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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.

Studies of vulval development in the nematode C. elegans have identified many genes that are involved in cell division and differentiation processes. Some of these encode components of conserved signal transduction pathways mediated by EGF, Notch, and Wnt. To understand how developmental mechanisms change during evolution, we are doing a comparative analysis of vulva formation in C. briggsae, a species that is closely related to C. elegans. Here, we report 14 mutations in 7 Multivulva (Muv) genes in C. briggsae that inhibit inappropriate division of vulval precursors. We have developed a new efficient and cost-effective gene mapping method to localize Muv mutations to small genetic intervals on chromosomes, thus facilitating cloning and functional studies. We demonstrate the utility of our method by determining molecular identities of three of the Muv genes that include orthologs of Cel-lin-1 (ETS) and Cel-lin-31 (Winged-Helix) of the EGF-Ras pathway and Cel-pry-1 (Axin), of the Wnt pathway. The remaining four genes reside in regions that lack orthologs of known C. elegans Muv genes. Inhibitor studies demonstrate that the Muv phenotype of all four new genes is dependent on the activity of the EGF pathway kinase, MEK. One of these, Cbr-lin(gu167), shows modest increase in the expression of Cbr-lin-3/EGF compared to wild type. These results argue that while Cbr-lin(gu167) may act upstream of Cbr-lin-3/EGF, the other three genes influence the EGF pathway downstream or in parallel to Cbr-lin-3. Overall, our findings demonstrate that the genetic program underlying a conserved developmental process includes both conserved and divergent functional contributions., (© 2014 Wiley Periodicals, Inc.)

Unlabelled: Vulvar cancer accounts for about 3-5% of all female genital carcinomas. TGF-P protein is a member of a superfamily of cytokines that regulate cell functions. A correlation between this protein and many neoplastic processes was reported., Objectives: In our study we analyzed TGF-β expression in vulvar tumor among patients with diagnosed squamous cell carcinoma (with and without inguinal nodes metastases)., Material and Methods: Paraffin embedded blocks obtained from vulvar tissues and inguinal nodes (from 31 patients with vulvar carcinoma FIGO ll-IV) were prepared. Next, the hematoxylin and eosin staining was performed. Monoclonal antibody NCL-TGF-beta was used for immunohistochemical tests., Results: Higher expression of TGF-beta in cancer cells corresponds to more advanced cancer stages (FIGO). A positive correlation between TGF-beta and metastases, as well as a number of inguinal nodes metastases was observed. The ratio between the number of stained cells in vulvar tumor and of inflammatory cells proved to be higher in FIGO stage III than IV Possibly TGF-beta increase in vulvar tumor contributes to the breakdown of immunological processes limiting cancer progression. Higher TGF-beta expression leads to metastasis in regional lymphatic nodes., Conclusions: TGF-beta overproduction is observed in vulvar neoplastic processes. In early stages of carcinogenesis TGF-beta inhibits cancer cell proliferation, but in more advanced stages it accelerates cancer progression by inhibiting the immunological response.

LET-23 Epidermal Growth Factor Receptor (EGFR) signaling specifies the vulval cell fates during C. elegans larval development. LET-23 EGFR localization on the basolateral membrane of the vulval precursor cells (VPCs) is required to engage the LIN-3 EGF-like inductive signal. The LIN-2 Cask/LIN-7 Veli/LIN-10 Mint (LIN-2/7/10) complex binds LET-23 EGFR, is required for its basolateral membrane localization, and therefore, vulva induction. Besides the LIN-2/7/10 complex, the trafficking pathways that regulate LET-23 EGFR localization have not been defined. Here we identify vh4, a hypomorphic allele of agef-1, as a strong suppressor of the lin-2 mutant Vulvaless (Vul) phenotype. AGEF-1 is homologous to the mammalian BIG1 and BIG2 Arf GTPase guanine nucleotide exchange factors (GEFs), which regulate secretory traffic between the Trans-Golgi network, endosomes and the plasma membrane via activation of Arf GTPases and recruitment of the AP-1 clathrin adaptor complex. Consistent with a role in trafficking we show that AGEF-1 is required for protein secretion and that AGEF-1 and the AP-1 complex regulate endosome size in coelomocytes. The AP-1 complex has previously been implicated in negative regulation of LET-23 EGFR, however the mechanism was not known. Our genetic data indicate that AGEF-1 is a strong negative regulator of LET-23 EGFR signaling that functions in the VPCs at the level of the receptor. In line with AGEF-1 being an Arf GEF, we identify the ARF-1.2 and ARF-3 GTPases as also negatively regulating signaling. We find that the agef-1(vh4) mutation results in increased LET-23 EGFR on the basolateral membrane in both wild-type and lin-2 mutant animals. Furthermore, unc-101(RNAi), a component of the AP-1 complex, increased LET-23 EGFR on the basolateral membrane in lin-2 and agef-1(vh4); lin-2 mutant animals. Thus, an AGEF-1/Arf GTPase/AP-1 ensemble functions opposite the LIN-2/7/10 complex to antagonize LET-23 EGFR basolateral membrane localization and signaling.

