not be aware of the experimental groups. Allocation concealment for data collection will not be possible, but data extraction will rely on technology (pit tag readers and thermal cameras) which output that cannot be interfered with by the researcher. Performance bias will be addressed by randomization of order by which each litter and each pup within each litter is assessed.

Software assisted (Excel RAND() function) randomization of treatments to each litter (the order to perform each method/procedure); to each pup of each litter - each pup will have an assigned number that will be randomized; to the cage position in the rack - the cages are also going to be identified with labels and randomized each time.

References: 1. Grigg, G. et al. Whole‐body endothermy: ancient, homologous and widespread among the ancestors of mammals, birds and crocodylians. Biol Rev Camb Philos Soc 97, 766–801 (2022). 2. Ivanov, K. P. The development of the concepts of homeothermy and thermoregulation. Journal of Thermal Biology 31, 24–29 (2006). 3. McNab, B. K. The Evolution of Endothermy in the Phylogeny of Mammals. The American Naturalist 112, 1–21 (1978). 4. Guschina, I. A. & Harwood, J. L. Mechanisms of temperature adaptation in poikilotherms. FEBS Letters 580, 5477–5483 (2006). 5. Bastos, B., Pradhan, N., Tarroso, P., Brito, J. C. & Boratyński, Z. Environmental determinants of minimum body temperature in mammals. fozo.1 70, 21004.1 (2021). 6. McKechnie, A. E. & Mzilikazi, N. Heterothermy in Afrotropical Mammals and Birds: A Review. Integrative and Comparative Biology 51, 349–363 (2011). 7. Ruf, T. & Geiser, F. Daily torpor and hibernation in birds and mammals. Biological Reviews 90, 891–926 (2015). 8. Geiser Fritz. Ontogeny and phylogeny of endothermy and torpor in mammals and birds. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 150, 176–180 (2008). 9. Hollis, L. M. Thermoregulation by big brown bats (Eptesicus fuscus): Ontogeny, proximate mechanisms, and dietary influences. 1 (2004). 10. Dzal, Y. A. & Milsom, W. K. Effects of hypoxia on the respiratory and metabolic responses to progressive cooling in newborn rodents that range in heterothermic expression. Experimental Physiology 106, 1005–1023 (2021). 11. Barnes, E. B. M. & Carey, H. V. Life in the cold - Evolution, Mechanisms, Adaptation, and Application. 609 (2004). 12. Fong, A. Y. Postnatal changes in the cardiorespiratory response and ability to autoresuscitate from hypoxic and hypothermic exposure in mammals. Respiratory Physiology & Neurobiology 174, 146–155 (2010). 13. Hiestand, W. A., Stemler, F. W. & Wiebers, J. E. Gasping Patterns of the Isolated Respiratory Centers of Birds and Mammals during Anoxia, Their Phylogenetic Significance, and Implication in Hibernation. Physiological Zoology 26, 167–173 (1953). 14. Dzal, Y. A. & Milsom, W. K. Hypoxia alters the thermogenic response to cold in adult homeothermic and heterothermic rodents. The Journal of Physiology 597, 4809–4829 (2019). 15. Hudson, J. W. & Scott, I. M. Daily Torpor in the Laboratoryoratory Mouse, Mus musculus Var. Albino. Physiological Zoology 52, 205–218 (1979). 16. Sieck, G. C. Molecular biology of thermoregulation. Journal of Applied Physiology 92, 1365–1366 (2002). 17. Nakamura, K. Central circuitries for body temperature regulation and fever. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 301, R1207–R1228 (2011). 18. Nakamura, K. & Morrison, S. F. A thermosensory pathway that controls body temperature. Nat Neurosci 11, 62–71 (2008). 19. Nakamura, K. & Morrison, S. F. A thermosensory pathway that controls body temperature. Nat Neurosci 11, 62–71 (2008). 20. Massoud, D. et al. Divergent Seasonal Reproductive Patterns in Syntopic Populations of Two Murine Species in Southern Spain, Mus spretus and Apodemus sylvaticus. Animals 11, 243 (2021).

We decided best to finish the experiment at P35, since our hypothesis aims to the weaning period. Thus the final sampling timepoint would change to P35.

Legally our veterinarian department only allowed to insert the PIT tags on the pups at P10