Comparative transcriptional profiling-based identification of raphanusanin-inducible genes

<p>Abstract</p> <p>Background</p> <p>Raphanusanin (Ra) is a light-induced growth inhibitor involved in the inhibition of hypocotyl growth in response to unilateral blue-light illumination in radish seedlings. Knowledge of the roles of Ra still remains elusive. To understand the roles of Ra and its functional coupling to light signalling, we constructed the Ra-induced gene library using the Suppression Subtractive Hybridisation (SSH) technique and present a comparative investigation of gene regulation in radish seedlings in response to short-term Ra and blue-light exposure.</p> <p>Results</p> <p>The predicted gene ontology (GO) term revealed that 55% of the clones in the Ra-induced gene library were associated with genes involved in common defence mechanisms, including thirty four genes homologous to <it>Arabidopsis </it>genes implicated in R-gene-triggered resistance in the programmed cell death (PCD) pathway. Overall, the library was enriched with transporters, hydrolases, protein kinases, and signal transducers. The transcriptome analysis revealed that, among the fifty genes from various functional categories selected from 88 independent genes of the Ra-induced library, 44 genes were up-regulated and 4 were down-regulated. The comparative analysis showed that, among the transcriptional profiles of 33 highly Ra-inducible genes, 25 ESTs were commonly regulated by different intensities and duration of blue-light irradiation. The transcriptional profiles, coupled with the transcriptional regulation of early blue light, have provided the functional roles of many genes expected to be involved in the light-mediated defence mechanism.</p> <p>Conclusions</p> <p>This study is the first comprehensive survey of transcriptional regulation in response to Ra. The results described herein suggest a link between Ra and cellular defence and light signalling, and thereby contribute to further our understanding of how Ra is involved in light-mediated mechanisms of plant defence.</p

Detection of revertant mosaicism in epidermolysis bullosa through Cas9-targeted long-read sequencing

Revertant mosaicism (RM) is a phenomenon in which inherited mutations are spontaneously corrected in somatic cells. RM occurs in some congenital skin diseases, but genetic validation of RM in clinically revertant skin has been challenging, especially when homologous recombination (HR) is responsible for RM. Here, we introduce nanopore Cas9-targeted sequencing (nCATS) for identifying HR in clinically revertant skin. We took advantage of compound heterozygous COL7A1 mutations in a patient with recessive dystrophic epidermolysis bullosa who showed revertant skin spots. Cas9-mediated enrichment of genomic DNA (gDNA) covering the two mutation sites (>8 kb) in COL7A1 and subsequent MinION sequencing successfully detected intragenic crossover in the epidermis of the clinically revertant skin. This method enables the discernment of haplotypes of up to a few tens of kilobases of gDNA. Moreover, it is devoid of polymerase chain reaction amplification, which can technically induce recombination. We, therefore, propose that nCATS is a powerful tool for understanding complicated gene modifications, including RM

Type XVII collagen interacts with the aPKC-PAR complex and maintains epidermal cell polarity

Type XVII collagen (COL17) is a transmembrane protein expressed in the basal epidermis. COL17 serves as a niche for epidermal stem cells, and although its reduction has been implicated in altering cell polarity and ageing of the epidermis, it is unknown how COL17 affects epidermal cell polarity. Here, we uncovered COL17 as a binding partner of the aPKC-PAR complex, which is a key regulating factor of cell polarity. Immunoprecipitation-immunoblot assay and protein-protein binding assay revealed that COL17 interacts with aPKC and PAR3. COL17 deficiency or epidermis-specific aPKC lambda deletion destabilized PAR3 distribution in the epidermis, while aPKC zeta knockout did not. Asymmetrical cell division was pronounced in COL17-null neonatal paw epidermis. These results show that COL17 is pivotal for maintaining epidermal cell polarity. Our study highlights the previously unrecognized role of COL17 in the basal keratinocytes

Wnt/beta-Catenin Signaling Stabilizes Hemidesmosomes in Keratinocytes

Hemidesmosomes (HDs) are adhesion complexes that promote epithelial-stromal attachment in stratified and complex epithelia, including the epidermis. In various biological processes, such as differentiation and migration of epidermal keratinocytes during wound healing or carcinoma invasion, quick assembly and disassembly of HDs are prerequisites. In this study, we show that inhibition of Wnt/0-catenin signaling disturbs HD organization in keratinocytes. Screening with inhibitors identified the depletion of HD components and HD-like structures through Wnt inhibition, but keratinocyte differentiation was not affected. Wnt inhibition significantly diminished plectin and type XVII collagen expression in the basal side of Wnt-inhibited cells and the dermo-epidermal junction of the Wnt-inactive murine basal epidermis. Similar to Wnt inhibition, PLEC- knockout cells or cells with plectin-type XVII collagen binding defects showed type XVII collagen reduction in the basal side of the cells, implying the possible involvement of Wnt/beta-catenin signaling in HD assembly. Atypical protein kinase C inhibition ameliorated the phenotypes of Wnt-inhibited cells. These findings show that Wnt/beta-catenin signaling regulates the localization of HD components in keratinocytes and that the atypical protein kinase C pathway is involved in Wnt inhibition-induced HD disarrangement. Our study suggests that the Wnt signaling pathway could be a potential therapeutic target for treating HD-defective diseases, such as epidermolysis bullosa

Collagen XVII deficiency alters epidermal patterning

Vertebrates exhibit patterned epidermis, exemplified by scales/interscales in mice tails and grooves/ridges on the human skin surface (microtopography). Although the role of spatiotemporal regulation of stem cells (SCs) has been implicated in this process, the mechanism underlying the development of such epidermal patterns is poorly understood. Here, we show that collagen XVII (COL17), a niche for epidermal SCs, helps stabilize epidermal patterns. Gene knockout and rescue experiments revealed that COL17 maintains the width of the murine tail scale epidermis independently of epidermal cell polarity. Skin regeneration after wounding was associated with slender scale epidermis, which was alleviated by overexpression of human COL17. COL17-negative skin in human junctional epidermolysis bullosa showed a distinct epidermal pattern from COL17-positive skin that resulted from revertant mosaicism. These results demonstrate that COL17 contributes to defining mouse tail scale shapes and human skin microtopography. Our study sheds light on the role of the SC niche in tissue pattern formation. Mammals exhibit epidermal patterning, as seen in mouse tail scales and human skin microtopography. In this article, the authors demonstrate that type XVII collagen (COL17), a niche for epidermal stem cells, modulate epidermal patterning in mice and humans. COL17 further prevents wound-induced epidermal deformation. This study paves the way for elucidating the role of the stem cell niche in tissue pattern formation

Hair follicle stem cell progeny heal blisters while pausing skin development

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that HF development can be sacrificed for the sake of epidermal wound regeneration. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases

A computational model of the epidermis with the deformable dermis and its application to skin diseases

The skin barrier is provided by the organized multi-layer structure of epidermal cells, which is dynamically maintained by a continuous supply of cells from the basal layer. The epidermal homeostasis can be disrupted by various skin diseases, which often cause morphological changes not only in the epidermis but in the dermis. We present a three-dimensional agent-based computational model of the epidermis that takes into account the deformability of the dermis. Our model can produce a stable epidermal structure with well-organized layers. We show that its stability depends on the cell supply rate from the basal layer. Modeling the morphological change of the dermis also enables us to investigate how the stiffness of the dermis affects the structure and barrier functions of the epidermis. Besides, we show that our model can simulate the formation of a corn (clavus) by assuming hyperproliferation and rapid differentiation. We also provide experimental data for human corn, which supports the model assumptions and the simulation result