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Ras genes are important oncogenes that are frequently mutated in cancer. Human oncogenic variants exhibit functional distinctions in terms of their representation in different cancer types, impact on cellular targets and sensitivity to pharmacological treatments. However, how these distinct variants influence and respond to the cellular networks in which they are embedded is poorly understood. To identify novel participants in the complex interplay between Ras genotype and cell interaction networks in vivo, we have developed and tested an experimental framework using a simple vulva-development assay in the nematode C. elegans. Using this system, we evaluated a set of Ras oncogenic substitution changes at G12, G13 and Q61. We found that these variants fall into distinct groups based on phenotypic differences, sensitivity to gene dosage and inhibition of the downstream kinase MEK and their response to genetic modulators that influence Ras activity in a non-autonomous manner. Together, our results demonstrated that oncogenic C. elegans Ras variants exhibit clear distinctions in how they interface with the vulva-development network and showed that extracellular modulators yield variant-restricted effects in vivo., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The acquisition of the post-mitotic state is crucial for the execution of many terminally differentiated cell behaviors during organismal development. However, the mechanisms that maintain the post-mitotic state in this context remain poorly understood. To gain insight into these mechanisms, we used the genetically and visually accessible model of C. elegans anchor cell (AC) invasion into the vulval epithelium. The AC is a terminally differentiated uterine cell that normally exits the cell cycle and enters a post-mitotic state before initiating contact between the uterus and vulva through a cell invasion event. Here, we set out to identify the set of negative cell cycle regulators that maintain the AC in this post-mitotic, invasive state. Our findings revealed a critical role for CKI-1 (p21 CIP1 /p27 KIP1 ) in redundantly maintaining the post-mitotic state of the AC, as loss of CKI-1 in combination with other negative cell cycle regulators-including CKI-2 (p21 CIP1 /p27 KIP1 ), LIN-35 (pRb/p107/p130), FZR-1 (Cdh1/Hct1), and LIN-23 (β-TrCP)-resulted in proliferating ACs. Remarkably, time-lapse imaging revealed that these ACs retain their ability to invade. Upon examination of a node in the gene regulatory network controlling AC invasion, we determined that proliferating, invasive ACs do so by maintaining aspects of pro-invasive gene expression. We therefore report that the requirement for a post-mitotic state for invasive cell behavior can be bypassed following direct cell cycle perturbation., (Copyright © 2024 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Introduction: Vulva squamous cell carcinoma (VSCC) develops through two separate molecular pathways-one involving high-risk human papilloma virus infection (HPV-associated), and the other without HPV infection (HPV-independent) often involving TP53 mutation. HPV-associated VSCC generally has a better progression-free survival than HPV-independent VSCC. The aim of this study was to determine TP53 mutation status using immunohistochemistry, compare different methods of HPV detection and correlate both with survival in a retrospective cohort of 123 patients with VSCC., Material and Methods: Immunohistochemistry for p53, Ki67 and p16 INK4A (a surrogate marker for HPV infection) was performed on formalin-fixed paraffin-embedded tissues from a cohort of surgically treated VSCC patients to identify molecular subtypes of VSCC. Presence of HPV infection was detected by HPV DNA PCR and HPV mRNA in situ hybridization (ISH). The Pearson chi-square test and multivariable Cox regression model were used to investigate the association of different parameters with progression-free survival and disease-specific survival (DSS), and Kaplan-Meier curves were used to show the association of different parameters with survival., Results: The results of p53 and p16 INK4A immunohistochemistry confirmed three VSCC subtypes associated with different prognosis. The TP53 mutation status was identified as an independent prognostic factor of worse progression-free survival (p = 0.024) after adjustment for FIGO stage. p16 INK4A immunohistochemistry, mRNA ISH, and DNA PCR had excellent concordance in terms of HPV detection. According to the multivariable Cox regression model, the presence of hrHPV mRNA correlated significantly with increased progression-free survival (p = 0.040) and DSS (p = 0.045), after adjustment for other confounders., Conclusions: p53 and p16 INK4A immunohistochemistry stratify VSCC cohort into three subtypes with TP53mutated patients having the worst prognosis. The detection of hrHPV mRNA by ISH was an independent predictor of increased survival. Thus, the combined detection of p53 and HPV mRNA might improve risk stratification in VSCC., (© 2023 The Authors. Acta Obstetricia et Gynecologica Scandinavica published by John Wiley & Sons Ltd on behalf of Nordic Federation of Societies of Obstetrics and Gynecology (NFOG).)

hox genes encode a conserved family of homeodomain transcription factors that are essential to determine the identity of body segments during embryogenesis and maintain adult somatic stem cells competent to regenerate organs. In contrast to higher organisms, somatic cells in C. elegans irreversibly exit the cell cycle after completing their cell lineage and the adult soma cannot regenerate. Here, we show that hox gene expression levels in C. elegans determine the temporal competence of somatic cells to proliferate. Down-regulation of the central hox gene lin-39 in dividing vulval cells results in their premature cell cycle exit, whereas constitutive lin-39 expression causes precocious Pn.p cell and sex myoblast divisions and prolongs the proliferative phase of the vulval cells past their normal point of arrest. Furthermore, ectopic expression of hox genes in the quiescent anchor cell re-activates the cell cycle and induces proliferation until young adulthood. Thus, constitutive expression of a single hox transcription factor is sufficient to prolong somatic cell proliferation beyond the restriction imposed by the cell lineage. The down-regulation of hox gene expression in most somatic cells at the end of larval development may be one cause for the absence of cell proliferation in adult C. elegans., (© 2023. The Author(s).)

Background: p24/transmembrane Emp24 domain (TMED) proteins are a set of evolutionarily conserved, single pass transmembrane proteins that have been shown to facilitate protein secretion and selection of cargo proteins to transport vesicles in the cellular secretion pathway. However, their functions in animal development are incompletely understood., Results: The C. elegans genome encodes eight identified TMED genes, with at least one member from each defined subfamily (α, β, γ, δ). TMED gene mutants exhibit a shared set of defects in embryonic viability, animal movement, and vulval morphology. Two γ subfamily genes, tmed-1 and tmed-3, exhibit the ability to compensate for each other, as defects in movement and vulva morphology are only apparent in double mutants. TMED mutants also exhibit a delay in breakdown of basement membrane during vulva development., Conclusions: The results establish a genetic and experimental framework for the study of TMED gene function in C. elegans, and argue that a functional protein from each subfamily is important for a shared set of developmental processes. A specific function for TMED genes is to facilitate breakdown of the basement membrane between the somatic gonad and vulval epithelial cells, suggesting a role for TMED proteins in tissue reorganization during animal development., (© 2023 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background/aim: The expression of the cyclin-dependent kinase inhibitor p16 correlates with the presence of human papillomavirus. The purpose of this investigation was to assess the prognostic relevance of p16 expression in patients with vulvar squamous cell carcinoma (VSCC) treated with radical surgery followed by adjuvant (chemo) radiation in selected cases., Patients and Methods: Seventy-eight patients were analyzed retrospectively., Results: Positive p16 immunostaining was detected in 19 (24.4%) patients. Five-year disease-free survival (DFS) and 5-year overall survival (OS) were better in p16-positive compared to p16-negative patients (83.9% versus 37.3% p=0.002 and 91.7% versus 57.6%, p=0.003, respectively). p16 expression retained prognostic relevance at multivariate analysis for both DFS and OS., Conclusion: p16 expression was detected in 24.4% of patients with VSCC and was found to be an independent prognostic variable for both DFS and OS., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Development of the Caenorhabditis elegans reproductive tract is orchestrated by the anchor cell (AC). This occurs in part through a cell invasion event that connects the uterine and vulval tissues. Several key transcription factors regulate AC invasion, such as EGL-43, HLH-2, and NHR-67. Specifically, these transcription factors function together to maintain the post-mitotic state of the AC, a requirement for AC invasion. Recently, a mechanistic connection has been made between loss of EGL-43 and AC cell-cycle entry. The current model states that EGL-43 represses LIN-12 (Notch) expression to prevent AC proliferation, suggesting that Notch signaling has mitogenic effects in the invasive AC. To reexamine the relationship between EGL-43 and LIN-12, we first designed and implemented a heterologous co-expression system called AIDHB that combines the auxin-inducible degron (AID) system of plants with a live cell-cycle sensor based on human DNA helicase B (DHB). After validating AIDHB using AID-tagged GFP, we sought to test it by using AID-tagged alleles of egl-43 and lin-12. Auxin-induced degradation of either EGL-43 or LIN-12 resulted in the expected AC phenotypes. Lastly, we seized the opportunity to pair AIDHB with RNAi to co-deplete LIN-12 and EGL-43, respectively, which revealed that LIN-12 is not required for AC proliferation following loss of EGL-43., Competing Interests: Competing interests D.Q.M. is a paid employee of Arcadia Science., (© 2022. Published by The Company of Biologists Ltd.)

