Identification of the regulatory circuit governing corneal epithelial fate determination and disease.

The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human LSCs from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors (TFs) that define specific cell fates and established their regulatory hierarchy. Single-cell RNA-seq (scRNA-seq) analyses of the cornea and the epidermis confirmed these shared and cell type-specific TFs. Notably, the shared and LSC-specific TFs can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the LSC fate and its association with corneal opacity.

The DEAD-box RNA helicase Dhx15 controls glycolysis and arbovirus replication in Aedes aegypti mosquito cells.

Aedes aegypti mosquitoes are responsible for the transmission of arthropod-borne (arbo)viruses including dengue and chikungunya virus (CHIKV) but in contrast to human hosts, arbovirus-infected mosquitoes are able to efficiently control virus replication to sub-pathological levels. Yet, our knowledge of the molecular interactions of arboviruses with their mosquito hosts is incomplete. Here, we aimed to identify and characterize novel host genes that control arbovirus replication in Aedes mosquitoes. RNA binding proteins (RBPs) are well-known to regulate immune signaling pathways in all kingdoms of life. We therefore performed a knockdown screen targeting 461 genes encoding predicted RBPs in Aedes aegypti Aag2 cells and identified 15 genes with antiviral activity against Sindbis virus. Amongst these, the three DEAD-box RNA helicases AAEL004419/Dhx15, AAEL008728, and AAEL004859 also acted as antiviral factors in dengue and CHIKV infections. Here, we explored the mechanism of Dhx15 in regulating an antiviral transcriptional response in mosquitoes by silencing Dhx15 in Aag2 cells followed by deep-sequencing of poly-A enriched RNAs. Dhx15 knockdown in uninfected and CHIKV-infected cells resulted in differential expression of 856 and 372 genes, respectively. Interestingly, amongst the consistently downregulated genes, glycolytic process was the most enriched gene ontology (GO) term as the expression of all core enzymes of the glycolytic pathway was reduced, suggesting that Dhx15 regulates glycolytic function. A decrease in lactate production indicated that Dhx15 silencing indeed functionally impaired glycolysis. Modified rates of glycolytic metabolism have been implicated in controlling the replication of several classes of viruses and strikingly, infection of Aag2 cells with CHIKV by itself also resulted in the decrease of several glycolytic genes. Our data suggests that Dhx15 regulates replication of CHIKV, and possibly other arboviruses, by controlling glycolysis in mosquito cells.

Arbovirus-vector protein interactomics identifies Loquacious as a co-factor for dengue virus replication in Aedes mosquitoes.

Efficient virus replication in Aedes vector mosquitoes is essential for the transmission of arboviral diseases such as dengue virus (DENV) in human populations. Like in vertebrates, virus-host protein-protein interactions are essential for viral replication and immune evasion in the mosquito vector. Here, 79 mosquito host proteins interacting with DENV non-structural proteins NS1 and NS5 were identified by label-free mass spectrometry, followed by a functional screening. We confirmed interactions with host factors previously observed in mammals, such as the oligosaccharyltransferase complex, and we identified protein-protein interactions that seem to be specific for mosquitoes. Among the interactors, the double-stranded RNA (dsRNA) binding protein Loquacious (Loqs), an RNA interference (RNAi) cofactor, was found to be essential for efficient replication of DENV and Zika virus (ZIKV) in mosquito cells. Loqs did not affect viral RNA stability or translation of a DENV replicon and its proviral activity was independent of its RNAi regulatory activity. Interestingly, Loqs colocalized with DENV dsRNA replication intermediates in infected cells and directly interacted with high affinity with DENV RNA in the 3' untranslated region in vitro (KD = 48-62 nM). Our study provides an interactome for DENV NS1 and NS5 and identifies Loqs as a key proviral host factor in mosquitoes. We propose that DENV hijacks a factor of the RNAi mechanism for replication of its own RNA.

Single-cell RNA-seq identifies a reversible mesodermal activation in abnormally specified epithelia of p63 EEC syndrome.

