Low thyroxine serves as an upstream regulator of ecophysiological adaptations in Ansell's mole-rats.

Introduction: About 10% of all rodent species have evolved a subterranean way of life, although life in subterranean burrows is associated with harsh environmental conditions that would be lethal to most animals living above ground. Two key adaptations for survival in subterranean habitats are low resting metabolic rate (RMR) and core body temperature (Tb). However, the upstream regulation of these traits was unknown thus far. Previously, we have reported exceptionally low concentrations of the thyroid hormone (TH) thyroxine (T4), and peculiarities in TH regulating mechanisms in two African mole-rat species, the naked mole-rat and the Ansell's mole-rat. Methods: In the present study, we treated Ansell's mole-rats with T4 for four weeks and analyzed treatment effects on the tissue and whole organism level with focus on metabolism and thermoregulation. Results: We found RMR to be upregulated by T4 treatment but not to the extent that was expected based on serum T4 concentrations. Our data point towards an extraordinary capability of Ansell's mole-rats to effectively downregulate TH signaling at tissue level despite very high serum TH concentrations, which most likely explains the observed effects on RMR. On the other hand, body weight was decreased in T4-treated animals and Tb was upregulated by T4 treatment. Moreover, we found indications of the hypothalamus-pituitary-adrenal axis potentially influencing the treatment effects. Conclusion: Taken together, we provide the first experimental evidence that the low serum T4 concentrations of Ansell's mole-rats serve as an upstream regulator of low RMR and Tb. Thus, our study contributes to a better understanding of the ecophysiological evolution of the subterranean lifestyle in African mole-rats.

Hypoxic and hypercapnic burrow conditions lead to downregulation of free triiodothyronine and hematocrit in Ansell's mole-rats (Fukomys anselli).

African mole-rats live in self-dug burrow systems under hypoxic and hypercapnic conditions. Adaptations to hypoxia include suppression of resting metabolic rate (RMR) and core body temperature (Tb). Because the thyroid hormones (THs) thyroxine (T4) and triiodothyronine (T3) are positive regulators of RMR and Tb, we hypothesized that serum TH concentrations would also be downregulated under hypoxic conditions. To test this hypothesis, we kept Ansell's mole-rats (Fukomys anselli) in terraria filled with soil in which they were allowed to construct underground burrows to achieve chronic intermittent hypoxia and hypercapnia. The animals stayed in these hypoxic and hypercapnic burrows voluntarily, although given the choice to stay aboveground. We collected blood samples before and after treatment to measure serum T4 and T3 concentrations as well as hematological parameters. The free fraction of the transcriptionally-active T3 was significantly decreased after treatment, indicating that cellular TH signaling was downregulated via peripheral mechanisms, consistent with the assumption that aerobic metabolism is downregulated under hypoxic conditions. Furthermore, we found that hematocrit and hemoglobin concentrations were also downregulated after treatment, suggesting that oxygen demand decreases under hypoxia, presumably due to the metabolic shift towards anaerobic metabolism. Taken together, we have identified a potential upstream regulator of physiological adaptations to hypoxia in these highly hypoxia-tolerant animals.

Mesenchymal-epithelial transition in lymph node metastases of oral squamous cell carcinoma is accompanied by ZEB1 expression.

BACKGROUND: Oral squamous cell carcinoma (OSCC), an HPV-negative head and neck cancer, frequently metastasizes to the regional lymph nodes but only occasionally beyond. Initial phases of metastasis are associated with an epithelial-mesenchymal transition (EMT), while the consolidation phase is associated with mesenchymal-epithelial transition (MET). This dynamic is referred to as epithelial-mesenchymal plasticity (EMP). While it is known that EMP is essential for cancer cell invasion and metastatic spread, less is known about the heterogeneity of EMP states and even less about the heterogeneity between primary and metastatic lesions. METHODS: To assess both the heterogeneity of EMP states in OSCC cells and their effects on stromal cells, we performed single-cell RNA sequencing (scRNAseq) of 5 primary tumors, 9 matching metastatic and 5 tumor-free lymph nodes and re-analyzed publicly available scRNAseq data of 9 additional primary tumors. For examining the cell type composition, we performed bulk transcriptome sequencing. Protein expression of selected genes were confirmed by immunohistochemistry. RESULTS: From the 23 OSCC lesions, the single cell transcriptomes of a total of 7263 carcinoma cells were available for in-depth analyses. We initially focused on one lesion to avoid confounding inter-patient heterogeneity and identified OSCC cells expressing genes characteristic of different epithelial and partial EMT stages. RNA velocity and the increase in inferred copy number variations indicated a progressive trajectory towards epithelial differentiation in this metastatic lesion, i.e., cells likely underwent MET. Extension to all samples revealed a less stringent but essentially similar pattern. Interestingly, MET cells show increased activity of the EMT-activator ZEB1. Immunohistochemistry confirmed that ZEB1 was co-expressed with the epithelial marker cornifin B in individual tumor cells. The lack of E-cadherin mRNA expression suggests this is a partial MET. Within the tumor microenvironment we found immunomodulating fibroblasts that were maintained in primary and metastatic OSCC. CONCLUSIONS: This study reveals that EMP enables different partial EMT and epithelial phenotypes of OSCC cells, which are endowed with capabilities essential for the different stages of the metastatic process, including maintenance of cellular integrity. During MET, ZEB1 appears to be functionally active, indicating a more complex role of ZEB1 than mere induction of EMT.