Background: Ryk is a subfamily of receptor tyrosine kinases, which along with Frizzled and Ror, function as Wnt receptors. Vertebrate Ryk intracellular domain (ICD) is released from the cell membrane by a proteolytic cleavage in the transmembrane region and localizes to the nucleus. In C. elegans, Ryk is encoded by the lin-18 gene and regulates the polarity of the P7.p vulval cell., Results: Based on Western blots, we were unable to detect the presence of the cleaved LIN-18 ICD fragment. Functional assays found that LIN-18 intracellular domain is not absolutely required for LIN-18 function, consistent with previous results. However, overexpression of the LIN-18 intracellular domain fragment (LIN-18ICD) weakly enhanced the phenotype of lin-18 loss-of-function mutants. Furthermore, this activity was specific to the serine-rich juxtamembrane region. We also found that the nuclear localization of LIN-18ICD fragment can be regulated by Wnt pathway components including CAM-1/Ror, and by PAR-5/14-3-3., Conclusions: Release of LIN-18ICD by cleavage at the membrane is not the main mechanism of LIN-18 signaling in vulval cells. However, our results suggest that LIN-18 intracellular domain interacts with Wnt pathway components and a 14-3-3 protein and likely plays a minor role in LIN-18 signaling., (© 2014 Wiley Periodicals, Inc.)

Sebaceous gland hyperplasia is a common skin condition, very rarely reported in the female genital region. We present 13 cases from 12 patients, the first case series of sebaceous gland hyperplasia of the vulva. Differences in age at presentation and clinical presentation compared with classic sebaceous gland hyperplasia from the head and neck region were noted. Also, it was rarely included in the clinical differential diagnosis. Immunohistochemical studies to determine any possible association with the Muir-Torre syndrome were performed and mismatch repair protein loss was not identified.

Large gaps in basement membrane (BM) occur during organ remodelling and cancer cell invasion. Whether dividing cells, which temporarily reduce their attachment to BM, influence these breaches is unknown. Here we analyse uterine-vulval attachment during development across 21 species of rhabditid nematodes and find that the BM gap that forms between these organs is always bounded by a non-dividing vulval cell. Through cell cycle manipulation and live cell imaging in Caenorhabditis elegans, we show that actively dividing vulval cells facilitate enlargement of this breach by promoting BM movement. In contrast, targeted cell cycle arrest halts BM movement and limits gap opening. Further, we demonstrate that the BM component laminin accumulates at the BM gap edge and promotes increased integrin levels in non-dividing vulval cells, stabilizing gap position. Together, these studies reveal that cell division can be used as a mechanism to regulate BM breaches, thus controlling the exchange of cells between tissues.

