A spatial portrait of the human sebaceous gland transcriptional program.

Sebaceous glands (SG) and their oily secretion (sebum) are indispensable for maintaining skin structure and function, and their deregulation causes skin disorders including but not limited to acne. Recent studies also indicate that sebum may have important immunomodulatory activities and may influence whole-body energy metabolism. However, the progressive transcriptional changes of sebocytes that lead to sebum production have never been characterized in detail. Here, we exploited the high cellular resolution provided by sebaceous hyperplasia and integrated spatial transcriptomics, pseudo time analysis, RNA velocity, and functional enrichment to map the landscape of sebaceous differentiation. Our results were validated by comparison with published SG transcriptome data and further corroborated by assessing the protein expression pattern of a subset of the transcripts in the public repository Human Protein Atlas. Departing from four sebocyte differentiation stages generated by unsupervised clustering, we demonstrate consecutive modulation of cellular functions associable with specific gene sets, from cell proliferation and oxidative phosphorylation via lipid synthesis to cell death. Both validation methods confirmed the biological significance of our results. Our report is complemented by a freely available and browsable online tool. Our data provide the first high-resolution spatial portrait of the SG transcriptional landscape and deliver starting points for experimentally assessing novel candidate molecules for regulating SG homeostasis in health and disease.

Skin single-cell transcriptomics reveals a core of sebaceous gland-relevant genes shared by mice and humans.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) has been widely applied to dissect cellular heterogeneity in normal and diseased skin. Sebaceous glands, essential skin components with established functions in maintaining skin integrity and emerging roles in systemic energy metabolism, have been largely neglected in scRNA-seq studies. METHODS: Departing from mouse and human skin scRNA-seq datasets, we identified gene sets expressed especially in sebaceous glands with the open-source R-package oposSOM. RESULTS: The identified gene sets included sebaceous gland-typical genes as Scd3, Mgst1, Cidea, Awat2 and KRT7. Surprisingly, however, there was not a single overlap among the 100 highest, exclusively in sebaceous glands expressed transcripts in mouse and human samples. Notably, both species share a common core of only 25 transcripts, including mitochondrial and peroxisomal genes involved in fatty acid, amino acid, and glucose processing, thus highlighting the intense metabolic rate of this gland. CONCLUSIONS: This study highlights intrinsic differences in sebaceous lipid synthesis between mice and humans, and indicates an important role for peroxisomal processes in this context. Our data also provides attractive starting points for experimentally addressing novel candidates regulating sebaceous gland homeostasis.

The role of cell-cell and cell-matrix junctional complexes in sebaceous gland homeostasis and differentiation.

Sebaceous glands (SG) are essential for maintaining skin integrity, as their lipid-rich secretion (sebum) lubricates and protects the epidermis and hairs. In addition, these glands have an emerging role in immunomodulation and may affect whole-body energy metabolism, besides being an appealing model for research in topics as lipogenesis, stem cell biology and tumorigenesis. In spite of the increasing interest in studying SGs pathophysiology, sebocyte cell-cell and cell-matrix adhesion processes have been only superficially examined, and never in a systematic way. This is regrettable considering the key role of cellular adhesion in general, the specific expression pattern of indivdual junctional complexes, and the reports of structural changes in SGs after altered expression of adhesion-relevant proteins. Here, we review the available information on structural and functional aspects of cell-cell and cell-matrix junctions in sebocytes, and how these processes change under pathological conditions. This information will contribute for better understanding sebocyte differentiation and sebum secretion, and may provide hints for novel therapeutic strategies for skin diseases.

Mammalian VPS45 orchestrates trafficking through the endosomal system.

Vacuolar protein sorting 45 homolog (VPS45), a member of the Sec1/Munc18 (SM) family, has been implicated in the regulation of endosomal trafficking. VPS45 deficiency in human patients results in congenital neutropenia, bone marrow fibrosis, and extramedullary renal hematopoiesis. Detailed mechanisms of the VPS45 function are unknown. Here, we show an essential role of mammalian VPS45 in maintaining the intracellular organization of endolysosomal vesicles and promoting recycling of cell-surface receptors. Loss of VPS45 causes defective Rab5-to-Rab7 conversion resulting in trapping of cargos in early endosomes and impaired delivery to lysosomes. In this context, we demonstrate aberrant trafficking of the granulocyte colony-stimulating factor receptor in the absence of VPS45. Furthermore, we find that lack of VPS45 in mice is not compatible with embryonic development. Thus, we identify mammalian VPS45 as a critical regulator of trafficking through the endosomal system and early embryogenesis of mice.