Comparative analysis of thyroid hormone systems in rodents with subterranean lifestyle.

African mole-rats are subterranean rodents inhabiting underground burrows. This habitat entails risks of overheating, hypoxia, and scarce food availability. Consequently, many subterranean species have evolved low basal metabolism and low body temperature, but the regulation of these traits at the molecular level were unknown. Measurements of serum thyroid hormone (TH) concentrations in African mole-rats have revealed a unique TH phenotype, which deviates from the typical mammalian pattern. Since THs are major regulators of metabolic rate and body temperature, we further characterised the TH system of two African mole-rat species, the naked mole-rat (Heterocephalus glaber) and the Ansell's mole-rat (Fukomys anselli) at the molecular level in a comparative approach involving the house mouse (Mus musculus) as a well-studied laboratory model in TH research. Most intriguingly, both mole-rat species had low iodide levels in the thyroid and naked mole-rats showed signs of thyroid gland hyperplasia. However, contrary to expectations, we found several species-specific differences in the TH systems of both mole-rat species, although ultimately resulting in similar serum TH concentrations. These findings indicate a possible convergent adaptation. Thus, our study adds to our knowledge for understanding adaptations to the subterranean habitat.

Evoked auditory potentials from African mole-rats and coruros reveal disparity in subterranean rodent hearing.

Hearing in subterranean rodents exhibits numerous peculiarities, including low sensitivity and restriction to a narrow range of comparatively low frequencies. Past studies provided two conflicting hypotheses explaining how these derived traits evolved: structural degeneration and adaptive specialization. To further elucidate this issue, we recorded auditory brainstem responses from three species of social subterranean rodents that differ in the degree of specialization to the underground habitat: the naked mole-rat (Heterocephalus glaber) and the Mashona mole-rat (Fukomys darlingi), which represent the ancient lineage of African mole-rats (Bathyergidae), and the coruro (Spalacopus cyanus), a South American rodent (Octodontidae) that adopted a subterranean lifestyle in more recent geological time. Additionally, we measured call amplitudes of social vocalizations to study auditory vocal coupling. We found elevated auditory thresholds and severe hearing range restrictions in the African mole-rats, with hearing in naked mole-rats tending to be more sensitive than in Mashona mole-rats, in which hearing notably deteriorated with increasing age. In contrast, hearing in coruros was similar to that of epigeic rodents, with its range extending into ultrasonic frequencies. However, as in the mole-rats, the coruros' region of best hearing was located at low frequencies close to 1 kHz. We argue that the auditory sensitivity of African mole-rats, although remarkably poor, has been underestimated by recent studies, whereas data on coruros conform to previous results. Considering the available evidence, we propose to be open to both degenerative and adaptive interpretations of hearing physiology in subterranean mammals, as each may provide convincing explanations for specific auditory traits observed.

Mutational Landscape of Virus- and UV-Associated Merkel Cell Carcinoma Cell Lines Is Comparable to Tumor Tissue.

Merkel cell carcinoma (MCC) is a rare, highly aggressive cutaneous malignancy that is either associated with the integration of the Merkel cell polyomavirus or chronic UV exposure. These two types of carcinogenesis are reflected in characteristic mutational features present in MCC tumor lesions. However, the genomic characteristics of MCC cell lines used as preclinical models are not well established. Thus, we analyzed the exomes of three virus-negative and six virus-positive MCC cell lines, all showing a classical neuroendocrine growth pattern. Virus-negative cell lines are characterized by a high tumor mutational burden (TMB), UV-light-induced DNA damage, functionally relevant coding mutations, e.g., in RB1 and TP53, and large amounts of copy number variations (CNVs). In contrast, virus-positive cell lines have a low TMB with few coding mutations and lack prominent mutational signatures, but harbor characteristic CNVs. One of the virus-negative cell lines has a local MYC amplification associated with high MYC mRNA expression. In conclusion, virus-positive and -negative MCC cell lines with a neuroendocrine growth pattern resemble mutational features observed in MCC tissue samples, which strengthens their utility for functional studies.

Audiograms of three subterranean rodent species (genus Fukomys) determined by auditory brainstem responses reveal extremely poor high-frequency hearing.

Life underground has shaped the auditory sense of subterranean mammals, shifting their hearing range to low frequencies. Mole-rats of the genus Fukomys have, however, been suggested to hear at frequencies up to 18.5 kHz, unusually high for a subterranean rodent. We present audiograms of three mole-rat species, Fukomys anselli, Fukomys micklemi and the giant mole-rat Fukomys mechowii, based on evoked auditory brainstem potentials. All species showed low sensitivity and restricted hearing ranges at 60 dB SPL extending from 125 Hz to 4 kHz (5 octaves) with most-sensitive hearing between 0.8 kHz and 1.4 kHz. The high-frequency cut-offs are the lowest found in mammals to date. In contrast to predictions from middle ear morphology, F. mechowii did not show higher sensitivity than F. anselli in the low-frequency range. These data suggest that the hearing range of Fukomys mole-rats is highly restricted to low frequencies and similar to that of other subterranean mammals.