The proper formation and function of an organ is dependent on the specification and integration of multiple cell types and tissues. An example of this is the Caenorhabditis elegans hermaphrodite egg-laying system, which requires coordination between the vulva, uterus, neurons, and musculature. While the genetic constituents of the first three components have been well studied, little is known about the molecular mechanisms underlying the specification of the egg-laying musculature. The egg-laying muscles are non-striated in nature and consist of sixteen cells, four each of type I and type II vulval muscles and uterine muscles. These 16 non-striated muscles exhibit distinct morphology, location, synaptic connectivity and function. Using an RNAi screen targeting the putative transcription factors in the C. elegans genome, we identified a number of novel factors important for the diversification of these different types of egg-laying muscles. In particular, we found that RNAi knockdown of lag-1, which encodes the sole C. elegans ortholog of the transcription factor CSL (CBF1, Suppressor of Hairless, LAG-1), an effector of the LIN-12/Notch pathway, led to the production of extra type I vulval muscles. Similar phenotypes were also observed in animals with down-regulation of the Notch receptor LIN-12 and its DSL (Delta, Serrate, LAG-2) ligand LAG-2. The extra type I vulval muscles in animals with reduced LIN-12/Notch signaling resulted from a cell fate transformation of type II vulval muscles to type I vulval muscles. We showed that LIN-12/Notch was activated in the undifferentiated type II vulval muscle cells by LAG-2/DSL that is likely produced by the anchor cell (AC). Our findings provide additional evidence highlighting the roles of LIN-12/Notch signaling in coordinating the formation of various components of the functional C. elegans egg-laying system. We also identify multiple new factors that play critical roles in the proper specification of the different types of egg-laying muscles., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Hox genes encode master regulators of regional fate specification during early metazoan development. Much is known about the initiation and regulation of Hox gene expression in Drosophila and vertebrates, but less is known in the non-arthropod invertebrate model system, C. elegans. The C. elegans Hox gene lin-39 is required for correct fate specification in the midbody region, including the Vulval Precursor Cells (VPCs). To better understand lin-39 regulation and function, we aimed to identify transcription factors necessary for lin-39 expression in the VPCs, and in particular sought factors that initiate lin-39 expression in the embryo., Results: We used the yeast one-hybrid (Y1H) method to screen for factors that bound to 13 fragments from the lin-39 region: twelve fragments contained sequences conserved between C. elegans and two other nematode species, while one fragment was known to drive reporter gene expression in the early embryo in cells that generate the VPCs. Sixteen transcription factors that bind to eight lin-39 genomic fragments were identified in yeast, and we characterized several factors by verifying their physical interactions in vitro, and showing that reduction of their function leads to alterations in lin-39 levels and lin-39::GFP reporter expression in vivo. Three factors, the orphan nuclear hormone receptor NHR-43, the hypodermal fate regulator LIN-26, and the GATA factor ELT-6 positively regulate lin-39 expression in the embryonic precursors to the VPCs. In particular, ELT-6 interacts with an enhancer that drives GFP expression in the early embryo, and the ELT-6 site we identified is necessary for proper embryonic expression. These three factors, along with the factors ZTF-17, BED-3 and TBX-9, also positively regulate lin-39 expression in the larval VPCs., Conclusions: These results significantly expand the number of factors known to directly bind and regulate lin-39 expression, identify the first factors required for lin-39 expression in the embryo, and hint at a positive feedback mechanism involving GATA factors that maintains lin-39 expression in the vulval lineage. This work indicates that, as in other organisms, the regulation of Hox gene expression in C. elegans is complicated, redundant and robust.

Aims: To investigate cyclin D1 and fascin immunoreactivity in normal, reactive and neoplastic vulvar skin correlating the findings with p16 protein and Ki67 expression., Methods: 66 vulvar biopsy or resection specimens demonstrating normal appearances, reactive epidermal changes, usual-type vulvar intraepithelial neoplasia (uVIN), differentiated-type VIN (dVIN), p16-positive squamous cell carcinoma (SCC) and p16-negative SCC were examined immunohistochemically for cyclin D1, fascin, Ki67 and p16 protein. Where applicable, expression patterns were compared in microanatomically distinct areas, particularly at the invasive front (deep tumour margin) of SCC., Results: Normal epidermis showed parabasal Ki67 and cyclin D1 staining while fascin labelled cells in the lower one-third of the epithelium. Reactive and dVIN specimens demonstrated mildly increased Ki67 and cyclin D1 expression that maintained parabasal polarity, whereas uVIN and p16-positive SCC were characterised by loss of cyclin D1 staining. However, in 14 of 20 p16-positive SCC small infiltrative tumour groups and single infiltrating cells at the invasive front showed a cyclin D1-positive/ Ki67-negative phenotype. In contrast, p16-negative SCC generally showed diffuse and concordant cyclin D1 and Ki67 labelling, including at the invasive margin. Fascin expression was increased in all VIN and SCC lesions., Conclusions: Variations in cyclin D1 and Ki67 expression between p16-positive and p16-negative vulvar SCCs suggest different mechanisms of invasion in these tumour subgroups. Fascin is upregulated in vulvar squamous neoplasia but immunostaining does not discriminate in situ from invasive lesions nor putative human papilloma virus (HPV)-associated and HPV-independent SCCs.