Vulvar squamous cell carcinomas and their precursors are currently classified by the World Health Organization based on their association with high-risk human papillomavirus (HPV). HPV independent lesions often harbor driver alterations in TP53, usually seen in the setting of chronic vulvar inflammation. However, a group of pre-invasive vulvar squamous lesions is independent from both HPV and mutant TP53. The lesions described within this category feature marked acanthosis, verruciform growth and altered squamous maturation, and over the last two decades several studies have added to their characterization. They have a documented association with verrucous carcinoma and conventional squamous cell carcinoma of the vulva, suggesting a precursor role. They also harbor recurrent genomic alterations in several oncogenes, mainly PIK3CA and HRAS, indicating a neoplastic nature. In this review, we provide a historical perspective and a comprehensive description of these lesions. We also offer an appraisal of the terminology used over the years, going from Vulvar Acanthosis with Altered Differentiation and Verruciform Lichen Simplex Chronicus to Differentiated Exophytic Vulvar Intraepithelial Lesion and Vulvar Aberrant Maturation, the latter term having been recently proposed by the International Society for the Study of Vulvovaginal Diseases. In line with the recognition of these lesions by the 2020 World Health Organization Classification of Tumours as a neoplastic precursor, we herein propose the term HPV-independent, p53-wild-type verruciform acanthotic Vulvar Intraepithelial Neoplasia (HPVi(p53wt) vaVIN), which better conveys not only the pathology but also the neoplastic nature and the biologic risk inherent to these uncommon and challenging lesions. We outline strict morphologic and immunohistochemical criteria for its diagnosis and distinction from mimickers. Immunohistochemistry for p16 and p53 should be performed routinely in the diagnostic work-up of these lesions, and the morphologic alternative term vaVIN should be reserved for instances in which p16/HPV/p53 status is unknown. We also discuss management considerations and the need to further explore precursors within and beyond the spectrum of verruciform acanthotic vulvar intraepithelial neoplasia., (© 2022. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Communication between mesodermal cells and epithelial cells is fundamental to normal animal development and is frequently disrupted in cancer. However, the genes and processes that mediate this communication are incompletely understood. To identify genes that mediate this communication and alter the proliferation of cells with an oncogenic Ras genotype, we carried out a tissue-specific genome-wide RNAi screen in Caenorhabditis elegans animals bearing a let-60(n1046gf) (RasG13E) allele. The screen identifies 24 genes that, when knocked down in adjacent mesodermal tissue, suppress the increased vulval epithelial cell proliferation defect associated with let-60(n1046gf). Importantly, gene knockdown reverts the mutant animals to a wild-type phenotype. Using chimeric animals, we genetically confirm that 2 of the genes function nonautonomously to revert the let-60(n1046gf) phenotype. The effect is genotype restricted, as knockdown does not alter development in a wild type (let-60(+)) or activated EGF receptor (let-23(sa62gf)) background. Although many of the genes identified encode proteins involved in essential cellular processes, including chromatin formation, ribosome function, and mitochondrial ATP metabolism, knockdown does not alter the normal development or function of targeted mesodermal tissues, indicating that the phenotype derives from specific functions performed by these cells. We show that the genes act in a manner distinct from 2 signal ligand classes (EGF and Wnt) known to influence the development of vulval epithelial cells. Altogether, the results identify genes with a novel function in mesodermal cells required for communicating with and promoting the proliferation of adjacent epithelial cells with an activated Ras genotype., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Development of the Caenorhabditis elegans vulva is a classic model of organogenesis. This system, which starts with 6 equipotent cells, encompasses diverse types of developmental event, including developmental competence, multiple signaling events to control precise and faithful patterning of three cell fates, execution and proliferation of specific cell lineages, and a series of sophisticated morphogenetic events. Early events have been subjected to extensive mutational and genetic investigations and later events to cell biological analyses. We infer the existence of dramatically changing profiles of gene expression that accompanies the observed changes in development. Yet, except from serendipitous discovery of several transcription factors expressed in dynamic patterns in vulval lineages, our knowledge of the transcriptomic landscape during vulval development is minimal. This study describes the composition of a vulva-specific transcriptome. We used tissue-specific harvesting of mRNAs via immunoprecipitation of epitope-tagged poly(A) binding protein, PAB-1, heterologously expressed by a promoter known to express GFP in vulval cells throughout their development. The identified transcriptome was small but tightly interconnected. From this data set, we identified several genes with identified functions in development of the vulva and validated more with promoter-GFP reporters of expression. For one target, lag-1, promoter-GFP expression was limited but a fluorescent tag of the endogenous protein revealed extensive expression. Thus, we have identified a transcriptome of C. elegans vulval lineages as a launching pad for exploration of functions of these genes in organogenesis., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

The Caenorhabditis elegans E protein ortholog HLH-2 is required for the specification and function of the anchor cell, a unique, terminally differentiated somatic gonad cell that organizes uterine and vulval development. Initially, 4 cells-2 α cells and their sisters, the β cells-have the potential to be the sole anchor cell. The β cells rapidly lose anchor cell potential and invariably become ventral uterine precursor cells, while the 2 α cells interact via LIN-12/Notch to resolve which will be the anchor cell and which will become another ventral uterine precursor cell. HLH-2 protein stability is dynamically regulated in cells with anchor cell potential; initially present in all 4 cells, HLH-2 is degraded in presumptive ventral uterine precursor cells while remaining stable in the anchor cell. Here, we demonstrate that stability of HLH-2 protein is regulated by the activity of lin-12/Notch in both α and β cells. Our analysis provides evidence that activation of LIN-12 promotes degradation of HLH-2 as part of a negative feedback loop during the anchor cell/ventral uterine precursor cell decision by the α cells, and that absence of lin-12 activity in β cells increases HLH-2 stability and may account for their propensity to adopt the anchor cell fate in a lin-12 null background. We also performed an RNA interference screen of 232 ubiquitin-related genes and identified 7 genes that contribute to HLH-2 degradation in ventral uterine precursor cells; however, stabilizing HLH-2 by depleting ubiquitin ligases in a lin-12(+) background does not result in supernumerary anchor cells, suggesting that LIN-12 activation does not oppose hlh-2 activity solely by causing HLH-2 protein degradation. Finally, we provide evidence for lin-12-independent transcriptional regulation of hlh-2 in β cells that correlates with known differences in POP-1/TCF levels and anchor cell potential between α and β cells. Together, our results indicate that hlh-2 activity is regulated at multiple levels to restrict the anchor cell fate to a single cell., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

In this issue of PLOS Biology, Lattmann and colleagues report a new function for proteins of the DNA prereplication complex promoting the anchor cell to invade through the basement membrane and initiate vulval development in Caenorhabditis elegans., Competing Interests: The authors have declared that no competing interests exist.

Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes overexpressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC genes function cell-autonomously in the G1-arrested AC to promote invasion, independently of their role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC, or a part of it, acts in the postmitotic AC as a transcriptional regulator that facilitates the switch to an invasive phenotype., Competing Interests: The authors have declared that no competing interests exist.

The Axin family of scaffolding proteins regulates a wide array of developmental and post-developmental processes in eukaryotes. Studies in the nematode Caenorhabditis elegans have shown that the Axin homolog PRY-1 plays essential roles in multiple tissues. To understand the genetic network of pry-1, we focused on a set of genes that are differentially expressed in the pry-1-mutant transcriptome and are linked to reproductive structure development. Knocking down eight of the genes (spp-1, clsp-1, ard-1, rpn-7, cpz-1, his-7, cdk-1, and rnr-1) via RNA interference efficiently suppressed the multivulva phenotype of pry-1 mutants. In all cases, the ectopic induction of P3.p vulval precursor cell was also inhibited. The suppressor genes are members of known gene families in eukaryotes and perform essential functions. Our genetic interaction experiments revealed that in addition to their role in vulval development, these genes participate in one or more pry-1-mediated biological events. Whereas four of them (cpz-1, his-7, cdk-1, and rnr-1) function in both stress response and aging, two (spp-1 and ard-1) are specific to stress response. Altogether, these findings demonstrate the important role of pry-1 suppressors in regulating developmental and post-developmental processes in C. elegans. Given that the genes described in this study are conserved, future investigations of their interactions with Axin and their functional specificity promises to uncover the genetic network of Axin in metazoans., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

During Caenorhabditis elegans vulval development, the uterine anchor cell (AC) first secretes an epidermal growth factor (EGF) to specify the vulval cell fates and then invades the underlying vulval epithelium. By doing so, the AC establishes direct contact with the invaginating primary vulF cells and attaches the developing uterus to the vulva. The signals involved and the exact sequence of events joining these two organs are not fully understood. Using a conditional let-23 EGF receptor (EGFR) allele along with novel microfluidic short- and long-term imaging methods, we discovered a specific function of the EGFR in the AC during vulval lumen morphogenesis. Tissue-specific inactivation of let-23 in the AC resulted in imprecise alignment of the AC with the primary vulval cells, delayed AC invasion and disorganized adherens junctions at the contact site forming between the AC and the dorsal vulF toroid. We propose that EGFR signaling, activated by a reciprocal EGF cue from the primary vulval cells, positions the AC at the vulval midline, guides it during invasion and assembles a cytoskeletal scaffold organizing the adherens junctions that connect the developing uterus to the dorsal vulF toroid. Thus, EGFR signaling in the AC ensures the precise alignment of the two developing organs., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

During Caenorhabditis elegans larval development, an inductive signal mediated by the LET-23 EGFR (epidermal growth factor receptor), specifies three of six vulva precursor cells (VPCs) to adopt vulval cell fates. An evolutionarily conserved complex consisting of PDZ domain-containing scaffold proteins LIN-2 (CASK), LIN-7 (Lin7 or Veli), and LIN-10 (APBA1 or Mint1) (LIN-2/7/10) mediates basolateral LET-23 EGFR localization in the VPCs to permit signal transmission and development of the vulva. We recently found that the LIN-2/7/10 complex likely forms at Golgi ministacks; however, the mechanism through which the complex targets the receptor to the basolateral membrane remains unknown. Here we found that overexpression of LIN-10 or LIN-7 can compensate for loss of their complex components by promoting LET-23 EGFR signaling through previously unknown complex-independent and receptor-dependent pathways. In particular, LIN-10 can independently promote basolateral LET-23 EGFR localization, and its complex-independent function uniquely requires its PDZ domains that also regulate its localization to Golgi. These studies point to a novel complex-independent function for LIN-7 and LIN-10 that broadens our understanding of how this complex regulates targeted sorting of membrane proteins.

The evolutionarily conserved LIN-2 (CASK)/LIN-7 (Lin7A-C)/LIN-10 (APBA1) complex plays an important role in regulating spatial organization of membrane proteins and signaling components. In Caenorhabditis elegans , the complex is essential for the development of the vulva by promoting the localization of the sole Epidermal growth factor receptor (EGFR) ortholog LET-23 to the basolateral membrane of the vulva precursor cells where it can specify the vulval cell fate. To understand how the LIN-2/7/10 complex regulates receptor localization, we determined its expression and localization during vulva development. We found that LIN-7 colocalizes with LET-23 EGFR at the basolateral membrane, whereas the LIN-2/7/10 complex colocalizes with LET-23 EGFR at cytoplasmic punctae that mostly overlap with the Golgi. Furthermore, LIN-10 recruits LIN-2, which in turn recruits LIN-7. We demonstrate that the complex forms in vivo with a particularly strong interaction and colocalization between LIN-2 and LIN-7, consistent with them forming a subcomplex. Thus, the LIN-2/7/10 complex forms on the Golgi on which it likely targets LET-23 EGFR trafficking to the basolateral membrane rather than functioning as a tether., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Characterizing the consequences of mutated Ras/LET-60 on the development of the C. elegans vulva has provided critical insights into the role of Ras in normal animal development. Furthermore, double mutant analysis revealed the role of Ras relative to other components of growth factor signal transduction. Here we describe the combined use of principles of parallelism and epistasis to investigate the use of different Ras effectors, Raf and RalGEF > Ral, during the development of the vulva and other tissues. We additionally describe the use of these principles to delineate the function of the close Ras relative, RAP-1. The worm continues to lead the way in clarifying otherwise poorly understood functions of Ras during animal development.

