HDAC1/2 and HDAC3 play distinct roles in controlling adult Meibomian gland homeostasis.

PURPOSE: To investigate the roles of HDAC1/2 and HDAC3 in adult Meibomian gland (MG) homeostasis. METHODS: HDAC1/2 or HDAC3 were inducibly deleted in MG epithelial cells of adult mice. The morphology of MG was examined. Proliferation, apoptosis, and expression of MG acinus and duct marker genes, meibocyte differentiation genes, and HDAC target genes, were analyzed via immunofluorescence, TUNEL assay, and RNA in situ hybridization. RESULTS: Co-deletion of HDAC1/2 in MG epithelium caused gradual loss of acini and formation of cyst-like structures in the central duct. These phenotypes required homozygous deletion of both HDAC1 and HDAC2, indicating that they function redundantly in the adult MG. Short-term deletion of HDAC1/2 in MG epithelium had little effect on meibocyte maturation but caused decreased proliferation of acinar basal cells, excessive DNA damage, ectopic apoptosis, and increased p53 acetylation and p16 expression in the MG. By contrast, HDAC3 deletion in MG epithelium caused dilation of central duct, atrophy of acini, defective meibocyte maturation, increased acinar basal cell proliferation, and ectopic apoptosis and DNA damage. Levels of p53 acetylation and p21 expression were elevated in HDAC3-deficient MGs, while the expression of the differentiation regulator PPARγ and the differentiation markers PLIN2 and FASN was downregulated. CONCLUSIONS: HDAC1 and HDAC2 function redundantly in adult Meibomian gland epithelial progenitor cells and are essential for their proliferation and survival, but not for acinar differentiation, while HDAC3 is required to limit acinar progenitor cell proliferation and permit differentiation. HDAC1/2 and HDAC3 have partially overlapping roles in maintaining survival of MG cells.

The correlation between serum asprosin and left ventricular diastolic dysfunction in elderly patients with type 2 diabetes mellitus in the community.

AIMS/INTRODUCTION: Serum asprosin is expected to become a screening indicator in early-stage diabetic heart disease. The relationship between serum asprosin and left ventricular diastolic dysfunction (LVDD) was studied in elderly patients with type 2 diabetes mellitus in the community. MATERIALS AND METHODS: A total of 252 elderly patients with type 2 diabetes mellitus were recruited from Zhuoma Community Care Station and Chengbei West Street Community Care Service Center in Changzhi City of Shanxi Province from November 2019 to July 2021. Patients were divided into the LVDD group (n = 195) and the non-LVDD group (n = 57). The t-test, Mann-Whitney U test, and χ2 test were used to compare indicators between the LVDD group and the non-LVDD group. Pearson or Spearman correlation analysis was adopted to evaluate the correlation between serum asprosin and other clinical data. Multivariate logistic regression analysis was applied to analyze the influencing factors on LVDD. RESULTS: Compared with patients without LVDD, patients with LVDD had a higher level of low-density lipoprotein cholesterol (LDL-C), fasting blood glucose (FPG), and asprosin, but a lower level of early diastolic movement speed (A) to diastolic movement velocity (E) (E/A). Asprosin was positively associated with waist circumference (WC), body mass index (BMI), creatinine, triglycerides (P < 0.05), and negatively associated with E/A and high density lipoprotein cholesterol HDL-C (P < 0.05). The risk of LVDD increased with elevated asprosin levels after adjustment for age, systolic blood pressure (SBP), BMI, FPG, and LDL-C. Compared with patients in the lowest tertile of serum asprosin (<275.25 pg/mL), a serum level of asprosin between 275.25-355.08 pg/mL [OR (95% CI) is 2.368 (1.169-4.796), P < 0.05] and asprosin >355.08 pg/mL [OR (95% CI) is 2.549 (1.275-5.095), P < 0.05] patients have a higher risk of left ventricular diastolic dysfunction. CONCLUSIONS: Serum asprosin was positively associated with left ventricular diastolic dysfunction, and the risk of LVDD increased significantly with increased serum levels of asprosin.

FZD2 regulates limb development by mediating ß-catenin-dependent and -independent Wnt signaling pathways.