Background: The role of p16INK4A expression in uterine cervix cancer is well established. In the remaining female lower genital tract cancers, the importance of p16INK4A up-regulation is less clear. In our study, we analyzed the role of p16INK4A expression and HPV infection in carcinomas of the vulva and the vagina in Tunisian women., Materials and Methods: We conducted a retrospective study of 30 carcinomas including 15 vulvar squamous cell carcinomas (SCCs) and 15 vaginal SCCs. Immunohistochemistry was used to determine p16INK4A expression. HPV detection and typing was by in situ hybridization., Results: p16INK4A expression was detected in 86.7% of vaginal SCCs with a strong and diffuse immunostaining in 60% of cases, and also in 73.3% of vulvar SCCs with focal immunoreactivity in 53.3% The association between p16INK4A expression and HPV infection was significant in vaginal SCCs (p=0.001) but not vulvar SCCs (p>0.05)., Conclusions: p16INK4A expression could be used as a useful marker for HPV positivity in vaginal SCCs similar to that described in uterine cervix cancers. However, our data support the presence of 2 different mechanisms for p16INK4A expression in HPV-related and HPV-unrelated vulvar carcinomas.

Context: Shortly after birth, pituitary gonadotropin secretion transiently activates in both sexes, and this surge is more robust in preterm (PT) than in full-term (FT) infants. In boys, the gonadotropin surge is associated with testicular activity and is considered an important part of normal reproductive development. In contrast, gonadal activation and its consequences in infant girls are poorly understood., Objective: Our objective was to evaluate the association of postnatal ovarian activity with simultaneous changes in estrogen target tissues in FT and PT girls., Patients and Methods: We measured urinary estradiol (E₂) levels in 29 FT and 34 PT girls using a mass spectrometric method from 1 week (D7) to 6 months of age (M1-M6). To assess the contribution of ovarian E₂ on urinary E₂ levels, the levels in girls were compared with the levels of boys of similar cohorts (29 FT and 33 PT boys). E₂ levels were compared with simultaneous changes in estrogenic target tissues including mammary glands in both sexes and uterus and vulvar epithelium in girls., Results: Median urinary E₂ levels increased after D7 in girls, but not in boys. Mammary gland diameter was larger in girls than in boys from M4 in FT (P < .001) and M2 in PT infants (P < .0001). In PT girls, E₂ levels increased at term and were then higher than those in FT girls (P < .0001). Urinary E₂ levels in PT girls were positively associated with mammary gland and uterine growth., Conclusions: These findings indicate that gonadal steroidogenesis activates during the postnatal gonadotropin surge in girls. In addition, the resulting elevated E₂ levels affect target tissues, suggesting that postnatal pituitary-ovarian activation plays a role in normal female reproductive development.

Unregulated expression of microRNAs is well known and has already been demonstrated in many tumor types. However, in vulvar carcinoma this field has been unknown territory. Our study characterizes microRNA in vulvar tumors through an expression profile of 754 miRNAs, relating this with clinical and anatomopathologic data, and presence of HPV infection. Twenty HPV-negative and 20 HPV-positive samples, genotyped for high-risk HPVs (HPV16, 18, 31, 33) and a pool of seven normal vulvar skin samples were used for the identification of differentially expressed miRNAs by TLDA Quantitative Real Time PCR (qRT-PCR). Twenty-five differentially expressed microRNAs between HPV-positive and HPV-negative groups and 79 differentially expressed on the tumor compared with normal samples were obtained. A network between microRNA expression profiles and putative target mRNAs predicted by target prediction algorithms and previously demonstrated as relevant in vulvar carcinomas, such as TP53, RB, PTEN, and EGFR was constructed. Downregulation of both miR-223-5p and miR-19-b1-5p were correlated with the presence of lymph node metastasis; downregulation of miR-100-3p and miR-19-b1-5p were correlated with presence of vascular invasion; overexpression of miR-519b and miR-133a were associated with advanced FIGO staging. In conclusion, our study demonstrates that microRNAs may be clinically important in vulvar carcinomas and our findings may help for further studies on functional implications of miRNA deregulation in this type of cancer., (©2013 AACR.)