Activation of a canonical EGFR-Ras-Raf-ERK cascade initiates patterning of multipotent vulval precursor cells (VPCs) of Caenorhabditis elegans We have previously shown that this pathway includes a negative-feedback component in which MPK-1/ERK activity targets the upstream kinase LIN-45/Raf for degradation by the SEL-10/FBXW7 E3 ubiquitin ligase. This regulation requires a Cdc4 phosphodegron (CPD) in LIN-45 that is conserved in BRAF. Here, we identify and characterize the minimal degron that encompasses the CPD and is sufficient for SEL-10-mediated, MPK-1-dependent protein degradation. A targeted screen of conserved protein kinase-encoding genes yielded gsk-3 (an ortholog of human GSK3B) and cdk-2 (a CDK2-related kinase) as required for LIN-45 degron-mediated turnover. Genetic analysis revealed that LIN-45 degradation is blocked at the second larval stage due to cell cycle quiescence, and that relief of this block during the third larval stage relies on activation of CDKs. Additionally, activation of MPK-1 provides spatial pattern to LIN-45 degradation but does not bypass the requirement for gsk-3 and cdk-2 This analysis supports a model whereby MPK-1/ERK, GSK-3/GSK3 and CDK-2/CDK2, along with SEL-10/FBXW7, constitute a regulatory network that exerts spatial and temporal control of LIN-45/Raf degradation during VPC patterning., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Mitochondria are the central hub for many metabolic processes, including the citric acid cycle, oxidative phosphorylation, and fatty acid oxidation. Recent studies have identified a new mitochondrial protein family, Fam210, that regulates bone metabolism and red cell development in vertebrates. The model organism Caenorhabditis elegans has a Fam210 gene, y56a3a.22, but it lacks both bones and red blood cells. In this study, we report that Y56A3A.22 plays a crucial role in regulating mitochondrial protein homeostasis and reproduction. The nematode y56a3a.22 is expressed in various tissues, including the intestine, muscle, hypodermis, and germline, and its encoded protein is predominantly localized in mitochondria. y56a3a.22 deletion mutants are sterile owing to impaired oogenesis. Loss of Y56A3A.22 induced mitochondrial unfolded protein response (UPR mt ), which is mediated through the ATFS-1-dependent pathway, in tissues such as the intestine, germline, hypodermis, and vulval muscle. We further show that infertility and UPR mt induces by Y56A3A.22 deficiency are not attributed to systemic iron deficiency. Together, our study reveals an important role of Y56A3A.22 in regulating mitochondrial protein homeostasis and oogenesis and provides a new genetic tool for exploring the mechanisms regulating mitochondrial metabolism and reproduction as well as the fundamental role of the Fam210 family., (Copyright © 2020 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

The C. elegans vulva is an excellent model for the study of developmental biology and cell-cell signaling. The developmental induction of vulval precursor cells (VPCs) to assume the 3°-3°-2°-1°-2°-3° patterning of cell fates occurs with 99.8% accuracy. During C. elegans vulval development, an EGF signal from the anchor cell initiates the activation of Ras LET-60 > Raf LIN-45 > MEK MEK-2 > ERK MPK-1 signaling cascade to induce the 1° cell. The presumptive 1° cell signals its two neighboring cells via Notch LIN-12 to develop 2° cells. In addition, Ras LET-60 switches effectors to RalGEF RGL-1 > Ral RAL-1 to promote 2° fate. Shin et al. (2019) showed that RalGEF RGL-1 is a dual-function protein in VPCs fate patterning. RalGEF RGL-1 functions as a scaffold for PDK PDK-1 > Akt AKT-1/2 modulatory signaling to promote 1° fate in addition to propagating the Ras LET-60 modulatory signal through Ral RAL-1 to promote 2° fate. The deletion of RalGEF RGL-1 increases the frequency of VPC patterning errors 15-fold compared to the wild-type control. We speculate that RalGEF RGL-1 represents an "insulated switch", whereby the promotion of one signaling activity curtails the promotion of the opposing activity. This property might increase the impact of the switch on fidelity more than two separately encoded proteins could. Understanding how developmental fidelity is controlled will help us to better understand the origins of cancer and birth defects, which occur in part due to the misspecification of cell fates.

The purpose of this study was to investigate the therapeutic effects of focused ultrasound on the expression of notch1, c-fos and transforming growth factor-β3 (TGF-β3) in genital skin of SD rats with vulvar lichen simplex chronicus (LSC). Fifty-six female SD rats with LSC were randomly divided into therapy and sham groups. The therapy group was exposed to focused ultrasound. The sham group received the same therapy with an instrument that had no power output. Four wk after a singly focused ultrasound therapy, histologic analyses revealed that recovered SD rats accounted for 75% of SD rats in the therapy group and 10.7% in the sham group. Total collagen fiber density in the superficial layer of dermis in the therapy group was significantly lower than that in the sham group. Notch1 and c-fos protein expression in the therapy group was significantly lower than that in the sham group, with the opposite effect present for TGF-β3. Focused ultrasound therapy may inhibit superficial collagen fibrosis in the dermis by affecting expression of notch1, c-fos and TGF-β3 in vulvar skin tissue and consequently reduce the recurrence rate of LSC., Competing Interests: Conflict of interest disclosure The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Long-term survival of an animal species depends on development being robust to environmental variations and climate changes. We used C. elegans to study how mechanisms that sense environmental changes trigger adaptive responses that ensure animals develop properly. In water, the nervous system induces an adaptive response that reinforces vulval development through an unknown backup signal for vulval induction. This response involves the heterotrimeric G-protein EGL-30//G αq acting in motor neurons. It also requires body-wall muscle, which is excited by EGL-30-stimulated synaptic transmission, suggesting a behavioral function of neurons induces backup signal production from muscle. We now report that increased acetylcholine during liquid growth activates an EGL-30-Rho pathway, distinct from the synaptic transmission pathway, that increases Wnt production from motor neurons. We also provide evidence that this neuronal Wnt contributes to EGL-30-stimulated vulval development, with muscle producing a parallel developmental signal. As diverse sensory modalities stimulate motor neurons via acetylcholine, this mechanism enables broad sensory perception to enhance Wnt-dependent development. Thus, sensory perception improves animal fitness by activating distinct neuronal functions that trigger adaptive changes in both behavior and developmental processes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The anchor cell (AC) in C. elegans secretes an epidermal growth factor (EGF) homolog that induces adjacent vulval precursor cells (VPCs) to differentiate. The EGF receptor in the nearest VPC sequesters the limiting EGF amounts released by the AC to prevent EGF from spreading to distal VPCs. Here, we show that not only EGFR localization in the VPCs but also EGF polarity in the AC is necessary for robust fate specification. The AC secretes EGF in a directional manner towards the nearest VPC. Loss of AC polarity causes signal spreading and, when combined with MAPK pathway hyperactivation, the ectopic induction of distal VPCs. In a screen for genes preventing distal VPC induction, we identified sra-9 and nlp-26 as genes specifically required for polarized EGF secretion. sra-9(lf) and nlp-26(lf) mutants exhibit errors in vulval fate specification, reduced precision in VPC to AC alignment and increased variability in MAPK activation. sra-9 encodes a seven-pass transmembrane receptor acting in the AC and nlp-26 a neuropeptide-like protein expressed in the VPCs. SRA-9 and NLP-26 may transduce a feedback signal to channel EGF secretion towards the nearest VPC., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Sudden activation of the stress axis by a lipopolysaccharide endotoxin (LPS) significantly reduces ewes' sexual attractivity to rams by delaying all signs of oestrous behaviour. To understand mechanisms involved in attracting male interest, we examined c-Fos (nuclear activation), ELOVL5 (enzyme involved in pheromone synthesis) and oestradiol receptors (ER) using immunohistochemistry on ewe vulval tissue at 0, 31 and 40 hr in the ovarian follicular phase with or without exposure to LPS at 28 hr (5 groups of 4 ewes per group). While there was intense staining for immunoreactive (IR)-c-Fos and IR-ELOVL5 in the vulval epithelium and sebaceous glands, there were no differences in intensity between groups of ewes. The absence of IR-ER staining in vulval epithelium and sebaceous/sweat glands was unexpected. Differences in ram behaviour towards ewes in the ovarian follicular phase and after LPS treatment do not appear to involve quantitative changes in vulval c-Fos, ELOVL5 or ER, but subtle qualitative differences in individual-specific compounds (attraction pheromones) remain an option., (© 2019 Blackwell Verlag GmbH.)

