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.

Wnt/ß-catenin signaling controls mouse eyelid growth by mediating epithelial-mesenchymal interactions.

PURPOSE: To investigate the role of Wnt/ß-catenin signaling in mouse eyelid development. METHODS: Wnt/ß-catenin signaling was disrupted by deleting supraorbital mesenchymal ß-catenin or epithelial Wls. p63 was removed to determine whether the expression of Wnts is affected. The eyelid morphology was examined at different stages. Proliferation, apoptosis, and expression of Wnt ligands and their target genes were analyzed via immunofluorescence staining, TUNEL assay, and in situ hybridization. RESULTS: Deletion of ß-catenin in supraorbital mesenchyme abolishes eyelid growth by causing decreased proliferation in supraorbital epithelium and underlying mesenchyme. Inhibition of Wnt secretion by deleting Wls in supraorbital epithelium results in failure of eyelid development, similar to the effects of deleting mesenchymal ß-catenin. Knockout of p63 results in formation of hypoplastic eyelids and reduced expression of several Wnt ligands in eyelid epithelium. CONCLUSIONS: Epithelial Wnt ligands activate mesenchymal Wnt/ß-catenin signaling to control eyelid growth and their expression is partially regulated by p63.

Dedifferentiation maintains melanocyte stem cells in a dynamic niche.

For unknow reasons, the melanocyte stem cell (McSC) system fails earlier than other adult stem cell populations1, which leads to hair greying in most humans and mice2,3. Current dogma states that McSCs are reserved in an undifferentiated state in the hair follicle niche, physically segregated from differentiated progeny that migrate away following cues of regenerative stimuli4-8. Here we show that most McSCs toggle between transit-amplifying and stem cell states for both self-renewal and generation of mature progeny, a mechanism fundamentally distinct from those of other self-renewing systems. Live imaging and single-cell RNA sequencing revealed that McSCs are mobile, translocating between hair follicle stem cell and transit-amplifying compartments where they reversibly enter distinct differentiation states governed by local microenvironmental cues (for example, WNT). Long-term lineage tracing demonstrated that the McSC system is maintained by reverted McSCs rather than by reserved stem cells inherently exempt from reversible changes. During ageing, there is accumulation of stranded McSCs that do not contribute to the regeneration of melanocyte progeny. These results identify a new model whereby dedifferentiation is integral to homeostatic stem cell maintenance and suggest that modulating McSC mobility may represent a new approach for the prevention of hair greying.

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.

Verification of Bound Aminoguanidine as the Marker Residue for the Banned Antibiotic, Nitrovin, in Pig Tissues.

Nitrovin (NTV) belongs to a class of antibiotics called nitrofurans, which are classified as nonallowed pharmacologically active substances that do not have a maximum residue limit listed in EU legislation. The objectives of this study were to confirm aminoguanidine (AGN) as a suitable marker residue to monitor NTV abuse and to investigate its persistence in porcine tissues. In this work, pigs were fed with NTV-medicated feed (50 mg/kg), and tissues (kidney, muscle, and liver) and plasma were collected on different withdrawal days. All samples were analyzed for bound AGN, total AGN, and the parent drug NTV itself. The highest concentrations of AGN residues were found in the liver, while the lowest were in muscle. Parent NTV was only detected in the kidney at low levels on day 0 of withdrawal. The findings are in support of using AGN as the marker residue for monitoring the illegal use of NTV in animal-derived products.

NF-κB perturbation reveals unique immunomodulatory functions in Prx1+ fibroblasts that promote development of atopic dermatitis.

Skin is composed of diverse cell populations that cooperatively maintain homeostasis. Up-regulation of the nuclear factor κB (NF-κB) pathway may lead to the development of chronic inflammatory disorders of the skin, but its role during the early events remains unclear. Through analysis of single-cell RNA sequencing data via iterative random forest leave one out prediction, an explainable artificial intelligence method, we identified an immunoregulatory role for a unique paired related homeobox-1 (Prx1)+ fibroblast subpopulation. Disruption of Ikkb-NF-κB under homeostatic conditions in these fibroblasts paradoxically induced skin inflammation due to the overexpression of C-C motif chemokine ligand 11 (CCL11; or eotaxin-1) characterized by eosinophil infiltration and a subsequent TH2 immune response. Because the inflammatory phenotype resembled that seen in human atopic dermatitis (AD), we examined human AD skin samples and found that human AD fibroblasts also overexpressed CCL11 and that perturbation of Ikkb-NF-κB in primary human dermal fibroblasts up-regulated CCL11. Monoclonal antibody treatment against CCL11 was effective in reducing the eosinophilia and TH2 inflammation in a mouse model. Together, the murine model and human AD specimens point to dysregulated Prx1+ fibroblasts as a previously unrecognized etiologic factor that may contribute to the pathogenesis of AD and suggest that targeting CCL11 may be a way to treat AD-like skin lesions.