Ospemifene is a unique tissue-selective estrogen agonist/antagonist (also known as a selective estrogen receptor modulator [SERM]) with demonstrated efficacy in Phase 3 studies of postmenopausal women with vulvar and vaginal atrophy (VVA). This report describes preclinical studies on the effects of ospemifene in the ovariectomized (OVX) rat model of menopause. Ospemifene (10mg/kg/day) and the SERM comparator, raloxifene (10mg/kg/day) were administered for 2 weeks and both increased vaginal weight; ospemifene was more effective than raloxifene. In addition, ospemifene had a greater effect on increasing vaginal epithelial height compared with raloxifene. The effect on uterine weight was less pronounced for both ospemifene and raloxifene. The ED50 of ospemifene on vaginal epithelial height was 0.39mg/kg/day and the magnitude was nearly the same as was seen with the positive control, 17α-ethinyl estradiol (EE2). In a histological analysis of ospemifene-treated rat vaginas, basal cells were overlaid by 2 to 3 cell layers of thickened goblet-like mucified cells apically; however, the cornification observed with EE2 was absent. Estrogenic activity of ospemifene was confirmed by upregulation of progesterone receptors in vaginal epithelium and stroma. Ospemifene showed similar affinity for estrogen receptor (ER)-α and ER-β, but an overall lower affinity than estradiol. Ospemifene antagonized estrogen response element (ERE)-mediated transactivation on MCF-7 cells, confirming its anti-estrogenic activity in breast cancer cells. The dose response for ospemifene in the rat is consistent with that observed in clinical studies of ospemifene 30 and 60mg, showing that the OVX rat is a highly predictive model of SERM activity in postmenopausal VVA., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Vulvar and vaginal atrophy (VVA) is a chronic, progressive medical condition prevalent among postmenopausal women, which produces symptoms such as dyspareunia, vaginal dryness, and vaginal irritation. Currently, the only prescription options are systemic and vaginal estrogen therapies that may be limited by concerns about long-term safety and breast cancer risk. Ospemifene is a tissue-selective estrogen agonist/antagonist (a selective estrogen receptor modulator) recently approved by the US Food and Drug Administration for treatment of dyspareunia, a symptom of VVA, due to menopause. Ospemifene, the first nonestrogen oral treatment for this indication, may provide an alternative to treatment with estrogen. Animal models with ospemifene suggest an inhibitory effect on growth of malignant breast tissue, but animal data cannot necessarily be extrapolated to humans. Clinical trials, including 3 long-term studies assessing the overall safety of ospemifene, support that ospemifene is generally well tolerated, with beneficial effects on the vagina, neutral effects on the breast, and minimal effects on the endometrium.

Paraganglioma is a neuroendocrine neoplasm, which is extremely rare in the vulva and only one case has been reported. Here we present a case of vulvar paraganglioma in a 48-year-old woman and a literature review. The patient found a lump located in the genitals below the symphysis pubis 3 months before presentation when she complained that the lump was increasing in size. A 3.2 cm x 2.3 cm x 1.5 cm nodule was excised from subcutaneous soft tissue in the vulva. Microscopy showed a diversity of cell morphologies and structures in the rich vascular network of the tumor separated the chief cells into round cell nests (Zellballen pattern). Some areas of the tumor presented epithelioid and spindle-shaped cells with increased cell density and indistinct structural characteristics. Hyaline degeneration of collagen fibers or mucoid degeneration was found in tumor interstitium. Immunohistochemical staining showed diffused expression of synaptophysin in the chief cells, focal expression of S-100 protein in the sustentacular cells and high expression of CD34 in the vascular components. Based on morphological and immunohistochemical results, a rare paraganglioma of the vulva was diagnosed.

The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The Caenorhabditis elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. Here, we show that the fibroblast growth factor (FGF) pathway acts in concert with the Frizzled homolog LIN-17 to influence the localization of SYS-1, a component of the Wnt/β-catenin asymmetry pathway, indirectly through the regulation of cwn-1. The source of the FGF ligand is the primary vulval precursor cell (VPC) P6.p, which controls the orientation of the neighboring secondary VPC P7.p by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt CWN-1 is expressed in the posterior body wall muscle of the worm as well as in the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the highest Wnt signal. Using single molecule fluorescence in situ hybridization, we show that the FGF pathway regulates the expression of cwn-1 in the SMs. These results demonstrate an interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity, and highlight the promiscuous nature of Wnts and the importance of Wnt gradient directionality within C. elegans.