Human Robinow syndrome (RS) and dominant omodysplasia type 2 (OMOD2), characterized by skeletal limb and craniofacial defects, are associated with heterozygous mutations in the Wnt receptor FZD2. However, as FZD2 can activate both canonical and non-canonical Wnt pathways, its precise functions and mechanisms of action in limb development are unclear. To address these questions, we generated mice harboring a single-nucleotide insertion in Fzd2 (Fzd2em1Smill), causing a frameshift mutation in the final Dishevelled-interacting domain. Fzd2em1Smill mutant mice had shortened limbs, resembling those of RS and OMOD2 patients, indicating that FZD2 mutations are causative. Fzd2em1Smill mutant embryos displayed decreased canonical Wnt signaling in developing limb mesenchyme and disruption of digit chondrocyte elongation and orientation, which is controlled by the ß-catenin-independent WNT5A/planar cell polarity (PCP) pathway. In line with these observations, we found that disruption of FZD function in limb mesenchyme caused formation of shortened bone elements and defects in Wnt/ß-catenin and WNT5A/PCP signaling. These findings indicate that FZD2 controls limb development by mediating both canonical and non-canonical Wnt pathways and reveal causality of pathogenic FZD2 mutations in RS and OMOD2 patients.

Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection.

Angiotensin-converting enzyme 2 (ACE2) is the cell-surface receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). While its central role in Coronavirus Disease 2019 (COVID-19) pathogenesis is indisputable, there remains significant debate regarding the role of this transmembrane carboxypeptidase in the disease course. These include the role of soluble versus membrane-bound ACE2, as well as ACE2-independent mechanisms that may contribute to viral spread. Testing these roles requires in vivo models. Here, we report humanized ACE2-floxed mice in which hACE2 is expressed from the mouse Ace2 locus in a manner that confers lethal disease and permits cell-specific, Cre-mediated loss of function, and LSL-hACE2 mice in which hACE2 is expressed from the Rosa26 locus enabling cell-specific, Cre-mediated gain of function. Following exposure to SARS-CoV-2, hACE2-floxed mice experienced lethal cachexia, pulmonary infiltrates, intravascular thrombosis and hypoxemia-hallmarks of severe COVID-19. Cre-mediated loss and gain of hACE2 demonstrate that neuronal infection confers lethal cachexia, hypoxemia, and respiratory failure in the absence of lung epithelial infection. In this series of genetic experiments, we demonstrate that ACE2 is absolutely and cell-autonomously required for SARS-CoV-2 infection in the olfactory epithelium, brain, and lung across diverse cell types. Therapies inhibiting or blocking ACE2 at these different sites are likely to be an effective strategy towards preventing severe COVID-19.

SARS-CoV-2 infection of olfactory epithelial cells and neurons drives acute lung injury and lethal COVID-19 in mice.

Lethal COVID-19 is associated with respiratory failure that is thought to be caused by acute respiratory distress syndrome (ARDS) secondary to pulmonary infection. To date, the cellular pathogenesis has been inferred from studies describing the expression of ACE2, a transmembrane protein required for SARS-CoV-2 infection, and detection of viral RNA or protein in infected humans, model animals, and cultured cells. To functionally test the cellular mechanisms of COVID-19, we generated hACE2 fl animals in which human ACE2 (hACE2) is expressed from the mouse Ace2 locus in a manner that permits cell-specific, Cre-mediated loss of function. hACE2 fl animals developed lethal weight loss and hypoxemia within 7 days of exposure to SARS-CoV-2 that was associated with pulmonary infiltrates, intravascular thrombosis and patchy viral infection of lung epithelial cells. Deletion of hACE2 in lung epithelial cells prevented viral infection of the lung, but not weight loss, hypoxemia or death. Inhalation of SARS-CoV-2 by hACE2 fl animals resulted in early infection of sustentacular cells with subsequent infection of neurons in the neighboring olfactory bulb and cerebral cortexâ€" events that did not require lung epithelial cell infection. Pharmacologic ablation of the olfactory epithelium or Foxg1 Cre mediated deletion of hACE2 in olfactory epithelial cells and neurons prevented lethality and neuronal infection following SARS-CoV-2 infection. Conversely, transgenic expression of hACE2 specifically in olfactory epithelial cells and neurons in Foxg1 Cre ; LSL- hACE2 mice was sufficient to confer neuronal infection associated with respiratory failure and death. These studies establish mouse loss and gain of function genetic models with which to genetically dissect viral-host interactions and demonstrate that lethal disease due to respiratory failure may arise from extrapulmonary infection of the olfactory epithelium and brain. Future therapeutic efforts focused on preventing olfactory epithelial infection may be an effective means of protecting against severe COVID-19.

Author Correction: MiR-31 promotes mammary stem cell expansion and breast tumorigenesis by suppressing Wnt signaling antagonists.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

How a Bird Gets Its Feathers: Insights from Chromatin Looping.