HDAC1/2 Control Proliferation and Survival in Adult Epidermis and Pre‒Basal Cell Carcinoma through p16 and p53.

HDAC inhibitors show therapeutic promise for skin malignancies; however, the roles of specific HDACs in adult epidermal homeostasis and in disease are poorly understood. We find that homozygous epidermal codeletion of Hdac1 and Hdac2 in adult mouse epidermis causes reduced basal cell proliferation, apoptosis, inappropriate differentiation, and eventual loss of Hdac1/2-null keratinocytes. Hdac1/2-deficient epidermis displays elevated acetylated p53 and increased expression of the senescence gene p16. Loss of p53 partially restores basal proliferation, whereas p16 deletion promotes long-term survival of Hdac1/2-null keratinocytes. In activated GLI2-driven pre-basal cell carcinoma, Hdac1/2 deletion dramatically reduces proliferation and increases apoptosis, and knockout of either p53 or p16 partially rescues both proliferation and basal cell viability. Topical application of the HDAC inhibitor romidepsin to the normal epidermis or to GLI2ΔN-driven lesions produces similar defects to those caused by genetic Hdac1/2 deletion, and these are partially rescued by loss of p16. These data reveal essential roles for HDAC1/2 in maintaining proliferation and survival of adult epidermal and basal cell carcinoma progenitors and suggest that the efficacy of therapeutic HDAC1/2 inhibition will depend in part on the mutational status of p53 and p16.

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.

20 years of Developmental Cell: Looking back.

In our 20th anniversary year, we reflect on how the cell and developmental biology fields have changed since the publication of Developmental Cell's first few issues. In this collection of Voices, authors who published in our early issues discuss the advances that helped shape their field over the past two decades.

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.

FAM20B-catalyzed glycosaminoglycans control murine tooth number by restricting FGFR2b signaling.

BACKGROUND: The formation of supernumerary teeth is an excellent model for studying the molecular mechanisms that control stem/progenitor cell homeostasis needed to generate a renewable source of replacement cells and tissues. Although multiple growth factors and transcriptional factors have been associated with supernumerary tooth formation, the regulatory inputs of extracellular matrix in this regenerative process remains poorly understood. RESULTS: In this study, we present evidence that disrupting glycosaminoglycans (GAGs) in the dental epithelium of mice by inactivating FAM20B, a xylose kinase essential for GAG assembly, leads to supernumerary tooth formation in a pattern reminiscent of replacement teeth. The dental epithelial GAGs confine murine tooth number by restricting the homeostasis of Sox2(+) dental epithelial stem/progenitor cells in a non-autonomous manner. FAM20B-catalyzed GAGs regulate the cell fate of dental lamina by restricting FGFR2b signaling at the initial stage of tooth development to maintain a subtle balance between the renewal and differentiation of Sox2(+) cells. At the later cap stage, WNT signaling functions as a relay cue to facilitate the supernumerary tooth formation. CONCLUSIONS: The novel mechanism we have characterized through which GAGs control the tooth number in mice may also be more broadly relevant for potentiating signaling interactions in other tissues during development and tissue homeostasis.

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.

Identifying Treatments for Taste and Smell Disorders: Gaps and Opportunities.