Shedding light into the black box: Advances in in vitro systems for studying implantation.

Implantation represents a critical step for embryonic development and pregnancy. Its success depends on the complex interplay between a receptive endometrium and a competent embryo. Implantation-related events remain hardly accessible, making implantation a true "black box" in developmental biology. Improved in vitro models are becoming useful experimental tools, as they are considerably more accessible than in vivo models, easier to manipulate, and permit the use of human cells or tissues, thus increasing the translational value of the studies. In this Review, we briefly summarize the relevant cell types and structures involved into the process of implantation, in order to outline which compartments are indispensable for creating the perfect in vitro model. We also critically address advantages and limitations of available models and assess their application potential. Moreover, we examine the chances and challenges brought by the latest approaches to recapitulate the endometrial compartment, as well as by peri-implantational embryoids.

Von Kossa and his staining technique.

One hundred and twenty years ago, the Hungarian physician Julius von Kossa developed a now classical staining method for detecting mineral deposits in animal tissues. Since then, this method has been widely adapted and combined with different counterstains, but still bears the name of its original inventor, who, if alive, would have turned 150 in 2015. As a rather inexpensive technique that does not require special instrumentation, von Kossa's staining method became extremely popular for demonstrating mineralized tissues in vivo and in vitro. This article pays tribute to von Kossa and to his handy staining method.

Applicability of organ-on-chip systems in toxicology and pharmacology.

Organ-on-chip (OoC) systems are microfabricated cell culture devices designed to model functional units of human organs by harboring an in vitro generated organ surrogate. In the present study, we reviewed issues and opportunities related to the application of OoC in the safety and efficacy assessment of chemicals and pharmaceuticals, as well as the steps needed to achieve this goal. The relative complexity of OoC over simple in vitro assays provides advantages and disadvantages in the context of compound testing. The broader biological domain of OoC potentially enhances their predictive value, whereas their complexity present issues with throughput, standardization and transferability. Using OoCs for regulatory purposes requires detailed and standardized protocols, providing reproducible results in an interlaboratory setting. The extent to which interlaboratory standardization of OoC is feasible and necessary for regulatory application is a matter of debate. The focus of applying OoCs in safety assessment is currently directed to characterization (the biology represented in the test) and qualification (the performance of the test). To this aim, OoCs are evaluated on a limited scale, especially in the pharmaceutical industry, with restricted sets of reference substances. Given the low throughput of OoC, it is questionable whether formal validation, in which many reference substances are extensively tested in different laboratories, is feasible for OoCs. Rather, initiatives such as open technology platforms, and collaboration between OoC developers and risk assessors may prove an expedient strategy to build confidence in OoCs for application in safety and efficacy assessment.

Sebaceous gland: Milestones of 30-year modelling research dedicated to the "brain of the skin".

In 2018, Schneider and Zouboulis analysed the available tools for studying sebaceous gland pathophysiology in vitro. Since then, the interest in this field remains unbroken, as demonstrated by recent reviews on sebaceous gland physiology, endocrinology and neurobiology, the role of sebaceous glands beyond acne, and several original works on different areas of sebaceous gland function, including sebaceous lipogenesis. Landmark developments in the first part of the 30-year modelling research dedicated to the sebaceous gland, which is considered by several scientists as the brain of the skin, were the short-term culture of human sebaceous glands, the culture of human sebaceous gland cells and the development of immortalized sebaceous gland cell lines exhibiting characteristics of normal sebocytes. On the other hand, current developments represent the establishment of sebaceous gland spheroids, the 3D-SeboSkin model of viable skin explants ex vivo, the combination of culture-expanded epidermal stem cells of mice and adult humans to form de novo hair follicles and sebaceous glands, when they are transplanted into excisional wounds in mice, and 3D-printed scaffolds coated with decellularized matrix of adipose-derived mesenchymal stromal cells and SZ95 sebocytes. These novel tools may become useful platforms for better understanding of cellular and molecular mechanisms governing sebocyte biology and sebaceous gland homeostasis, such as the changes in sebum synthesis and composition, the infundibular differentiation and the influence of the innate immunity and the cutaneous microbiome and for identifying potential therapeutic targets of skin diseases affecting the sebaceous glands.