Mutations in transcription factor p63 are associated with developmental disorders that manifest defects in stratified epithelia including the epidermis. The underlying cellular and molecular mechanism is however not yet understood. We established an epidermal commitment model using human induced pluripotent stem cells (iPSCs) and characterized differentiation defects of iPSCs derived from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients carrying p63 mutations. Transcriptome analyses revealed stepwise cell fate transitions during epidermal commitment: Specification from multipotent simple epithelium to basal stratified epithelia and ultimately to the mature epidermal fate. Differentiation defects of EEC iPSCs caused by p63 mutations occurred during the specification switch from the simple epithelium to the basal-stratified epithelial fate. Single-cell transcriptome and pseudotime analyses of cell states identified mesodermal activation that was associated with the deviated commitment route of EEC iPSCs. Integrated analyses of differentially regulated genes and p63-dependent dynamic genomic enhancers during epidermal commitment suggest that p63 directly controls epidermal gene activation at the specification switch and has an indirect effect on mesodermal gene repression. Importantly, inhibitors of mesodermal induction enhanced epidermal commitment of EEC iPSCs. Our findings demonstrate that p63 is required for specification of stratified epithelia, and that epidermal commitment defects caused by p63 mutations can be reversed by repressing mesodermal induction. This study provides insights into disease mechanisms underlying stratified epithelial defects caused by p63 mutations and suggests potential therapeutic strategies for the disease.

Terminal keratinocyte differentiation in vitro is associated with a stable DNA methylome.

The epidermal compartment of the skin is regenerated constantly by proliferation of epidermal keratinocytes. Differentiation of a subset of these keratinocytes allows the epidermis to retain its barrier properties. Regulation of keratinocyte fate-whether to remain proliferative or terminally differentiate-is complex and not fully understood. The objective of our study was to assess if DNA methylation changes contribute to the regulation of keratinocyte fate. We employed genome-wide MethylationEPIC beadchip array measuring approximately 850 000 probes combined with RNA sequencing of in vitro cultured non-differentiated and terminally differentiated adult human primary keratinocytes. We did not observe a correlation between methylation status and transcriptome changes. Moreover, only two differentially methylated probes were detected, of which one was located in the TRIM29 gene. Although TRIM29 knock-down resulted in lower expression levels of terminal differentiation genes, these changes were minor. From these results, we conclude that-in our in vitro experimental setup-it is unlikely that changes in DNA methylation have an important regulatory role in terminal keratinocyte differentiation.

An FEVR-associated mutation in ZNF408 alters the expression of genes involved in the development of vasculature.

Familial exudative vitreoretinopathy (FEVR) is an inherited retinal disorder hallmarked by an abnormal development of retinal vasculature. A missense mutation in ZNF408 (p.H455Y) was reported to underlie autosomal dominant FEVR in a large Dutch family, and ZNF408 was shown to play a role in the development of vasculature. Nonetheless, little is known about the molecular mechanism of ZNF408-associated FEVR. To investigate this, an in vitro model of ZNF408-associated FEVR was generated by overexpressing wild-type and p.H455Y ZNF408 in human umbilical vein endothelial cells. Cells overexpressing mutant ZNF408 were unable to form a capillary-like network in an in vitro tube formation assay, thereby mimicking the clinical feature observed in patients with FEVR. Intriguingly, transcriptome analysis revealed that genes involved in the development of vasculature were deregulated by the p.H455Y mutation. Chromatin immunoprecipitation showed that p.H455Y ZNF408 has reduced DNA-binding ability, as compared to the wild-type protein. The fact that the p.H455Y mutation disrupts the expression of genes important for the development of vasculature sheds further light on the molecular mechanisms underlying ZNF408-associated FEVR.

Modeling of Aniridia-Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein.