Mechanisms controlling skin heterogeneity are poorly understood. In this issue of Developmental Cell, Liang et al. show that in chicken, the difference in ß-keratin genes expressed in feathered and scaly skin is regulated via typical enhancers, while differential expression within individual feathers correlates with chromatin looping within the gene cluster.

HDAC3 ensures stepwise epidermal stratification via NCoR/SMRT-reliant mechanisms independent of its histone deacetylase activity.

Chromatin modifiers play critical roles in epidermal development, but the functions of histone deacetylases in this context are poorly understood. The class I HDAC, HDAC3, is of particular interest because it plays divergent roles in different tissues by partnering with tissue-specific transcription factors. We found that HDAC3 is expressed broadly in embryonic epidermis and is required for its orderly stepwise stratification. HDAC3 protein stability in vivo relies on NCoR and SMRT, which function redundantly in epidermal development. However, point mutations in the NCoR and SMRT deacetylase-activating domains, which are required for HDAC3's enzymatic function, permit normal stratification, indicating that HDAC3's roles in this context are largely independent of its histone deacetylase activity. HDAC3-bound sites are significantly enriched for predicted binding motifs for critical epidermal transcription factors including AP1, GRHL, and KLF family members. Our results suggest that among these, HDAC3 operates in conjunction with KLF4 to repress inappropriate expression of Tgm1, Krt16, and Aqp3 In parallel, HDAC3 suppresses expression of inflammatory cytokines through a Rela-dependent mechanism. These data identify HDAC3 as a hub coordinating multiple aspects of epidermal barrier acquisition.

Regional Control of Hairless versus Hair-Bearing Skin by Dkk2.

Haired skin is a defining characteristic of mammals. However, some specialized skin regions, such as human palms, soles and ventral wrist, and mouse plantar foot, are entirely hairless. Using mouse plantar skin as a model system, we show that the endogenous secreted Wnt inhibitor DKK2 suppresses plantar hair follicle development and permits the formation of hairless skin. Plantar skin retains all of the mechanistic components needed for hair follicle development, as genetic deletion of Dkk2 permits formation of fully functional plantar hair follicles that give rise to external hair, contain sebaceous glands and a stem cell compartment, and undergo regenerative growth. In the absence of Dkk2, Wnt/ß-catenin signaling activity is initially broadly elevated in embryonic plantar skin and gradually becomes patterned, mimicking follicular development in normally haired areas. These data provide a paradigm in which regionally restricted expression of a Wnt inhibitor underlies specification of hairless versus hairy skin.

MiR-31 promotes mammary stem cell expansion and breast tumorigenesis by suppressing Wnt signaling antagonists.

MicroRNA-mediated post-transcriptional regulation plays key roles in stem cell self-renewal and tumorigenesis. However, the in vivo functions of specific microRNAs in controlling mammary stem cell (MaSC) activity and breast cancer formation remain poorly understood. Here we show that miR-31 is highly expressed in MaSC-enriched mammary basal cell population and in mammary tumors, and is regulated by NF-κB signaling. We demonstrate that miR-31 promotes mammary epithelial proliferation and MaSC expansion at the expense of differentiation in vivo. Loss of miR-31 compromises mammary tumor growth, reduces the number of cancer stem cells, as well as decreases tumor-initiating ability and metastasis to the lung, supporting its pro-oncogenic function. MiR-31 modulates multiple signaling pathways, including Prlr/Stat5, TGFß and Wnt/ß-catenin. Particularly, it activates Wnt/ß-catenin signaling by directly targeting Wnt antagonists, including Dkk1. Importantly, Dkk1 overexpression partially rescues miR31-induced mammary defects. Together, these findings identify miR-31 as the key regulator of MaSC activity and breast tumorigenesis.

Cytoplasmic chromatin triggers inflammation in senescence and cancer.

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

ß-catenin is required for taste bud cell renewal and behavioral taste perception in adult mice.