Sebaceous lipids are essential for water repulsion, protection against UVB-induced apoptosis and ocular integrity in mice.

Sebocytes, which are characterized by lipid accumulation that leads to cell disruption, can be found in hair follicle-associated sebaceous glands (SGs) or in free SGs such as the Meibomian glands in the eyelids. Because genetic tools that allow targeting of sebocytes while maintaining intact epidermal lipids are lacking, the relevance of sebaceous lipids in health and disease remains poorly understood. Using Scd3, which is expressed exclusively in mature sebocytes, we established a mouse line with sebocyte-specific expression of Cre recombinase. Both RT-PCR analysis and crossing into Rosa26-lacZ reporter mice and Kras(G12D) mice confirmed Cre activity specifically in SGs, with no activity in other skin compartments. Importantly, loss of SCD3 function did not cause detectable phenotypical alterations, endorsing the usefulness of Scd3-Cre mice for further functional studies. Scd3-Cre-induced, diphtheria chain A toxin-mediated depletion of sebaceous lipids resulted in impaired water repulsion and thermoregulation, increased rates of UVB-induced epidermal apoptosis and caused a severe pathology of the ocular surface resembling Meibomian gland dysfunction. This novel mouse line will be useful for further investigating the roles of sebaceous lipids in skin and eye integrity.

Cortactin is a scaffolding platform for the E-cadherin adhesion complex and is regulated by protein kinase D1 phosphorylation.

Dynamic regulation of cell-cell adhesion by the coordinated formation and dissolution of E-cadherin-based adherens junctions is crucial for tissue homeostasis. The actin-binding protein cortactin interacts with E-cadherin and enables F-actin accumulation at adherens junctions. Here, we were interested to study the broader functional interactions of cortactin in adhesion complexes. In line with literature, we demonstrate that cortactin binds to E-cadherin, and that a posttranslational modification of cortactin, RhoA-induced phosphorylation by protein kinase D1 (PKD1; also known as PRKD1) at S298, impairs adherens junction assembly and supports their dissolution. Two new S298-phosphorylation-dependent interactions were also identified, namely, that phosphorylation of cortactin decreases its interaction with ß-catenin and the actin-binding protein vinculin. In addition, binding of vinculin to ß-catenin, as well as linkage of vinculin to F-actin, are also significantly compromised upon phosphorylation of cortactin. Accordingly, we found that regulation of cell-cell adhesion by phosphorylation of cortactin downstream of RhoA and PKD1 is vitally dependent on vinculin-mediated protein interactions. Thus, cortactin, unexpectedly, is an important integration node for the dynamic regulation of protein complexes during breakdown and formation of adherens junctions.

Primary sebocytes and sebaceous gland cell lines for studying sebaceous lipogenesis and sebaceous gland diseases.

Sebocytes, the major cell type in sebaceous glands, are differentiated epithelial cells that gradually accumulate lipids and eventually disrupt, releasing their content (sebum) in a secretory process known as holocrine secretion. Via the hair canal, sebum reaches the skin surface, where it has several known or postulated functions, including pheromonal, thermoregulatory, antimicrobial and antioxidant activities. Altered sebum secretion and/or structural sebaceous gland changes have also been involved in the pathogenesis of skin diseases, such as acne vulgaris and some forms of alopecia. Here, we assess how recent work employing primary sebocytes and sebaceous gland cell lines contributed for our understanding of sebaceous lipogenesis and its role in skin health and disease.

The receptor tyrosine kinase ERBB4 is expressed in skin keratinocytes and influences epidermal proliferation.