Heterozygous PAX6 gene mutations leading to haploinsufficiency are the main cause of congenital aniridia, a rare and progressive panocular disease characterized by reduced visual acuity. Up to 90% of patients suffer from aniridia-related keratopathy (ARK), caused by a combination of factors including limbal epithelial stem cell (LSC) deficiency, impaired healing response and abnormal differentiation of the corneal epithelium. It usually begins in the first decade of life, resulting in recurrent corneal erosions, sub-epithelial fibrosis, and corneal opacification. Unfortunately, there are currently no efficient treatments available for these patients and no in vitro model for this pathology. We used CRISPR/Cas9 technology to introduce into the PAX6 gene of LSCs a heterozygous nonsense mutation found in ARK patients. Nine clones carrying a p.E109X mutation on one allele were obtained with no off-target mutations. Compared with the parental LSCs, heterozygous mutant LSCs displayed reduced expression of PAX6 and marked slow-down of cell proliferation, migration and detachment. Moreover, addition to the culture medium of recombinant PAX6 protein fused to a cell penetrating peptide was able to activate the endogenous PAX6 gene and to rescue phenotypic defects of mutant LSCs, suggesting that administration of such recombinant PAX6 protein could be a promising therapeutic approach for aniridia-related keratopathy. More generally, our results demonstrate that introduction of disease mutations into LSCs by CRISPR/Cas9 genome editing allows the creation of relevant cellular models of ocular disease that should greatly facilitate screening of novel therapeutic approaches. Stem Cells 2018;36:1421-1429.

Computational Fluid Dynamics Study of Bifurcation Aneurysms Treated with Pipeline Embolization Device: Side Branch Diameter Study.

An intracranial aneurysm, abnormal swelling of the cerebral artery, may lead to undesirable rates of mortality and morbidity upon rupture. Endovascular treatment involves the deployment of a flow-diverting stent that covers the aneurysm orifice, thereby reducing the blood flow into the aneurysm and mitigating the risk of rupture. In this study, computational fluid dynamics analysis is performed on a bifurcation model to investigate the change in hemodynamics with various side branch diameters. The condition after the deployment of a pipeline embolization device is also simulated. Hemodynamic factors such as flow velocity, pressure, and wall shear stress are studied. Aneurysms with a larger side branch vessel might have greater risk after treatment in terms of hemodynamics. Although a stent could lead to flow reduction entering the aneurysm, it would drastically alter the flow rate inside the side branch vessel. This may result in side-branch hypoperfusion subsequent to stenting. In addition, two patient-specific bifurcation aneurysms are tested, and the results show good agreement with the idealized models. Furthermore, the peripheral resistance of downstream vessels is investigated by varying the outlet pressure conditions. This quantitative analysis can assist in treatment planning and therapeutic decision-making.

The Aryl Hydrocarbon Receptor Regulates Epidermal Differentiation through Transient Activation of TFAP2A.

The aryl hydrocarbon receptor (AHR) is an evolutionary conserved environmental sensor identified as an indispensable regulator of epithelial homeostasis and barrier organ function. Molecular signaling cascade and target genes upon AHR activation and their contribution to cell and tissue function are however not fully understood. Multiomics analyses using human skin keratinocytes revealed that upon ligand activation, AHR binds open chromatin to induce expression of transcription factors, for example, TFAP2A, as a swift response to environmental stimuli. The terminal differentiation program, including upregulation of barrier genes, FLG and keratins, was mediated by TFAP2A as a secondary response to AHR activation. The role of AHR-TFAP2A axis in controlling keratinocyte terminal differentiation for proper barrier formation was further confirmed using CRISPR/Cas9 in human epidermal equivalents. Overall, the study provides additional insights into the molecular mechanism behind AHR-mediated barrier function and identifies potential targets for the treatment of skin barrier diseases.

Chromatin profiling identifies transcriptional readthrough as a conserved mechanism for piRNA biogenesis in mosquitoes.

The piRNA pathway in mosquitoes differs substantially from other model organisms, with an expanded PIWI gene family and functions in antiviral defense. Here, we define core piRNA clusters as genomic loci that show ubiquitous piRNA expression in both somatic and germline tissues. These core piRNA clusters are enriched for non-retroviral endogenous viral elements (nrEVEs) in antisense orientation and depend on key biogenesis factors, Veneno, Tejas, Yb, and Shutdown. Combined transcriptome and chromatin state analyses identify transcriptional readthrough as a conserved mechanism for cluster-derived piRNA biogenesis in the vector mosquitoes Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles gambiae. Comparative analyses between the two Aedes species suggest that piRNA clusters function as traps for nrEVEs, allowing adaptation to environmental challenges such as virus infection. Our systematic transcriptome and chromatin state analyses lay the foundation for studies of gene regulation, genome evolution, and piRNA function in these important vector species.

