Genetic variation in haemoglobin is associated with evolved changes in breathing in high-altitude deer mice

Physiological systems often have emergent properties but the effects of genetic variation on physiology are often unknown, which presents a major challenge to understanding the mechanisms of phenotypic evolution. We investigated whether genetic variants in haemoglobin (Hb) that contribute to high-altitude adaptation in deer mice (Peromyscus maniculatus) are associated with evolved changes in control of breathing. We created F2 inter-population hybrids of highland and lowland deer mice to test for phenotypic associations of α- and β-globin variants on a mixed genetic background. Hb genotype had expected effects on Hb-O2 affinity that were associated with differences in arterial O2 saturation in hypoxia. However, high-altitude genotypes were also associated with breathing phenotypes that should contribute to enhancing O2 uptake in hypoxia. Mice with highland α-globin exhibited a more effective breathing pattern, with highland homozygotes breathing deeper but less frequently across a range of inspired O2, and this difference was comparable to the evolved changes in breathing pattern in deer mouse populations native to high altitude. The ventilatory response to hypoxia was augmented in mice that were homozygous for highland β-globin. The association of globin variants with variation in breathing phenotypes could not be recapitulated by acute manipulations of Hb-O2 affinity, because treatment with efaproxiral (a synthetic drug that acutely reduces Hb-O2 affinity) had no effect on breathing in normoxia or hypoxia. Therefore, adaptive variation in haemoglobin may have unexpected effects on physiology in addition to the canonical function of this protein in circulatory O2 transport.

Methylation studies in Peromyscus: aging, altitude adaptation, and monogamy

DNA methylation-based biomarkers of aging have been developed for humans and many other mammals and could be used to assess how stress factors impact aging. Deer mice (Peromyscus) are long-living rodents that have emerged as an informative model to study aging, adaptation to extreme environments, and monogamous behavior. In the present study, we have undertaken an exhaustive, genome-wide analysis of DNA methylation in Peromyscus, spanning different species, stocks, sexes, tissues, and age cohorts. We describe DNA methylation-based estimators of age for different species of deer mice based on novel DNA methylation data generated on highly conserved mammalian CpGs measured with a custom array. The multi-tissue epigenetic clock for deer mice was trained on 3 tissues (tail, liver, and brain). Two human-Peromyscus clocks accurately measure age and relative age, respectively. We present CpGs and enriched pathways that relate to different conditions such as chronological age, high altitude, and monogamous behavior. Overall, this study provides a first step towards studying the epigenetic correlates of monogamous behavior and adaptation to high altitude in Peromyscus. The human-Peromyscus epigenetic clocks are expected to provide a significant boost to the attractiveness of Peromyscus as a biological model.

A comparative analysis of differentially expressed mRNAs, miRNAs and circRNAs provides insights into the key genes involved in the high-altitude adaptation of yaks

Background Yaks that inhabit the Tibetan Plateau exhibit striking phenotypic and physiological differences from cattle and have adapted well to the extreme conditions on the plateau. However, the mechanisms used by these animals for the regulation of gene expression at high altitude are not fully understood. Results Here, we sequenced nine lung transcriptomes of yaks at altitudes of 3400, 4200 and 5000 m, and low-altitude Zaosheng cattle, which is a closely related species, served as controls. The analysis identified 21,764 mRNAs, 1377 circRNAs and 1209 miRNAs. By comparing yaks and cattle, 4975 mRNAs, 252 circRNAs and 75 miRNAs were identified differentially expressed. By comparing yaks at different altitudes, we identified 756 mRNAs, 64 circRNAs and 83 miRNAs that were differentially expressed (fold change ≥2 and P-value < 0.05). The pathways enriched in the mRNAs, circRNAs and miRNAs identified from the comparison of yaks and cattle were mainly associated with metabolism, including ‘glycosaminoglycan degradation’, ‘pentose and glucuronate interconversions’ and ‘flavone and flavonol biosynthesis’, and the mRNAs, circRNAs and miRNAs identified from the comparison of yaks at different altitude gradients were significantly enriched in metabolic pathways and immune and genetic information processing pathways. The core RNAs were identified from the mRNA-miRNA-circRNA networks constructed using the predominant differentially expressed RNAs. The core genes specific to the difference between yaks and cattle were associated with the endoplasmic reticulum and fat deposition, but those identified from the comparison among yaks at different altitude gradients were associated with maintenance of the normal biological functions of cells. Conclusions This study enhances our understanding of the molecular mechanisms involved in hypoxic adaptation in yaks and might contribute to improvements in the understanding and prevention of hypoxia-related diseases.

