Observation of yak alveolar tissue slices at different developmental stages
The alveolus is the functional unit of the lungs, and gas exchange in the lungs largely depends on the size of the respiratory area of the lungs [14]. With the growth and development of the yak, individual volume and surface area increase, and the number of alveoli per unit area decrease (Fig. 1B). The total number of alveoli increases, which increases the area of gas exchange in the lung, accelerates the rate of gas exchange in lung tissue [15], and improves the utilization rate of oxygen, enabling yaks to adapt quickly to low-oxygen and high-altitude environments. The ability of yaks to adapt to low oxygen at high altitudes has representative significance [16].
The elastic fibres have a retractive force in alveolar tissue, and the adult yaks tend to ripen; therefore, it is more beneficial for yaks to exchange air between the outside atmosphere and the blood in the lungs by using their own retraction force. This property helps blood vessels bear the pressure of the heartbeat and blood flow to keep the blood flow constant [17]. The proportion of elastic fibres in yak alveoli was observed to increase significantly after 30 days of age (P < 0.05) (Fig. 1D). Therefore, it can be seen that 30 days of age is the key period of yak alveolar development. The alveolar tissues of 180-day-old adults exhibit some ribbon alveolar septum structure of the semipermeable membrane, and the related literature presents similar reports [18]. The location of the elastic fibre that we observed was consistent with this translucent membrane structure, and it was inferred that it may be an elastic fibre.
Expression analysis of differentially expressed genes
By distinguishing the biological information of transcriptomic data between two age groups, it was found that the comparison of the 30-day-old group with the 180-day-old group yielded the highest level of differentially expressed genes (Fig. 2B). Consequently, the stage from 30 days old to 180 days old was indicated to involve many gene expression changes, as this period is a significant stage of yak lung tissue development. This result was consistent with our previous morphological observations.
GO and KEGG annotations
GO enrichment analysis showed that the biological process of differentially expressed genes in three age groups involved the development process, mainly in the membrane and nucleus. The main biological processes were biological regulation and metabolism, and the process involved the functions of protein binding and ion binding. The proportion of differentially expressed genes in the yak lung tissue was highest between the 30-day-old group and the 180-day-old group (Fig. 3B), which suggested that yak lung tissue underwent sustained development from 30 days of age to 180 days of age.
KEGG pathway analysis showed that the PI3K-Akt signalling pathway was an important cellular regulation pathway in three age groups and was related to the formation of fibres [19]. The signalling pathways involved in the formation of fibres were mostly observed between 30 days of age and 180 days of age [20]. Among these pathways, the PI3K-Akt signalling pathway accounted for the largest proportion (Fig. 4B) followed by MAPK, which was also closely related to cell growth and development [21].
Genes related to fibrogenesis
Thirty-four of the 36 genes involved in fibre formation were differentially expressed from 30 days of age to 180 days of age. It was found that a large number of fibres were formed in this stage. Moreover, the expression levels of the genes related to promoting fibrogenesis increased significantly between 30 days of age and 180 days of age (P < 0.05), while the expression levels of the genes related to inhibiting fibrogenesis decreased at this stage. The genes related to elastic fibrogenesis are FBN1, FBN2, EMILIN3, EMILIN2 and ELN; FBN1 and FBN2 belong to the fibrillin protein family [22]; and EMILIN3, EMILIN2 and ELN belong to the elastin family. The fibrillin family and the elastin family genes are closely related to the formation of elastic fibres [23]; therefore, we selected the ELN for analysis, and the expression level of ELN reached its maximum at 30 days of age (Fig. 5A); also, ELN was abundant in lung tissue. Elastin constitutes elastic fibre, and natural elastin is surrounded by a shell composed of microfibrils. Microfibrils are composed of some glycoproteins, and fibrillin is necessary to maintain the integrity of elastic fibres [24]. Elastic fibre is a stretched rubber-like fibre that can provide elasticity and tensile strength to tissues. Although collagen can provide strength and toughness to the extracellular matrix, it needs to be elastic for lung tissue, and the elasticity primarily depends on elastic fibres in the extracellular matrix.
There are 7 fibroblast-related genes, FGF1, FGF9, FGF18, FIBP, CNPY3, TLR3 and FN1, which can promote the formation of fibroblasts. Fibroblast growth factors have a wide range of biological activities that are closely related to cell proliferation and differentiation [25]. These factors can promote the mitosis of fibroblasts and the growth of mesodermal cells, stimulate the formation of blood vessels and play a role in wound healing and limb regeneration [26]. Fibroblast-related genes can promote the growth of fibroblasts and subsequently cause them to develop into fibroblasts [27]. The expression levels of related genes reached a maximum at 30 or 180 days of age (Fig. 5B), which shows that fibre formation is upregulated at this stage.
There are 20 genes that promote the formation of collagen fibres. The collagen family is primarily associated with cell composition, and other related genes mainly participate in fibre formation by inducing related growth factors and various cytokines [28]. COL3A1 gene expression reached the highest value at 30 days of age (Fig. 5C). Type III collagen is a kind of high-molecular-weight protein. Filamentous collagen fibres are the bonding materials of connective tissue, which can keep the skin firm and elastic, participate in the migration, differentiation and proliferation of cells, and promote the generation of collagen fibres [29]. Glutathione peroxidase can improve the survival rate of cells and ensure the integrity of genetic DNA [30]. The GPX1 gene reached its maximum expression level at 180 days of age (Fig. 5D), confirming that GPX can promote collagen fibre formation [31]. Four genes, ADAMTS2, ACAN, TGFβ2 and TGFβ1, repress the formation of collagen fibres. These genes are mainly involved in inducing (Fig. 5E) and inhibiting the effects of growth factors and cytokines to inhibit fibre formation.