The aryl hydrocarbon receptor regulates epidermal differentiation through transient activation of TFAP2A.

The aryl hydrocarbon receptor (AHR) is an evolutionary conserved environmental sensor identified as indispensable regulator of epithelial homeostasis and barrier organ function. Molecular signaling cascade and target genes upon AHR activation and their contribution to cell and tissue function are however not fully understood. Multi-omics analyses using human skin keratinocytes revealed that, upon ligand activation, AHR binds open chromatin to induce expression of transcription factors (TFs), e.g., Transcription Factor AP-2α (TFAP2A), as a swift response to environmental stimuli. The terminal differentiation program including upregulation of barrier genes, filaggrin and keratins, was mediated by TFAP2A as a secondary response to AHR activation. The role of AHR-TFAP2A axis in controlling keratinocyte terminal differentiation for proper barrier formation was further confirmed using CRISPR/Cas9 in human epidermal equivalents. Overall, the study provides novel insights into the molecular mechanism behind AHR-mediated barrier function and potential novel targets for the treatment of skin barrier diseases.

Fibroblast growth factor-2 bound to specific dermal fibroblast-derived extracellular vesicles is protected from degradation.

Fibroblast growth factor-2 (FGF2) has multiple roles in cutaneous wound healing but its natural low stability prevents the development of its use in skin repair therapies. Here we show that FGF2 binds the outer surface of dermal fibroblast (DF)-derived extracellular vesicles (EVs) and this association protects FGF2 from fast degradation. EVs isolated from DF cultured in the presence of FGF2 harbor FGF2 on their surface and FGF2 can bind purified EVs in absence of cells. Remarkably, FGF2 binding to EVs is restricted to a specific subpopulation of EVs, which do not express CD63 and CD81 markers. Treatment of DF with FGF2-EVs activated ERK and STAT signaling pathways and increased cell proliferation and migration. Local injection of FGF2-EVs improved wound healing in mice. We further demonstrated that binding to EVs protects FGF2 from both thermal and proteolytic degradation, thus maintaining FGF2 function. This suggests that EVs protect soluble factors from degradation and increase their stability and half-life. These results reveal a novel aspect of EV function and suggest EVs as a potential tool for delivering FGF2 in skin healing therapies.

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis.

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Distinct Pathogenic Genes Causing Intellectual Disability and Autism Exhibit a Common Neuronal Network Hyperactivity Phenotype.

Pathogenic mutations in either one of the epigenetic modifiers EHMT1, MBD5, MLL3, or SMARCB1 have been identified to be causative for Kleefstra syndrome spectrum (KSS), a neurodevelopmental disorder with clinical features of both intellectual disability (ID) and autism spectrum disorder (ASD). To understand how these variants lead to the phenotypic convergence in KSS, we employ a loss-of-function approach to assess neuronal network development at the molecular, single-cell, and network activity level. KSS-gene-deficient neuronal networks all develop into hyperactive networks with altered network organization and excitatory-inhibitory balance. Interestingly, even though transcriptional data reveal distinct regulatory mechanisms, KSS target genes share similar functions in regulating neuronal excitability and synaptic function, several of which are associated with ID and ASD. Our results show that KSS genes mainly converge at the level of neuronal network communication, providing insights into the pathophysiology of KSS and phenotypically congruent disorders.

p63 cooperates with CTCF to modulate chromatin architecture in skin keratinocytes.

The transcription factor p63 regulates epidermal genes and the enhancer landscape in skin keratinocytes. Its molecular function in controlling the chromatin structure is, however, not yet completely understood. Here, we integrated multi-omics profiles, including the transcriptome, transcription factor DNA-binding and chromatin accessibility, in skin keratinocytes isolated from EEC syndrome patients carrying p63 mutations, to examine the role of p63 in shaping the chromatin architecture. We found decreased chromatin accessibility in p63- and CTCF-bound open chromatin regions that potentially contributed to gene deregulation in mutant keratinocytes. Cooperation of p63 and CTCF seemed to assist chromatin interactions between p63-bound enhancers and gene promoters in skin keratinocytes. Our study suggests an intriguing model where cell type-specific transcription factors such as p63 cooperate with the genome organizer CTCF in the three-dimensional chromatin space to regulate the transcription program important for the proper cell identity.