The identification and characterization of age-related degenerative changes is a critical goal because it can elucidate mechanisms of aging biology and contribute to understanding interventions that promote longevity. Here, we document a novel, age-related degenerative change in C. elegans hermaphrodites, an important model system for the genetic analysis of longevity. Matricidal hatching--intra-uterine hatching of progeny that causes maternal death--displayed an age-related increase in frequency and affected ~70% of mated, wild-type hermaphrodites. The timing and incidence of matricidal hatching were largely independent of the levels of early and total progeny production and the duration of male exposure. Thus, matricidal hatching appears to reflect intrinsic age-related degeneration of the egg-laying system rather than use-dependent damage accumulation. Consistent with this model, mutations that extend longevity by causing dietary restriction significantly delayed matricidal hatching, indicating age-related degeneration of the egg-laying system is controlled by nutrient availability. To identify the underlying tissue defect, we analyzed serotonin signaling that triggers vulval muscle contractions. Mated hermaphrodites displayed an age-related decline in the ability to lay eggs in response to exogenous serotonin, indicating that vulval muscles and/or a further downstream function that is necessary for egg laying degenerate in an age-related manner. By characterizing a new, age-related degenerative event displayed by C. elegans hermaphrodites, these studies contribute to understanding a frequent cause of death in mated hermaphrodites and establish a model of age-related reproductive complications that may be relevant to the birthing process in other animals such as humans., (© 2013 John Wiley & Sons Ltd and the Anatomical Society.)

Though critical to normal development and cancer metastasis, how cells traverse basement membranes is poorly understood. A central impediment has been the challenge of visualizing invasive cell interactions with basement membrane in vivo. By developing live-cell imaging methods to follow anchor cell (AC) invasion in Caenorhabditis elegans, we identify F-actin-based invadopodia that breach basement membrane. When an invadopodium penetrates basement membrane, it rapidly transitions into a stable invasive process that expands the breach and crosses into the vulval tissue. We find that the netrin receptor UNC-40 (DCC) specifically enriches at the site of basement membrane breach and that activation by UNC-6 (netrin) directs focused F-actin formation, generating the invasive protrusion and the cessation of invadopodia. Using optical highlighting of basement membrane components, we further demonstrate that rather than relying solely on proteolytic dissolution, the AC's protrusion physically displaces basement membrane. These studies reveal an UNC-40-mediated morphogenetic transition at the cell-basement membrane interface that directs invading cells across basement membrane barriers.

The Caenorhabditis elegans vulval precursor cells (VPCs) offer a paradigm for investigating how multipotency of progenitor cells is maintained during periods of quiescence. The VPCs are born in the first larval stage. When hermaphrodites are grown under favorable conditions, the EGF-mediated "inductive" signal and the LIN-12/Notch-mediated "lateral" signal confer a precise spatial pattern of distinct vulval cell fates in the third larval stage, a day after hatching. Under adverse conditions, hermaphrodites undergo a prolonged quiescent period as dauer larvae, which can endure for several months with progenitor cells such as VPCs in developmental arrest. If favorable conditions ensue, larvae recover and resume development as postdauer third stage larvae, with the same VPC spatial-patterning events as in continuously developing third stage larvae. Here, we identify several consequences of dauer life history for VPC specification. In wild-type dauers, VPCs undergo a phenomenon reminiscent of natural direct reprogramming to maintain or reestablish multipotency; they acquire an active block to signal transduction by EGF receptor and LIN-12/Notch and have a different mechanism for regulating transcription of the lateral signal. Furthermore, DAF-16/FoxO, a target of insulin/insulin-like growth factor signaling, is required to promote VPC fate plasticity during dauer and for normal vulval patterning after passage through dauer, suggesting that DAF-16/FoxO coordinates environment and life history with plasticity of cell fate.

Biological systems may perform reproducibly to generate invariant outcomes, despite external or internal noise. One example is the C. elegans vulva, in which the final cell fate pattern is remarkably robust. Although this system has been extensively studied and the molecular network underlying cell fate specification is well understood, very little is known in quantitative terms. Here, through pathway dosage modulation and single molecule fluorescence in situ hybridization, we show that the system can tolerate a 4-fold variation in genetic dose of the upstream signaling molecule LIN-3/epidermal growth factor (EGF) without phenotypic change in cell fate pattern. Furthermore, through tissue-specific dosage perturbations of the EGF and Notch pathways, we determine the first-appearing patterning errors. Finally, by combining different doses of both pathways, we explore how quantitative pathway interactions influence system behavior. Our results highlight the feasibility and significance of launching experimental studies of robustness and quantitative network analysis in genetically tractable, multicellular eukaryotes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Metazoans display remarkable conservation of gene families, including growth factors, yet somehow these genes are used in different ways to generate tremendous morphological diversity. While variations in the magnitude and spatio-temporal aspects of signaling by a growth factor can generate different body patterns, how these signaling variations are organized and coordinated during development is unclear. Basic body plans are organized by the end of gastrulation and are refined as limbs, organs, and nervous systems co-develop. Despite their proximity to developing tissues, neurons are primarily thought to act after development, on behavior. Here, we show that in Caenorhabditis elegans, the axonal projections of neurons regulate tissue progenitor responses to Wnts so that certain organs develop with the correct morphology at the right axial positions. We find that foreshortening of the posteriorly directed axons of the two canal-associated neurons (CANs) disrupts mid-body vulval morphology, and produces ectopic vulval tissue in the posterior epidermis, in a Wnt-dependent manner. We also provide evidence that suggests that the posterior CAN axons modulate the location and strength of Wnt signaling along the anterior-posterior axis by employing a Ror family Wnt receptor to bind posteriorly derived Wnts, and hence, refine their distributions. Surprisingly, despite high levels of Ror expression in many other cells, these cells cannot substitute for the CAN axons in patterning the epidermis, nor can cells expressing a secreted Wnt inhibitor, SFRP-1. Thus, unmyelinated axon tracts are critical for patterning the C. elegans body. Our findings suggest that the evolution of neurons not only improved metazoans by increasing behavioral complexity, but also by expanding the diversity of developmental patterns generated by growth factors such as Wnts., Competing Interests: The authors have declared that no competing interests exist.

