MAPKKs, as one of the main members of the mitogen-activated protein kinase (MAPK) cascade pathway, are located in the middle of the cascade and are involved in many physiological processes of plant growth and development, as well as stress tolerance. Previous studies have found that StMAPKK5 is responsive to drought and salt stress. To further investigate the function and regulatory mechanism of StMAPKK5 in potato stress response, potato variety ‘Atlantic’ was subjected to drought and NaCl treatments, and the expression of the StMAPKK5 gene was detected by qRT-PCR. StMAPKK5 overexpression and RNA interference-mediated StMAPKK5 knockdown potato plants were constructed. The relative water content, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities, as well as proline (Pro) and malondialdehyde (MDA) contents of plant leaves, were also assayed under drought and NaCl stress. The StMAPKK5 interacting proteins were identified and validated by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). The results showed that the expression of StMAPKK5 was significantly up-regulated under drought and NaCl stress conditions. The StMAPKK5 protein was localized in the nucleus, cytoplasm, and cell membrane. The expression of StMAPKK5 affected the relative water content, the enzymatic activities of SOD, CAT, and POD, and the proline and MDA contents of potatoes under drought and salt stress conditions. These results suggest that StMAPKK5 plays a significant role in regulating drought and salt tolerance in potato crop. Yeast two-hybrid (Y2H) screening identified four interacting proteins: StMYB19, StZFP8, StPUB-like, and StSKIP19. BiFC confirmed the authenticity of the interactions. These findings suggest that StMAPKK5 is crucial for potato growth, development, and response to adversity.

Hair follicle stem cells (HFSCs) are resident stem cells within hair follicles (HFs) that possess self-renewal and differentiation capacities, serving as a critical model for regenerative medicine research. Their dynamic interaction with dermal papilla cells (DPCs) plays a decisive role in HF development and cycling. FGF22 is a paracrine fibroblast growth factor that can regulate the proliferation, differentiation and migration of epithelial cells. This study established a DPC-HFSC co-culture system, revealing that FGF22 overexpression in DPCs significantly upregulated FGFR1/FGFR2 mRNA expression levels in HFSCs (p < 0.05), with a 1.67-fold increase in EdU-positive cell proportion (p < 0.01). CCK-8 assays demonstrated markedly enhanced HFSC viability (p < 0.01), with a 17% reduction in HFSC apoptosis (p < 0.05). Conversely, FGF22 knockout downregulated FGFR1/FGFR2 expression (p < 0.05), reduced HFSC proliferation capacity by 25% (p < 0.01), and increased HFSC apoptosis levels by 1.81-fold (p < 0.05). In addition, FGF22 overexpression promotes the proliferation and differentiation of HFSCs by activating Wnt/β-Catenin, Sonic Hedgehog (Shh) and Notch signaling pathways, or inhibiting BMP signaling pathways. Knockout of FGF22 weakens these processes and inhibits the activation and differentiation of HFSCs. This study, through the DPCs-HFSCs co-culture system, revealed the regulatory mechanism of FGF22 secreted by DPCs on the proliferation and differentiation of HFSCs, thereby providing theoretical references for fields such as fine-wool sheep breeding, human regenerative medicine, and hair loss treatment.