BACKGROUND: The epidermal growth factor receptor (EGFR) and associated receptors ERBB2 and ERBB3 are important for skin development and homeostasis. To date, ERBB4 could not be unambiguously identified in the epidermis. The aim of this study was to analyze the ERBB-receptor family with a special focus on ERBB4 in vitro in human keratinocytes and in vivo in human and murine epidermis. METHODS: We compared the transcript levels of all ERBB-receptors and the seven EGFR-ligands in HaCaT and A431 cells. ERBB-receptor activity was analyzed after epidermal growth factor (EGF) stimulation by Western blot analysis. The location of the receptors was investigated by immunofluorescence in human keratinocytes and skin. Finally, we investigated the function of ERBB4 in the epidermis of skin-specific ERBB4-knockout mice. RESULTS: After EGF stimulation, all ligands were upregulated except for epigen. Expression levels of EGFR were unchanged, but all other ERBB-receptors were down-regulated after EGF stimulation, although all ERBB-receptors were phosphorylated. We detected ERBB4 at mRNA and protein levels in both human epidermal cell lines and in the basal layer of human and murine epidermis. Skin-specific ERBB4-knockout mice revealed a significantly reduced epidermal thickness with a decreased proliferation rate. CONCLUSIONS: ERBB4 is expressed in the basal layer of human epidermis and cultured keratinocytes as well as in murine epidermis. Moreover, ERBB4 is phosphorylated in HaCaT cells due to EGF stimulation, and its deletion in murine epidermis affects skin thickness by decreasing proliferation. GENERAL SIGNIFICANCE: ERBB4 is expressed in human keratinocytes and plays a role in murine skin homeostasis.

Betacellulin regulates schwann cell proliferation and myelin formation in the injured mouse peripheral nerve.

When a nerve fiber is cut or crushed, the axon segment that is separated from the soma degenerates distal from the injury in a process termed Wallerian degeneration (WD). C57BL/6OlaHsd-WldS (WldS ) mutant mice exhibit significant delays in WD. This results in considerably delayed Schwann cell and macrophage responses and thus in impaired nerve regenerations. In our previous work, thousands of genes were screened by DNA microarrays and over 700 transcripts were found to be differentially expressed in the injured sciatic nerve of WldS compared with wild-type (WT) mice. One of these transcripts, betacellulin (Btc), was selected for further analysis since it has yet to be characterized in the nervous system, despite being known as a ligand of the ErbB receptor family. We show that Btc mRNA is strongly upregulated in immature and dedifferentiated Sox2+ Schwann cells located in the sciatic nerve distal stump of WT mice, but not WldS mutants. Transgenic mice ubiquitously overexpressing Btc (Tg-Btc) have increased numbers of Schmidt-Lantermann incisures compared with WT mice, as revealed by Coherent anti-Stokes Raman scattering (CARS). Tg-Btc mice also have faster nerve conduction velocity. Finally, we found that deficiency in Btc reduces the proliferation of myelinating Schwann cells after sciatic nerve injury, while Btc overexpression induces Schwann cell proliferation and improves recovery of locomotor function. Taken together, these results suggest a novel regulatory role of Btc in axon-Schwann cell interactions involved in myelin formation and nerve repair. GLIA 2017 GLIA 2017;65:657-669.

Lipid droplets and associated proteins in sebocytes.

Mammalian skin is characterized by the presence of sebaceous glands (SGs), which develop with the hair follicle and whose predominant cell type is the sebocyte. Sebocytes are epithelial cells that progressively accumulate lipids and eventually release their content (sebum) by holocrine secretion as cells disrupt. In addition to thermoregulatory and pheromonal actions, numerous additional functions have been demonstrated or postulated for sebum, including antimicrobial and antioxidant activities. The SG has also been involved in the pathogenesis of skin diseases as acne vulgaris and some forms of alopecia. Although lipid accumulation culminating in cell disruption and content release is the hallmark of sebocyte differentiation, only a surprisingly low number of studies have so far focused on sebocyte lipid droplets and their associated proteins.

Genetically modified laboratory mice with sebaceous glands abnormalities.