The nematode Caenorhabditis briggsae is an excellent model organism for the comparative analysis of gene function and developmental mechanisms. To study the evolutionary conservation and divergence of genetic pathways mediating vulva formation, we screened for mutations in C. briggsae that cause the egg-laying defective (Egl) phenotype. Here, we report the characterization of 13 genes, including three that are orthologs of Caenorhabditis elegans unc-84 (SUN domain), lin-39 (Dfd/Scr-related homeobox), and lin-11 (LIM homeobox). Based on the morphology and cell fate changes, the mutants were placed into four different categories. Class 1 animals have normal-looking vulva and vulva-uterine connections, indicating defects in other components of the egg-laying system. Class 2 animals frequently lack some or all of the vulval precursor cells (VPCs) due to defects in the migration of P-cell nuclei into the ventral hypodermal region. Class 3 animals show inappropriate fusion of VPCs to the hypodermal syncytium, leading to a reduced number of vulval progeny. Finally, class 4 animals exhibit abnormal vulval invagination and morphology. Interestingly, we did not find mutations that affect VPC induction and fates. Our work is the first study involving the characterization of genes in C. briggsae vulva formation, and it offers a basis for future investigations of these genes in C. elegans.

Morphogenesis is a developmental phase during which cell fates are executed. Mechanical forces shaping individual cells play a key role during tissue morphogenesis. By investigating morphogenesis of the Caenorhabditis elegans hermaphrodite vulva, we show that the force-generating actomyosin network is differentially regulated by NOTCH and EGFR/RAS/MAPK signaling to shape the vulval tube. NOTCH signaling activates expression of the RHO kinase LET-502 in the secondary cell lineage through the ETS-family transcription factor LIN-1. LET-502 induces actomyosin-mediated contraction of the apical lumen in the secondary toroids, thereby generating a dorsal pushing force. In contrast, MAPK signaling in the primary lineage downregulates LET-502 RHO kinase expression to prevent toroid contraction and allow the gonadal anchor cell to expand the dorsal lumen of the primary toroids. The antagonistic action of the MAPK and NOTCH pathways thus controls vulval tube morphogenesis linking cell fate specification to morphogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cyclin D1 (CCND1) belongs to the family of D-type cyclins involved in cell cycle progression, transcriptional regulation, and cell migration. CCND1 was found to be amplified and overexpressed in a variety of cancers, including some vulvar carcinoma cell lines. To determine the relationship of CCND1 copy number changes and CCND1 protein expression with clinicopathologic features and prognosis, 183 vulvar carcinomas were analyzed on a tissue microarray. Amplification was observed in 32 (22.4%) vulvar cancer specimens and was statistically related to the presence of regional lymph node metastases (P < .001). Detectable CCND1 expression was found in 139 (83.2%) of vulvar carcinomas, and 76 (45.5%) exhibited a moderate or strong expression. Increased levels of CCND1 expression were significantly related to higher patient age (P = .013), positive pN category (P = .004), and negative human papillomavirus status (P < .001). Basaloid as well as verrucous, warty-type, and mixed vulvar carcinomas showed lower CCND1 expression levels than keratinizing or nonkeratinizing tumors (P < .001 and P = .032, respectively). Elevated CCND1 expression levels and amplification of the CCND1 gene were closely connected in the present analysis (P < .001). Patient prognosis was independent from CCND1 amplification status and expression level (P = .57 each). In conclusion, CCND1 is amplified and overexpressed in a substantial proportion of vulvar carcinomas and associated with the occurrence of locoregional lymph node metastases, especially in human papillomavirus-negative tumors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The spatial patterning of three cell fates in a row of competent cells is exemplified by vulva development in the nematode Caenorhabditis elegans. The intercellular signaling network that underlies fate specification is well understood, yet quantitative aspects remain to be elucidated. Quantitative models of the network allow us to test the effect of parameter variation on the cell fate pattern output. Among the parameter sets that allow us to reach the wild-type pattern, two general developmental patterning mechanisms of the three fates can be found: sequential inductions and morphogen-based induction, the former being more robust to parameter variation. Experimentally, the vulval cell fate pattern is robust to stochastic and environmental challenges, and minor variants can be detected. The exception is the fate of the anterior cell, P3.p, which is sensitive to stochastic variation and spontaneous mutation, and is also evolving the fastest. Other vulval precursor cell fates can be affected by mutation, yet little natural variation can be found, suggesting stabilizing selection. Despite this fate pattern conservation, different Caenorhabditis species respond differently to perturbations of the system. In the quantitative models, different parameter sets can reconstitute their response to perturbation, suggesting that network variation among Caenorhabditis species may be quantitative. Network rewiring likely occurred at longer evolutionary scales.