Dermal papilla cells (DPCs) serve as the signaling hub regulating hair follicle (HF) development and cyclical growth. This study aims to investigate the biological function and molecular mechanisms of TGFBR1 (transforming growth factor β receptor 1), a differentially expressed gene identified through single-cell transcriptomic sequencing (scRNA-seq) in the DPCs from fine-wool sheep. Primary DPCs were isolated and purified using a combination of enzymatic digestion and mechanical dissociation, followed by immunofluorescence identification (α-SMA and SOX2-positive). Following successful transfection with constructed TGFBR1 overexpression plasmids and siRNA interference vectors, cell proliferation was assessed via EDU staining and CCK-8 assays. mRNA expression of key genes in Wnt/β-catenin, BMP, and Notch signaling pathways (PCNA, CCND1, CTNNB1, SFRP2, BMP2, NOTCH3, SMAD4, etc.) was validated by RT-qPCR. Single-cell transcriptomics revealed significant downregulation of TGFBR1 in DPCs from fine-wool sheep. Functional validation demonstrated that TGFBR1 overexpression markedly suppressed DPC proliferation, whereas knockdown of TGFBR1 expression promoted DPC proliferation. Molecular mechanism studies showed that TGFBR1 overexpression significantly downregulated PCNA, CCND1, CTNNB1, NOTCH3, and SMAD4 while upregulating SFRP2, BMP2, and TGFB1 expression. These findings demonstrate that TGFBR1 acts as a negative regulator of DPCs proliferation by modulating the activity of multiple signaling pathways, including Wnt/β-catenin, BMP, and Notch, thereby suppressing the proliferative capacity of DPCs. This study not only provides new theoretical support for elucidating the role of the TGF-β signaling pathway in H development but also offers theoretical reference for in-depth research on molecular breeding in ultra -fine-wool sheep and the molecular mechanisms underlying HF development.

Various signalling molecules and pathways critical for wool production and quality regulate wool secondary follicle (SF) development. Circular RNAs (circRNAs) regulate SF morphogenesis through a competing endogenous RNA (ceRNA) mechanism; these novel cyclic non-coding RNAs are not known to regulate the development of SFs in the foetal period of fine-wool sheep. Here, we analysed circRNA expression profiles in the foetal skin of Gansu Alpine fine-wool sheep at 10 developmental stages (E87, E90, E93, E96, E99, E102, E105, E108, E111, and E138) using RNA sequencing. Among the differentially expressed circRNAs (DE circRNAs), 173 were significantly enriched in signalling pathways related to hair follicle (HF) development, such as Wnt/β-catenin, transforming growth factor-β/Smad, Notch, and mitogen-activated protein kinase. Six HF-development-related circRNAs were expressed at different stages and potentially regulated SF development through the ceRNA network. In total, 16 DE circRNAs and their targets, 44 miRNAs, and 65 mRNAs were screened, and 88 ceRNA regulatory network pathways related to SF development were constructed. These key DE circRNAs could be candidate genes for further exploration of the molecular HF development mechanism, providing an important theoretical basis for unravelling the regulatory network of SF development in fine-wool sheep and genetic wool trait improvement.

Wool fibre diameter is a key economic trait in wool-producing animals, and its formation is regulated by the synergistic interaction between the proliferation and differentiation of hair follicle stem cells (HFSCs) and signals from the microenvironment. Dermal papilla cells (DPCs), as a key signal source, regulate the fate of HFSCs via paracrine pathways; however, the key molecular mechanisms remain unclear. Single-cell RNA sequencing (scRNA-seq) and genome-wide association studies (GWAS) revealed that ZNF280B is specifically expressed in both DPCs and HFSCs, and its g.677G>A variant is significantly associated with wool fibre diameter. Functional studies indicate that overexpression of ZNF280B promotes the proliferation of DPCs and HFSCs and inhibits apoptosis, whilst knockdown has the opposite effect; mechanistically, ZNF280B alters mRNA levels of key marker genes of the Wnt/β-catenin signalling pathway while selectively changing transcripts of the TGF-β pathway components in DPCs, and differentially modulates key marker transcripts in both the Wnt/β-catenin and Notch pathways in HFSCs. In summary, ZNF280B mediates the paracrine signalling of DPCs to influence HFSC proliferation and differentiation. It achieves this by integrating the Wnt/β-catenin/TGF-β/Notch signalling axes. This reveals that ZNF280B acts as an important regulatory node mediating DPC-HFSC paracrine communication, and provides new insights into the developmental mechanisms of fine-wool hair follicles and molecular breeding for fibre diameter.