Sebaceous glands (SG) are exocrine glands that release their product by holocrine secretion, meaning that the whole cell becomes a secretion following disruption of the membrane. SG may be found in association with a hair follicle, forming the pilosebaceous unit, or as modified SG at different body sites such as the eyelids (Meibomian glands) or the preputial glands. Depending on their location, SG fulfill a number of functions, including protection of the skin and fur, thermoregulation, formation of the tear lipid film, and pheromone-based communication. Accordingly, SG abnormalities are associated with several diseases such as acne, cicatricial alopecia, and dry eye disease. An increasing number of genetically modified laboratory mouse lines develop SG abnormalities, and their study may provide important clues regarding the molecular pathways regulating SG development, physiology, and pathology. Here, we summarize in tabulated form the available mouse lines with SG abnormalities and, focusing on selected examples, discuss the insights they provide into SG biology and pathology. We hope this survey will become a helpful information source for researchers with a primary interest in SG but also as for researchers from unrelated fields that are unexpectedly confronted with a SG phenotype in newly generated mouse lines.

E-cadherin's role in development, tissue homeostasis and disease: Insights from mouse models: Tissue-specific inactivation of the adhesion protein E-cadherin in mice reveals its functions in health and disease.

Recent studies uncovered critical roles of the adhesion protein E-cadherin in health and disease. Global inactivation of Cdh1, the gene encoding E-cadherin in mice, results in early embryonic lethality due to an inability to form the trophectodermal epithelium. To unravel E-cadherin's functions beyond development, numerous mouse lines with tissue-specific disruption of Cdh1 have been generated. The consequences of E-cadherin loss showed great variability depending on the tissue in question, ranging from nearly undetectable changes to a complete loss of tissue structure and function. This review focuses on these studies and discusses how they provided important insights into E-cadherin's role in cell adhesion, proliferation and differentiation, and its consequences for biological processes as epithelial-to-mesenchymal transition, vascularization, and carcinogenesis. Lastly, we present some perspectives and possible approaches for future research.

Structure and function of epigen, the last EGFR ligand.

Epigen is the latest addition to the mammalian family of EGFR ligands. Epigen was initially identified as a novel expressed sequence tag with homology to the EGF family by high throughput sequencing of a mouse keratinocyte complementary DNA library, and received its name for its ability to act as an epithelial mitogen. In vitro studies attributed to epigen several unique features, such as persistent and potent biological actions involving low affinity receptor binding, as well as sub-maximal receptor activation and inactivation. Similarly to the other EGFR ligands, the expression of epigen is up-regulated by hormones and in certain cancer types. While the biological functions of epigen remain to be uncovered, it appears to play a role in epidermal structures, such as the mammary gland and the sebaceous gland. The latter organ, in particular, was greatly enlarged in transgenic mice overexpressing epigen. Interestingly, mice lacking epigen develop and grow normally, probably due to functional compensation by other EGFR ligands. Future studies are likely to reveal the biological roles of the unique receptor binding properties of epigen, as well as its potential harnessing during disease.

The ABC of BTC: structural properties and biological roles of betacellulin.

Betacellulin was initially detected as a growth-promoting factor in the conditioned medium of a mouse pancreatic ß-cell tumor cell line. Sequencing of the purified protein and of the cloned cDNA supported the assumption that betacellulin is a new ligand of the epidermal growth factor receptor (EGFR), which was later confirmed experimentally. As a typical EGFR ligand, betacellulin is expressed by a variety of cell types and tissues, and the soluble growth factor is proteolytically cleaved from a larger membrane-anchored precursor. Importantly, BTC can - in addition to the EGFR - bind and activate all possible heterodimeric combinations of the related ERBB receptors including the highly oncogenic ERBB2/3 dimer, as well as homodimers of ERBB4. While a large number of studies attest a role for betacellulin in the differentiation of pancreatic ß-cells, the last decade witnessed the association of betacellulin with a large number of additional biological processes, ranging from reproduction to the control of neural stem cells.

The molecular basis and reproductive function(s) of copulatory plugs.

In many animals, male ejaculates coagulate to form what has been termed a copulatory plug, a structure that varies in size and shape but often fills and seals the female's reproductive tract. The first published observation of a copulatory plug in a mammal was made more than 160 years ago, and questions about its formation and role in reproduction continue to endear evolutionary and population geneticists, behavioral ecologists, and molecular, reproductive, and developmental biologists alike. Here, we review the current knowledge of copulatory plugs, focusing on rodents and asking two main questions: how is it formed and what does it do? An evolutionary biology perspective helps us understand the latter, potentially leading to insights into the selective regimes that have shaped the diversity of this structure. Mol. Reprod. Dev. 83: 755-767, 2016 © 2016 Wiley Periodicals, Inc.