Taste stimuli are transduced by taste buds and transmitted to the brain via afferent gustatory fibers. Renewal of taste receptor cells from actively dividing progenitors is finely tuned to maintain taste sensitivity throughout life. We show that conditional ß-catenin deletion in mouse taste progenitors leads to rapid depletion of progenitors and Shh+ precursors, which in turn causes taste bud loss, followed by loss of gustatory nerve fibers. In addition, our data suggest LEF1, TCF7 and Wnt3 are involved in a Wnt pathway regulatory feedback loop that controls taste cell renewal in the circumvallate papilla epithelium. Unexpectedly, taste bud decline is greater in the anterior tongue and palate than in the posterior tongue. Mutant mice with this regional pattern of taste bud loss were unable to discern sweet at any concentration, but could distinguish bitter stimuli, albeit with reduced sensitivity. Our findings are consistent with published reports wherein anterior taste buds have higher sweet sensitivity while posterior taste buds are better tuned to bitter, and suggest ß-catenin plays a greater role in renewal of anterior versus posterior taste buds.

WNT10A mutation causes ectodermal dysplasia by impairing progenitor cell proliferation and KLF4-mediated differentiation.

Human WNT10A mutations are associated with developmental tooth abnormalities and adolescent onset of a broad range of ectodermal defects. Here we show that ß-catenin pathway activity and adult epithelial progenitor proliferation are reduced in the absence of WNT10A, and identify Wnt-active self-renewing stem cells in affected tissues including hair follicles, sebaceous glands, taste buds, nails and sweat ducts. Human and mouse WNT10A mutant palmoplantar and tongue epithelia also display specific differentiation defects that are mimicked by loss of the transcription factor KLF4. We find that ß-catenin interacts directly with region-specific LEF/TCF factors, and with KLF4 in differentiating, but not proliferating, cells to promote expression of specialized keratins required for normal tissue structure and integrity. Our data identify WNT10A as a critical ligand controlling adult epithelial proliferation and region-specific differentiation, and suggest downstream ß-catenin pathway activation as a potential approach to ameliorate regenerative defects in WNT10A patients.

Activation of ß-Catenin Signaling in CD133-Positive Dermal Papilla Cells Drives Postnatal Hair Growth.

The hair follicle dermal papilla (DP) contains a unique prominin-1/CD133-positive (CD133+) cell subpopulation, which has been shown to possess hair follicle-inducing capability. By assaying for endogenous CD133 expression and performing lineage tracing using CD133-CreERT2; ZsGreen1 reporter mice, we find that CD133 is expressed in a subpopulation of DP cells during the growth phase of the murine hair cycle (anagen), but is absent at anagen onset. However, how CD133+ DP cells interact with keratinocytes to induce hair regenerative growth remains unclear. Wnt/ß-catenin has long been recognized as a major signaling pathway required for hair follicle morphogenesis, development, and regeneration. Nuclear Wnt/ß-catenin activity is observed in the DP during the hair growth phase. Here we show that induced expression of a stabilized form of ß-catenin in CD133+ DP cells significantly accelerates spontaneous and depilation-induced hair growth. However, hair follicle regression is not affected in these mutants. Further analysis indicates that CD133+ DP-expressed ß-catenin increases proliferation and differentiation of epithelial matrix keratinocytes. Upregulated Wnt/ß-catenin activity in CD133+ DP cells also increases the number of proliferating DP cells in each anagen follicle. Our data demonstrate that ß-catenin signaling potentiates the capability of CD133+ DP cells to promote postnatal hair growth.

Activating ß-catenin signaling in CD133-positive dermal papilla cells increases hair inductivity.

Bioengineering hair follicles using cells isolated from human tissue remains a difficult task. Dermal papilla (DP) cells are known to guide the growth and cycling activities of hair follicles by interacting with keratinocytes. However, DP cells quickly lose their inductivity during in vitro passaging. Rodent DP cell cultures need external addition of growth factors, including WNT and BMP molecules, to maintain the hair inductive property. CD133 is expressed by a subpopulation of DP cells that are capable of inducing hair follicle formation in vivo. We report here that expression of a stabilized form of ß-catenin promoted clonal growth of CD133-positive (CD133+) DP cells in in vitro three-dimensional hydrogel culture while maintaining expression of DP markers, including alkaline phosphatase (AP), CD133, and integrin α8. After a 2-week in vitro culture, cultured CD133+ DP cells with up-regulated ß-catenin activity led to an accelerated in vivo hair growth in reconstituted skin compared to control cells. Further analysis showed that matrix cell proliferation and differentiation were significantly promoted in hair follicles when ß-catenin signaling was up-regulated in CD133+ DP cells. Our data highlight an important role for ß-catenin signaling in promoting the inductive capability of CD133+ DP cells for in vitro expansion and in vivo hair follicle regeneration, which could potentially be applied to cultured human DP cells.

Beta-catenin is essential for ameloblast movement during enamel development.