The chemical senses of taste and smell play a vital role in conveying information about ourselves and our environment. Tastes and smells can warn against danger and also contribute to the daily enjoyment of food, friends and family, and our surroundings. Over 12% of the US population is estimated to experience taste and smell (chemosensory) dysfunction. Yet, despite this high prevalence, long-term, effective treatments for these disorders have been largely elusive. Clinical successes in other sensory systems, including hearing and vision, have led to new hope for developments in the treatment of chemosensory disorders. To accelerate cures, we convened the "Identifying Treatments for Taste and Smell Disorders" conference, bringing together basic and translational sensory scientists, health care professionals, and patients to identify gaps in our current understanding of chemosensory dysfunction and next steps in a broad-based research strategy. Their suggestions for high-yield next steps were focused in 3 areas: increasing awareness and research capacity (e.g., patient advocacy), developing and enhancing clinical measures of taste and smell, and supporting new avenues of research into cellular and therapeutic approaches (e.g., developing human chemosensory cell lines, stem cells, and gene therapy approaches). These long-term strategies led to specific suggestions for immediate research priorities that focus on expanding our understanding of specific responses of chemosensory cells and developing valuable assays to identify and document cell development, regeneration, and function. Addressing these high-priority areas should accelerate the development of novel and effective treatments for taste and smell disorders.

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.

Fractionated head and neck irradiation impacts taste progenitors, differentiated taste cells, and Wnt/ß-catenin signaling in adult mice.

Head and neck cancer patients receiving conventional repeated, low dose radiotherapy (fractionated IR) suffer from taste dysfunction that can persist for months and often years after treatment. To understand the mechanisms underlying functional taste loss, we established a fractionated IR mouse model to characterize how taste buds are affected. Following fractionated IR, we found as in our previous study using single dose IR, taste progenitor proliferation was reduced and progenitor cell number declined, leading to interruption in the supply of new taste receptor cells to taste buds. However, in contrast to a single dose of IR, we did not encounter increased progenitor cell death in response to fractionated IR. Instead, fractionated IR induced death of cells within taste buds. Overall, taste buds were smaller and fewer following fractionated IR, and contained fewer differentiated cells. In response to fractionated IR, expression of Wnt pathway genes, Ctnnb1, Tcf7, Lef1 and Lgr5 were reduced concomitantly with reduced progenitor proliferation. However, recovery of Wnt signaling post-IR lagged behind proliferative recovery. Overall, our data suggest carefully timed, local activation of Wnt/ß-catenin signaling may mitigate radiation injury and/or speed recovery of taste cell renewal following fractionated IR.

Overexpression of Desmoglein 2 in a Mouse Model of Gorlin Syndrome Enhances Spontaneous Basal Cell Carcinoma Formation through STAT3-Mediated Gli1 Expression.

Activation of the hedgehog pathway is causative of virtually all sporadic and Gorlin syndrome-related basal cell carcinomas (BCCs), with loss of function of Ptc1 being the most common genomic lesion. Sporadic BCCs also overexpress Dsg2, a desmosomal cadherin normally found in the basal layer. Using a mouse model of Gorlin syndrome (Ptc1+/lacZ mice), we found that overexpressing Dsg2 in the basal layer (K14-Dsg2/Ptc1+/lacZ mice) or the superficial epidermis (Inv-Dsg2/Ptc1+/lacZ mice) resulted in increased spontaneous BCC formation at 3 and 6 months, respectively. The tumors did not show loss of heterozygosity of Ptc1, despite high levels of Gli1 and phosphorylated Stat3. A panel of sporadic human BCCs showed increased staining of both Dsg2 and phosphorylated Stat3 in all nine samples. Overexpression of Dsg2 in ASZ001 cells, a Ptc1-/- BCC cell line, induced Stat3 phosphorylation and further increased Gli1 levels, in both an autocrine and paracrine manner. Three different Stat3 inhibitors reduced viability and Gli1 expression in ASZ001 cells but not in HaCaT cells. Conversely, stimulation of Stat3 in ASZ001 cells with IL-6 increased Gli1 expression. Our results indicate that Dsg2 enhances canonical hedgehog signaling downstream of Ptc1 to promote BCC development through the activation of phosphorylated Stat3 and regulation of Gli1 expression.

Revitalizing Aging Skin through Diet.

Mechanisms underlying aging of the skin dermis are poorly understood. Now, two studies (Marsh et al., 2018; Salzer et al., 2018) describe complementary approaches to this question: Salzer et al. show that aging dermal fibroblasts lose defined identity in a diet-influenced fashion, and Marsh et al. reveal that fibroblast loss over time is compensated by membrane expansion rather than proliferation, resulting in decreased cellular density.