Genetic analysis of LIN-12/Notch signaling in C . elegans has provided many insights into human biology. Activating missense mutations in the Negative Regulatory Region (NRR) of the ectodomain of LIN-12/Notch were first described in C. elegans , and similar mutations in human Notch were later found to cause T-cell acute lymphoblastic leukemia (T-ALL). The ubiquitin ligase sel-10 /Fbw7 is the prototype of a conserved negative regulator of lin-12 /Notch that was first defined by loss-of-function mutations that enhance lin-12 NRR-missense activity in C. elegans , and then demonstrated to regulate Notch activity in mammalian cells and to be a bona fide tumor suppressor in T-ALL. Here, we report the results of an RNAi screen of 248 C. elegans protein kinase-encoding genes with human orthologs for enhancement of a weakly activating NRR-missense mutation of lin-12 in the Vulval Precursor Cells. We identified, and validated, thirteen kinase genes whose loss led to increase lin-12 activity; eleven of these genes have never been implicated previously in regulating Notch activity in any system. Depleting the activity of five kinase genes ( cdk-8 , wnk-1 , kin-3 , hpo-11 , and mig-15 ) also significantly enhanced the activity of a transgene in which heterologous sequences drive expression of the untethered intracellular domain of LIN-12, suggesting that they increase the activity or stability of the signal-transducing form of LIN-12/Notch. Precedents set by other regulators of lin-12 /Notch defined through genetic interactions in C. elegans suggest that this new set of genes may include negative regulators that are functionally relevant to mammalian development and cancer., (Copyright © 2019 Deng et al.)

The six C. elegans vulval precursor cells (VPCs) are induced to form the 3°-3°-2°-1°-2°-3° pattern of cell fates with high fidelity. In response to EGF signal, the LET-60/Ras-LIN-45/Raf-MEK-2/MEK-MPK-1/ERK canonical MAP kinase cascade is necessary to induce 1° fate and synthesis of DSL ligands for the lateral Notch signal. In turn, LIN-12/Notch receptor is necessary to induce neighboring cells to become 2°. We previously showed that, in response to graded EGF signal, the modulatory LET-60/Ras-RGL-1/RalGEF-RAL-1/Ral signal promotes 2° fate in support of LIN-12. In this study, we identify two key differences between RGL-1 and RAL-1. First, deletion of RGL-1 confers no overt developmental defects, while previous studies showed RAL-1 to be essential for viability and fertility. From this observation, we hypothesize that the essential functions of RAL-1 are independent of upstream activation. Second, RGL-1 plays opposing and genetically separable roles in VPC fate patterning. RGL-1 promotes 2° fate via canonical GEF-dependent activation of RAL-1. Conversely, RGL-1 promotes 1° fate via a non-canonical GEF-independent activity. Our genetic epistasis experiments are consistent with RGL-1 functioning in the modulatory 1°-promoting AGE-1/PI3-Kinase-PDK-1-AKT-1 cascade. Additionally, animals lacking RGL-1 experience 15-fold higher rates of VPC patterning errors compared to the wild type. Yet VPC patterning in RGL-1 deletion mutants is not more sensitive to environmental perturbations. We propose that RGL-1 functions to orchestrate opposing 1°- and 2°-promoting modulatory cascades to decrease developmental stochasticity. We speculate that such switches are broadly conserved but mostly masked by paralog redundancy or essential functions., Competing Interests: The authors have declared that no competing interests exist.

Genetic screens in the nematode Caenorhabditis elegan s identified the EGF/Ras and Notch pathways as central for vulval precursor cell fate patterning. Schematically, the anchor cell secretes EGF, inducing the P6.p cell to a primary (1°) vulval fate; P6.p in turn induces its neighbors to a secondary (2°) fate through Delta-Notch signaling and represses Ras signaling. In the nematode Oscheius tipulae , the anchor cell successively induces 2° then 1° vulval fates. Here, we report on the molecular identification of mutations affecting vulval induction in O. tipulae A single Induction Vulvaless mutation was found, which we identify as a cis -regulatory deletion in a tissue-specific enhancer of the O . tipulae lin-3 homolog, confirmed by clustered regularly interspaced short palindromic repeats/Cas9 mutation. In contrast to this predictable Vulvaless mutation, mutations resulting in an excess of 2° fates unexpectedly correspond to the plexin/semaphorin pathway. Hyperinduction of P4.p and P8.p in these mutants likely results from mispositioning of these cells due to a lack of contact inhibition. The third signaling pathway found by forward genetics in O. tipulae is the Wnt pathway; a decrease in Wnt pathway activity results in loss of vulval precursor competence and induction, and 1° fate miscentering on P5.p. Our results suggest that the EGF and Wnt pathways have qualitatively similar activities in vulval induction in C. elegans and O. tipulae , albeit with quantitative differences in the effects of mutation. Thus, the derived induction process in C. elegans with an early induction of the 1° fate appeared during evolution, after the recruitment of the EGF pathway for vulval induction., (Copyright © 2019 Vargas-Velazquez et al.)

The recent work of Besseling and Bringmann (2016) identified a molecular intervention for C. elegans in which premature segregation of maternal and paternal chromosomes in the fertilized oocyte can produce viable animals exhibiting a non-Mendelian inheritance pattern. Overexpression in embryos of a single protein regulating chromosome segregation (GPR-1) provides a germline derived clonally from a single parental gamete. We present a collection of strains and cytological assays to consistently generate and track non-Mendelian inheritance. These tools allow reproducible and high-frequency (>80%) production of non-Mendelian inheritance, the facile and simultaneous homozygosis for all nuclear chromosomes in a single generation, the precise exchange of nuclear and mitochondrial genomes between strains, and the assessments of non-canonical mitosis events. We show the utility of these strains by demonstrating a rapid assessment of cell lineage requirements (AB versus P1) for a set of genes (lin-2, lin-3, lin-12, and lin-31) with roles in C. elegans vulval development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Malignant lipomatous tumors of the vulva are extremely rare. We report the case of a 53-year-old patient with a nodule on her right labium majus whose histological and immunohistochemical profile (S100 and p16) confirmed a diagnosis of vulvar myxoid liposarcoma.

Solitary fibrous tumor (SFT) is a neoplasm of fibroblastic lineage that has been documented in almost every anatomic location. Vulval SFT is very rare with only 10 cases reported to date. We present 2 additional SFTs located in the vulva, in adult women of 59 and 25 yr of age. The first showed a classic morphology and immunophenotype with uniform and strong STAT6 nuclear expression. The other one was a spindle-cell de novo dedifferentiated SFT with heterogeneous nuclear and cytoplasmic STAT6 staining, which could only be correctly diagnosed after molecular analysis with demonstration of a NAB2-STAT6 gene fusion. This genetic aberration is considered to represent the major pathogenic driver in SFT and is highly specific for this neoplasm. The differential diagnosis of vulval SFT is wide and varies depending on the histologic SFT subtype. Molecular analysis is mandatory for a correct diagnosis in cases without the characteristic histopathologic and immunophenotypical features.