The Expression Regulatory Network in the Lung Tissue of Tibetan Pigs Provides Insight Into Hypoxia-Sensitive Pathways in High-Altitude Hypoxia

To adapt to a low-oxygen environment, Tibetan pigs have developed a series of unique characteristics and can transport oxygen more effectively; however, the regulation of the associated processes in high-altitude animals remains elusive. We performed mRNA-seq and miRNA-seq, and we constructed coexpression regulatory networks of the lung tissues of Tibetan and Landrace pigs. HBB, AGT, COL1A2, and EPHX1 were identified as major regulators of hypoxia-induced genes that regulate blood pressure and circulation, and they were enriched in pathways related to signal transduction and angiogenesis, such as HIF-1, PI3K-Akt, mTOR, and AMPK. HBB may promote the combination of hemoglobin and oxygen as well as angiogenesis for high-altitude adaptation in Tibetan pigs. The expression of MMP2 showed a similar tendency of alveolar septum thickness among the four groups. These results indicated that MMP2 activity may lead to widening of the alveolar wall and septum, alveolar structure damage, and collapse of alveolar space with remarkable fibrosis. These findings provide a perspective on hypoxia-adaptive genes in the lungs in addition to insights into potential candidate genes in Tibetan pigs for further research in the field of high-altitude adaptation.

De novo Assembly, Annotation, and Analysis of Transcriptome Data of the Ladakh Ground Skink Provide Genetic Information on High-Altitude Adaptation

The Himalayan Arc is recognized as a global biodiversity hotspot. Among its numerous cryptic and undiscovered organisms, this composite high-mountain ecosystem harbors many taxa with adaptations to life in high elevations. However, evolutionary patterns and genomic features have been relatively rarely studied in Himalayan vertebrates. Here, we provide the first well-annotated transcriptome of a Greater Himalayan reptile species, the Ladakh Ground skink Asymblepharus ladacensis (Squamata: Scincidae). Based on tissues from the brain, an embryonic disc, and pooled organ material, using pair-end Illumina NextSeq 500 RNAseq, we assembled ~77,000 transcripts, which were annotated using seven functional databases. We tested ~1600 genes, known to be under positive selection in anurans and reptiles adapted to high elevations, and potentially detected positive selection for 114 of these genes in Asymblepharus. Even though the strength of these results is limited due to the single-animal approach, our transcriptome resource may be valuable data for further studies on squamate reptile evolution in the Himalayas as a hotspot of biodiversity.

Identification of a miRNA–mRNA Regulatory Networks in Placental Tissue Associated With Tibetan High Altitude Adaptation

The Tibetan population has lived and successfully reproduced at high altitude for many generations. Studies have shown that Tibetans have various mechanisms for protection against high-altitude hypoxia, which are probably due, at least in part, to placental adaptation. However, comprehensive in silico analyses of placentas in Tibetans are lacking. We performed a microarray-based comparative transcriptome analysis of 10 Tibetan women from Yushu, Qinghai, CHN (∼3,780 m) and 10 European women living in Leadville, CO, United States (∼3,100 m) for less than three generations. Expression of HIF-1α, STAT3, EGFR, HSP5A, XBP1, and ATF6A mRNA was less in the Tibetan placentas as compared with European placentas. A total of 38 miRNAs were involved in regulating these genes. Differentially expressed genes were enriched for HIF1α signaling pathways, protein processing in the endoplasmic reticulum, PI3K-AKT signaling pathways, and MAPK signaling pathways. Based on the transcriptome profiles, the Tibetan population was distinct from the European population; placental tissues from the Tibetan population are lacking hypoxic responses, and “passivation” occurs in response to hypoxic stress. These results provide insights into the molecular signature of adaptation to high altitudes in these two populations.