Mutant p63 Affects Epidermal Cell Identity through Rewiring the Enhancer Landscape.

Transcription factor p63 is a key regulator of epidermal keratinocyte proliferation and differentiation. Mutations in the p63 DNA-binding domain are associated with ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome. However, the underlying molecular mechanism of these mutations remains unclear. Here, we characterized the transcriptome and epigenome of p63 mutant keratinocytes derived from EEC patients. The transcriptome of p63 mutant keratinocytes deviated from the normal epidermal cell identity. Epigenomic analyses showed an altered enhancer landscape in p63 mutant keratinocytes contributed by loss of p63-bound active enhancers and unexpected gain of enhancers. The gained enhancers were frequently bound by deregulated transcription factors such as RUNX1. Reversing RUNX1 overexpression partially rescued deregulated gene expression and the altered enhancer landscape. Our findings identify a disease mechanism whereby mutant p63 rewires the enhancer landscape and affects epidermal cell identity, consolidating the pivotal role of p63 in controlling the enhancer landscape of epidermal keratinocytes.

Complete mitochondrial genome of Costaria costata shows conservative evolution in Laminariales.

Costaria costata, with great commercial and industrial value, typically grows in low intertidal and subtidal retgions in East Asia. The complete mitochondrial genome of C. costata is determined as circular-mapping and AT-rich (65%). The 37,461 bp mitochondrial genome consists of 25 tRNAs, 38 genes (including ORFs) and 3 ribosome genes. The gene arrangement and component are identical to those of Laminaria mitochondrial genomes, which show highly conservative evolution in mitochondrial genomes within the Laminariales. Moreover, the C. costata mitogenome makes full use of nucleotide and genetic information by large amounts of gene overlappings for better adapting the evolution of small genomes.

Complete mitochondrial genome of Undaria pinnatifida (Alariaceae, Laminariales, Phaeophyceae).

Undaria pinnatifida is one of the most important economic marine algae and key components of coastal ecosystems. Undaria pinnatifida owns a typical heteromorphic, diplohaplontic life cycle. We present the complete sequence of mitochondrial genome of U. pinnatifida, focusing on genome organization and phylogenetic relationship between different brown algae lineages. The size of U. pinnatifida mitochondrial DNA is 37,402 bp, including 3 rRNAs, 25 tRNAs, 35 proteins, as well as 3 ORFs. No intron is found and most genes are encoded on the H-strand. The phylogenetic trees (BI) constructed on 35 protein-coding genes from 17 species proved that Saccharina has a closer relationship with Laminaria than that with Undaria. The results supported the conclusion that Alariaceae is sister genus to the Laminariaceae. Above researches will facilitate the understanding of evolutionary relationship within brown algae.

Discovery of a strongly-interrelated gene network in corals under constant darkness by correlation analysis after wavelet transform on complex network model.

Coral reefs occupy a relatively small portion of sea area, yet serve as a crucial source of biodiversity by establishing harmonious ecosystems with marine plants and animals. Previous researches mainly focused on screening several key genes induced by stress. Here we proposed a novel method--correlation analysis after wavelet transform of complex network model, to explore the effect of light on gene expression in the coral Acropora millepora based on microarray data. In this method, wavelet transform and the conception of complex network were adopted, and 50 key genes with large differences were finally captured, including both annotated genes and novel genes without accurate annotation. These results shed light on our understanding of coral's response toward light changes and the genome-wide interaction among genes under the control of biorhythm, and hence help us to better protect the coral reef ecosystems. Further studies are needed to explore how functional connections are related to structural connections, and how connectivity arises from the interactions within and between different systems. The method introduced in this study for analyzing microarray data will allow researchers to explore genome-wide interaction network with their own dataset and understand the relevant biological processes.