Studying how molecular pathways respond to ecologically relevant environmental variation is fundamental to understand organismal development and its evolution. Here we characterize how starvation modulates Caenorhabditis elegans vulval cell fate patterning - an environmentally sensitive process, with a nevertheless robust output. Past research has shown many vulval mutants affecting EGF-Ras-MAPK, Delta-Notch and Wnt pathways to be suppressed by environmental factors, such as starvation. Here we aimed to resolve previous, seemingly contradictory, observations on how starvation modulates levels of vulval induction. Using the strong starvation suppression of the Vulvaless phenotype of lin-3/egf reduction-of-function mutations as an experimental paradigm, we first tested for a possible involvement of the sensory system in relaying starvation signals to affect vulval induction: mutation of various sensory inputs, DAF-2/Insulin or DAF-7/TGF-β signaling did not abolish lin-3(rf) starvation suppression. In contrast, nutrient deprivation induced by mutation of the intestinal peptide transporter gene pept-1 or the TOR pathway component rsks-1 (the ortholog of mammalian P70S6K) very strongly suppressed lin-3(rf) mutant phenotypes. Therefore, physiologically starved animals induced by these mutations tightly recapitulated the effects of external starvation on vulval induction. While both starvation and pept-1 RNAi were sufficient to increase Ras and Notch pathway activities in vulval cells, the highly penetrant Vulvaless phenotype of a tissue-specific null allele of lin-3 was not suppressed by either condition. This and additional results indicate that partial lin-3 expression is required for starvation to affect vulval induction. These results suggest a cross-talk between nutrient deprivation, TOR-S6K and EGF-Ras-MAPK signaling during C. elegans vulval induction., (Copyright © 2018 Grimbert et al.)

Objective: The vulvar pain syndrome (VPS) is a multifactorial disease severely influencing the lifestyle of affected women. Among possible etiological factors, local injury, peripheral and/or central sensitization of the nervous system, and a chronic inflammatory status have been positively associated with the development of VPS. The identification of a constitutive altered local inflammatory profile in VPS women may represent an important point in the characterization of patients' phenotype as a useful marker influencing the vulvar micro-environment. The aim of this study was to investigative the possible role of the local cytokines production in women with VPS in comparison to healthy women., Study Design: In this study were collected vaginal swabs from 57 healthy women (HC) who never suffered from VPS and from 30 patients diagnosed with vulvodynia (VPS) by at least 3 years and currently symptomatic. All patients included in this study showed the absence of Sexually Transmitted (STD) diseases and Reproductive Tract Infection. Real-time PCR was performed to assess the genomic sequences of ST pathogens. The Luminex Bio-Plex platform was used for the analysis of a panel of 48 immune factors., Results: Eleven molecules, specifically involved in the pro-inflammatory pathway were significantly modulated in VPS patients in comparison to healthy women, suggesting a persistent inflammatory process., Conclusions: Therefore, these inflammatory factors could be possible biological markers involved in this disease. Nevertheless, other studies are needed to consider this specific immune profile as a valid marker of the vulvodynia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This work focused on immunohistochemistry markers of acute viral infections. Viral infected cells were detected by in situ based methods (reovirus, rabies virus) or cytologic changes (human papillomavirus, molloscum contagiosum virus, herpes simplex virus). Two proteins involved in nuclear trafficking, importin-β and exportin-5, were detected in the infected cells for each virus and not in the control tissues. A wide variety of other proteins, including caspase-3, and bcl-2 family members (bcl2, bclX, MCL1, BAK, BAX, BIM, BAD) showed wide variations in expression among the different viral infections. Specificity of the importin-β and exportin-5 signals varied greatly with different commercially available peroxidase conjugates. It is concluded that immunohistochemistry detection of importin-β and exportin-5 may be useful markers of acute viral infection, which suggests that increased nuclear trafficking may be an important concomitant of viral proliferation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Vulvar lichen sclerosis (VLS) is a chronic inflammatory skin disorder. Evidence is accumulating that microRNAs (miRNAs) exert crucial roles in initiation and development of a wide range of human diseases. MiR-155-5p has been frequently reported to be implicated in the tumorigenesis and progression of multiple types of cancers, however, its biological role in VLS remains unclear. This study aimed to explore the role of miR-155-5p in VLS and clarify the potential molecular mechanisms involved. In the present study, miR-155-5p was observed to be significantly upregulated in VLS tissues. Functional studies showed that miR-155-5p facilitated cell proliferation, accelerated cell cycle progression and inhibited forkhead box O (FOXO) signaling pathway in fibroblast cells. Mechanical studies demonstrated that miR-155-5p exerted its promoting effects on fibroblast cell proliferation via targeting both forkhead box O3 (FOXO3) and cyclin-dependent kinase inhibitor 1B (CDKN1B). Besides, Pearson's correlation analysis revealed that miR-155-5p expression was negatively correlated with the mRNA expression of FOXO3 and CDKN1B in VLS tissues. Taken together, our results indicate that miR-155-5p promotes fibroblast cell proliferation and inhibits FOXO signaling pathway by negative modulation of both FOXO3 and CDKN1B in VLS, and that miR-155-5p may be used to be a potential therapeutic target for VLS., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This report describes a rare case of invasive extramammary Paget disease of the vulva with signet ring cell morphology in a 58-yr-old woman with a history of signet ring cell carcinoma of the stomach. This case was initially misinterpreted as a metastatic gastric carcinoma to the vulva because an initial small, superficial biopsy specimen showed infiltration of signet ring cells in the dermis without intraepidermal Paget cells. However, a surgically resected specimen showed concordant immunophenotypes in both intraepidermal Paget cells and intradermal signet ring cell components with immunoreactivity to cytokeratin (CK) 7, CEA, and gross cystic disease fluid protein-15, and immunonegativity for CK20, MUC5AC, and MUC6. Gastric signet ring cell carcinoma showed immunoreactivity to CK7, CEA, MUC5AC, and MUC6, and immunonegativity for gross cystic disease fluid protein-15 and CK20. The diagnosis of primary invasive extramammary Paget disease of the vulva was also supported by a long interval after gastrectomy (7.5 yr), the solitary involvement of the vulva, and the absence of lymphovascular invasion. This case demonstrates that invasive extramammary Paget disease may have a signet ring cell morphology and immunohistochemical profile similar to those of gastric signet ring cell carcinoma, but the addition of gross cystic disease fluid protein-15 immunostain in the panel of markers is helpful in the differential diagnosis.

The aim of the study was to verify the efficacy of vulvar Visnadine spray in premenopausal women affected by female sexual arousal disorder (FSAD). Thirty-eight women aged 25-40 years affected by FSAD were enrolled in the randomized crossover study, by two possible sequences: on-demand, washout, daily (A sequence); and daily, washout, on-demand (B sequence). The Female Sexual Function Index (FSFI) and the Female Sexual Distress Scale (FSDS) were used to assess sexual function and sexual distress, respectively. Color Doppler ultrasonography was used to measure clitoral blood flow. The study had two follow-ups at 30 (T1) and 60 days (T2). Thirty-one women completed the study. Mean (SD) sexual activity and vulvar Visnadine spray usage was 1 ± 0.9 weekly during on-demand administration for both the sequences (Vs T0, p = NS). The mean sexual activity during daily usage was 2 ± 0.9 (Vs T0, p < .004) and 2 ± 0.8 (Vs T0, p < .001) for A and B sequences, respectively. FSFI total score, particularly genital arousal, improved more during the daily than during on-demand phases of both sequences (p < .001). Finally, clitoral blood flow improved significantly during daily usage of both the sequences (p < .001). Our study suggests that vulvar Visnadine spray could improve sexual performance of women affected by FSAD, producing changes in subjective and objective sexual aspects.