Beta-catenin is a multifunctional protein that plays key roles in cadherin-based cell adherens junctions and in the Wnt signaling pathway. The canonical Wnt/ß-catenin pathway can regulate transcription factors that control cell movement/invasion. We investigated whether ß-catenin regulates ameloblast movement through canonical Wnt signaling. The morphological and physical properties of enamel were assessed in enamel from control and ß-catenin conditional knockout (cKO) mice. Ameloblast-lineage cells (ALC) were used to investigate the potential roles of ß-catenin in cell migration and in E-cadherin expression. Compared with controls, incisors from ß-catenin cKO mice were short, blunt, and where enamel was present, it was soft and malformed. Scanning electron microscopy revealed a dysplastic rod pattern within the enamel of incisors from ß-catenin cKO mice, and Vickers microhardness measurements confirmed that mice with ß-catenin ablated from their enamel organ had enamel that was significantly softer than normal. Amelogenesis was disrupted in the absence of ß-catenin and the ameloblasts did not differentiate properly. We further demonstrated that migration of ALCs was inhibited in vitro and that E-cadherin expression was significantly up-regulated when ALCs were treated with the ß-catenin inhibitor, ICG-001. Beta-catenin ablation causes enamel malformation in mice and this phenotype may occur, in part, by a lack of ameloblast differentiation and/or movement necessary to form the decussating enamel rod structure.

ß-Catenin Signaling Biases Multipotent Lingual Epithelial Progenitors to Differentiate and Acquire Specific Taste Cell Fates.

Continuous taste bud cell renewal is essential to maintain taste function in adults; however, the molecular mechanisms that regulate taste cell turnover are unknown. Using inducible Cre-lox technology, we show that activation of ß-catenin signaling in multipotent lingual epithelial progenitors outside of taste buds diverts daughter cells from a general epithelial to a taste bud fate. Moreover, while taste buds comprise 3 morphological types, ß-catenin activation drives overproduction of primarily glial-like Type I taste cells in both anterior fungiform (FF) and posterior circumvallate (CV) taste buds, with a small increase in Type II receptor cells for sweet, bitter and umami, but does not alter Type III sour detector cells. Beta-catenin activation in post-mitotic taste bud precursors likewise regulates cell differentiation; forced activation of ß-catenin in these Shh+ cells promotes Type I cell fate in both FF and CV taste buds, but likely does so non-cell autonomously. Our data are consistent with a model where ß-catenin signaling levels within lingual epithelial progenitors dictate cell fate prior to or during entry of new cells into taste buds; high signaling induces Type I cells, intermediate levels drive Type II cell differentiation, while low levels may drive differentiation of Type III cells.

Distinct functions for Wnt/ß-catenin in hair follicle stem cell proliferation and survival and interfollicular epidermal homeostasis.

Wnt/ß-catenin signaling is a central regulator of adult stem cells. Variable sensitivity of Wnt reporter transgenes, ß-catenin's dual roles in adhesion and signaling, and hair follicle degradation and inflammation resulting from broad deletion of epithelial ß-catenin have precluded clear understanding of Wnt/ß-catenin's functions in adult skin stem cells. By inducibly deleting ß-catenin globally in skin epithelia, only in hair follicle stem cells, or only in interfollicular epidermis and comparing the phenotypes with those caused by ectopic expression of the Wnt/ß-catenin inhibitor Dkk1, we show that this pathway is necessary for hair follicle stem cell proliferation. However, ß-catenin is not required within hair follicle stem cells for their maintenance, and follicles resume proliferating after ectopic Dkk1 has been removed, indicating persistence of functional progenitors. We further unexpectedly discovered a broader role for Wnt/ß-catenin signaling in contributing to progenitor cell proliferation in nonhairy epithelia and interfollicular epidermis under homeostatic, but not inflammatory, conditions.

Polypyrimidine tract-binding protein 2 binds to selective, intronic messenger RNA and microRNA targets in the mouse testis.

Here we use an in vivo cross-linking and immunoprecipitation procedure to detect RNA targets of the multifunctional RNA-binding protein polypyrimidine tract-binding protein (PTBP) 2 in mouse testis. Eleven known mRNAs, including Ptbp2 mRNA, 28 RNAs matching intron sequences, and 12 small RNAs and repeat sequences are identified. The specificity of interaction between PTBP2 and its target RNAs was confirmed using RNA interference with mouse N2A cells. Reduction of PTBP2 levels led to decreases in 7 of 10 of the mRNAs, to the repression of alternative splicing of introns, and to reductions in specific miRNAs.