Six initially equivalent, multipotential Vulval Precursor Cells (VPCs) in Caenorhabditis elegans adopt distinct cell fates in a precise spatial pattern, with each fate associated with transcription of different target genes. The pattern is centered on a cell that adopts the "1°" fate through Epidermal Growth Factor Receptor (EGFR) activity, and produces a lateral signal composed of ligands that activate LIN-12/Notch in the two flanking VPCs to cause them to adopt "2°" fate. Here, we investigate orthologs of a transcription complex that acts in mammalian EGFR signaling- lin-1 /Elk1, sur-2/ Med23, and the Cdk8 Kinase module (CKM)-previously implicated in aspects of 1° fate in C. elegans and show they act in different combinations for different processes for 2° fate. When EGFR is inactive, the CKM, but not SUR-2, helps to set a threshold for LIN-12/Notch activity in all VPCs. When EGFR is active, all three factors act to resist LIN-12/Notch, as revealed by the reduced ability of ectopically-activated LIN-12/Notch to activate target gene reporters. We show that overcoming this resistance in the 1° VPC leads to repression of lateral signal gene reporters, suggesting that resistance to LIN-12/Notch helps ensure that P6.p becomes a robust source of the lateral signal. In addition, we show that sur-2/ Med23 and lin-1 /Elk1, and not the CKM, are required to promote endocytic downregulation of LIN-12-GFP in the 1° VPC. Finally, our analysis using cell fate reporters reveals that both EGFR and LIN-12/Notch signal transduction pathways are active in all VPCs in lin-1 /Elk1 mutants, and that lin-1 /Elk1 is important for integrating EGFR and lin-12 /Notch signaling inputs in the VPCs so that the proper gene complement is transcribed., (Copyright © 2017 by the Genetics Society of America.)

During epithelial tube morphogenesis, linear arrays of cells are converted into tubular structures through actomyosin-generated intracellular forces that induce tissue invagination and lumen formation. We have investigated lumen morphogenesis in the C. elegans vulva. The first discernible event initiating lumen formation is the apical constriction of the two innermost primary cells (VulF). The VulF cells thereafter constrict their lateral membranes along the apicobasal axis to extend the lumen dorsally. Lateral, but not apical, VulF constriction requires the prior invasion of the anchor cell (AC). The invading AC extends actin-rich protrusions toward VulF, resulting in the formation of a direct AC-VulF interface. The recruitment of the F-BAR-domain protein TOCA-1 to the AC-VulF interface induces the accumulation of force-generating actomyosin, causing a switch from apical to lateral membrane constriction and the dorsal extension of the lumen. Invasive cells may induce shape changes in adjacent cells to penetrate their target tissues., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Lichen sclerosus (LS) is a chronic and inflammatory disease causing sensory symptoms such as itch and pain and affecting most frequently genital skin of women. Intraepidermal nerve fiber density (IENFD) was examined immunohistologically in 20 vulvar skin biopsies of patients affected by LS and in 20 control vulvar skin biopsies, in order to determine if these sensory sensations originate in changes in the epidermal innervation. Obtained results show fewer protein gene product 9.5 (PGP 9.5) positive intraepidermal nerve fibers (IENF) in LS tissues compared to controls (P = 0.004), while the number of calcitonin gene-related peptide (CGRP) positive IENF in LS was increased compared to normal vulvar tissue (P = 0.03). No differences in the number of vasoactive intestinal peptide (VIP) expressing IENF could be observed. To our best knowledge, this is the first study to describe intraepidermal nerve fiber density in vulvar LS. Significant differences in IENFD between LS and control skin samples, which have been found, point to the damage to the small nerve fibers in the disease process of LS, which may contribute to pathogenesis of LS sensory symptoms.

It is a fundamental open question as to how embryos develop into complex adult organisms with astounding reproducibility, particularly because cells are inherently variable on the molecular level. During C. elegans vulva induction, the anchor cell induces cell fate in the vulva precursor cells in a distance-dependent manner. Surprisingly, we found that initial anchor cell position was highly variable and caused variability in cell fate induction. However, we observed that vulva induction was "canalized," i.e., the variability in anchor cell position and cell fate was progressively reduced, resulting in an invariant spatial pattern of cell fates at the end of induction. To understand the mechanism of canalization, we quantified induction dynamics as a function of anchor cell position during the canalization process. Our experiments, combined with mathematical modeling, showed that canalization required a specific combination of long-range induction, lateral inhibition, and cell migration that is also found in other developmental systems., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Epidermal growth factor receptor (EGFR)-mediated activation of the canonical Ras/MAPK signaling cascade is responsible for cell proliferation and cell growth. This signaling pathway is frequently overactivated in epithelial cancers; therefore, studying regulation of this pathway is crucial not only for our fundamental understanding of cell biology but also for our ability to treat EGFR-related disease. Genetic model organisms such as Caenorhabditis elegans, a hermaphroditic nematode, played a vital role in identifying components of the EGFR/Ras/MAPK pathway and delineating their order of function, and continues to play a role in identifying novel regulators of the pathway. Polarized activation of LET-23, the C. elegans homolog of EGFR, is responsible for induction of the vulval cell fate; perturbations in this signaling pathway produce either a vulvaless or multivulva phenotype. The translucent cuticle of the nematode facilitates in vivo visualization of the receptor, revealing that localization of LET-23 EGFR is tightly regulated and linked to its function. In this chapter, we review the methods used to harness vulva development as a tool for studying EGFR signaling and trafficking in C. elegans.

Background: Provoked vestibulodynia manifests as allodynia of the vulvar vestibular mucosa. The exact mechanisms that result in altered pain sensation are unknown. Recently, we demonstrated the presence of secondary lymphoid tissue, which is the vestibule-associated lymphoid tissue in the vestibular mucosa, and showed that this tissue becomes activated in provoked vestibulodynia., Objective: The purpose of this study was to examine whether expression of intraepithelial nerve fibers and nerve growth factor are related to immune activation in provoked vestibulodynia., Study Design: Vestibular mucosal specimens were obtained from 27 patients with severe provoked vestibulodynia that was treated by vestibulectomy and from 15 control subjects. We used antibodies against the protein gene product 9.5, the neuron specific neurofilament, and nerve growth factor for immunohistochemistry to detect intraepithelial nerve fibers and nerve growth factor expressing immune cells in the vestibular mucosa. For intraepithelial nerve fibers, we determined their linear density (fiber counts per millimeter of the outer epithelial surface, protein gene product 9.5) or presence (neuron specific neurofilament). Nerve growth factor was analyzed by counting the staining-positive immune cells. Antibodies against CD20 (B lymphocytes) and CD3 (T lymphocytes) were used to identify and locate mucosal areas with increased density of lymphocytes and the presence of germinal centers (ie, signs of immune activation). B-cell activation index was used to describe the overall intensity of B-cell infiltration., Results: We found more protein gene product 9.5-positive intraepithelial fibers in vestibulodynia than in the control samples (6.3/mm [range, 0.0-15.8] vs 2.0/mm [range, 0.0-12.0]; P=.006). Neuron specific neurofilament -positive intraepithelial fibers were found in 17 of 27 vestibulodynia cases (63.0%) and in none of the control cases. Protein gene product 9.5-positive intraepithelial fibers were more common in samples with more pronounced immune activation. The density of these fibers was higher in samples with than without germinal centers (6.1/mm [range, 4.3-15.8] vs 3.0/mm [range, 0.0-13.4]; P=.020). A positive correlation between the fiber density and B-cell activation index score of the sample was found (Spearman's Rho, 0.400; P=.004; R 2 =0.128). No significant difference, however, was found in the density or presence of nerve fibers between samples with high and low T-cell densities. We identified areas of minor and major vestibular glands in 16 of the patient samples and in 1 control sample. Protein gene product 9.5-positive nerve fibers were found more often in glandular epithelium surrounded by B-cell infiltration than in glands without B cells (P=.013). Also, the presence of neuron specific neurofilament-positive fibers in glandular epithelium was associated with B-cell infiltrates (P=.053). Nerve growth factor-positive immune cells were more common in mucosal areas with than without B-cell infiltration and intraepithelial nerve fibers., Conclusion: Excessive epithelial nerve growth in provoked vestibulodynia is associated with increased B-cell infiltration and the presence of germinal centers. This supports the fundamental role of immune activation in provoked vestibulodynia., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The development of the single cell layer skin or hypodermis of Caenorhabditis elegans is an excellent model for understanding cell fate specification and differentiation. Early in C. elegans embryogenesis, six rows of hypodermal cells adopt dorsal, lateral or ventral fates that go on to display distinct behaviors during larval life. Several transcription factors are known that function in specifying these major hypodermal cell fates, but our knowledge of the specification of these cell types is sparse, particularly in the case of the ventral hypodermal cells, which become Vulval Precursor Cells and form the vulval opening in response to extracellular signals. Previously, the gene pvl-4 was identified in a screen for mutants with defects in vulval development. We found by whole genome sequencing that pvl-4 is the Paired-box gene pax-3, which encodes the sole PAX-3 transcription factor homolog in C. elegans. pax-3 mutants show embryonic and larval lethality, and body morphology abnormalities indicative of hypodermal cell defects. We report that pax-3 is expressed in ventral P cells and their descendants during embryogenesis and early larval stages, and that in pax-3 reduction-of-function animals the ventral P cells undergo a cell fate transformation and express several markers of the lateral seam cell fate. Furthermore, forced expression of pax-3 in the lateral hypodermal cells causes them to lose expression of seam cell markers. We propose that pax-3 functions in the ventral hypodermal cells to prevent these cells from adopting the lateral seam cell fate. pax-3 represents the first gene required for specification solely of the ventral hypodermal fate in C. elegans providing insights into cell type diversification., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ribosome biogenesis takes place in the nucleolus, the size of which is often coordinated with cell growth and development. However, how metazoans control nucleolar size remains largely unknown. Caenorhabditis elegans provides a good model to address this question owing to distinct tissue distribution of nucleolar sizes and a mutant, ncl-1, which exhibits larger nucleoli than wild-type worms. Here, through a series of loss-of-function analyses, we report that the nucleolar size is regulated by a circuitry composed of microRNA let-7, translation repressor NCL-1, and a major nucleolar pre-rRNA processing protein FIB-1/fibrillarin. In cooperation with RNA binding proteins PUF and NOS, NCL-1 suppressed the translation of FIB-1/fibrillarin, while let-7 targeted the 3'UTR of ncl-1 and inhibited its expression. Consequently, the abundance of FIB-1 is tightly controlled and correlated with the nucleolar size. Together, our findings highlight a novel genetic cascade by which post-transcriptional regulators interplay in developmental control of nucleolar size and function.

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type-specific "mRNA tagging" to enrich for VPC and seam cell-specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type-specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells., (Copyright © 2015 Gorrepati et al.)

Objectives: The vulva is the primary site affected in lichen sclerosus, a chronic dermatosis in women that is histologically characterized by a zone of collagen remodeling in the superior dermis. The normal physiological properties of the vulva depend on the assembly of collagen types I (COLI), III (COLIII) and V (COLV), which form heterotypic fibers, and extracellular matrix protein interactions. COLV regulates the heterotypic fiber diameter, and the preservation of its properties is important for maintaining normal tissue architecture and function. In the current work, we analyzed the expression of COLV and its relationship with COLI, COLIII, elastic fibers and extracellular matrix protein 1 in vulvar biopsies from patients with lichen sclerosus., Methods: Skin biopsies from 21 patients with lichen sclerosus, classified according to Hewitt histological criteria, were studied and compared to clinically normal vulvar tissue (N=21). Morphology, immunohistochemistry, immunofluorescence, 3D reconstruction and morphometric analysis of COLI, COLIII, COLV deposition, elastic fibers and extracellular matrix 1 expression in a zone of collagen remodeling in the superior dermis were performed., Results: A significant decrease of elastic fibers and extracellular matrix 1 protein was present in the hyalinization zone of lichen sclerosus compared to healthy controls. The non-homogeneous distribution of collagen fibers visualized under immunofluorescence in the hyalinization zone of lichen sclerosus and control skin was confirmed by histomorphometry. Lichen sclerosus dermis shows a significant increase of COLI, COLIII and COLV expression compared to the healthy controls. Significant inverse associations were found between elastic fibers and COLV and between COLV and extracellular matrix 1 expression. A direct association was found between elastic fiber content and extracellular matrix 1 expression. Tridimensional reconstruction of the heterotypic fibers of the lichen sclerosus zone of collagen remodeling confirmed the presence of densely clustered COLV., Conclusions: Increased deposition of abnormal COLV and its correlation with extracellular matrix 1 and elastic fibers suggest that COLV may be a trigger in the pathogenesis of lichen sclerosus.

The let-7 microRNA (miRNA) regulates stemness in animals ranging from worms to humans. However, the cause of the dramatic vulval rupturing phenotype of let-7 mutant C. elegans has remained unknown. Consistent with the notion that miRNAs function by coordinately tuning the expression of many targets, bursting may result from joint dysregulation of several targets, possibly in the epidermis. Alternatively, overexpression of LET-60/RAS, a key vulva development gene and a phylogenetically conserved target of let-7, may be responsible. Here, we show that let-7 functions in the vulval-uterine system to ensure vulval integrity but that regulation of most targets of let-7, including LET-60/RAS, is dispensable. Using CRISPR-Cas9 to edit endogenous let-7 target sites, we found that regulation of LIN-41/TRIM71 alone is necessary and sufficient to prevent vulval rupturing. Hence, let-7 does not function to reduce gene expression noise broadly, but to direct vulval development through extensive regulation of a single, defined target., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Malignant melanoma of the vulva is a rare disease with aggressive behavior and poor prognosis. It consist < 5% of all cases of melanoma in females, as the ratio of its manifestation, compared with the cutaneous melanoma is 1:71. Higher risk of developing melanoma of the vulva is established in white women, as the peak of the incidence is between 60 and 70 years of age. Clinically, MM of the vulva manifests as asymptomatic pigmented, rarely a pigmented lesion, as the usual clinical form is superficial spreading MM and much less common nodular MM, which is associated with a poorer prognosis in. general. The diagnosis is confirmed by histological examination. Conduction of PCR and DNA analysis for detection of BRAF mutations, NRAS mutations and KIT amplification is also appropriate. Advanced age, black race, tumor size, tumor thickness, ulceration, presence of satellite lesions, involvement of adjacent organs (vagina, urethra), and the presence of regional or distant metastases are identified as the most important prognostic markers. Radical wide excision followed by bilateral lymphadenectomy id considered as the optimal therapeutic approach.