Several clinical-pathological parameters have been related to survival of patients with invasive squamous cell carcinoma of the vulva, whereas few studies have investigated the ability of biological variables to predict the clinical outcome of these patients. The present paper reviews the literature data on the prognostic relevance of lymph node-related parameters, primary tumor-related parameters, FIGO stage, blood variables, and tissue biological variables. Regarding these latter, the paper takes into account the analysis of DNA content, cell cycle-regulatory proteins, apoptosis-related proteins, epidermal growth factor receptor [EGFR], and proteins that are involved in tumor invasiveness, metastasis and angiogenesis. At present, the lymph node status and FIGO stage according to the new 2009 classification system are the main predictors for vulvar squamous cell carcinoma, whereas biological variables do not have yet a clinical relevance and their role is still investigational., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Developmental signaling networks are composed of dozens of components whose interactions are very difficult to quantify in an embryo. Geometric reasoning enumerates a discrete hierarchy of phenotypic models with a few composite variables whose parameters may be defined by in vivo data. Vulval development in the nematode Caenorhabditis elegans is a classic model for the integration of two signaling pathways; induction by EGF and lateral signaling through Notch. Existing data for the relative probabilities of the three possible terminal cell types in diverse genetic backgrounds as well as timed ablation of the inductive signal favor one geometric model and suffice to fit most of its parameters. The model is fully dynamic and encompasses both signaling and commitment. It then predicts the correlated cell fate probabilities for a cross between any two backgrounds/conditions. The two signaling pathways are combined additively, without interactions, and epistasis only arises from the nonlinear dynamical flow in the landscape defined by the geometric model. In this way, the model quantitatively fits genetic experiments purporting to show mutual pathway repression. The model quantifies the contributions of extrinsic vs. intrinsic sources of noise in the penetrance of mutant phenotypes in signaling hypomorphs and explains available experiments with no additional parameters. Data for anchor cell ablation fix the parameters needed to define Notch autocrine signaling.

The Caenorhabditis elegans vulva has served as a paradigm for how conserved developmental pathways, such as EGF-Ras-MAPK, Notch and Wnt signaling, participate in networks driving animal organogenesis. Here, we discuss an emerging direction in the field, which places vulva research in a quantitative and microevolutionary framework. The final vulval cell fate pattern is known to be robust to change, but only recently has the variation of vulval traits been measured under stochastic, environmental or genetic variation. Whereas the resulting cell fate pattern is invariant among rhabditid nematodes, recent studies indicate that the developmental system has accumulated cryptic variation, even among wild C. elegans isolates. Quantitative differences in the signaling network have emerged through experiments and modeling as the driving force behind cryptic variation in Caenorhabditis species. On a wider evolutionary scale, the establishment of new model species has informed about the presence of qualitative variation in vulval signaling pathways., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objectives: To evaluate the expression of different matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in vulvar lichen sclerosus (LS), a chronic dermatosis in women, histologically characterized by a zone of collagen remodeling in the superior dermis., Study Design: Analysis of the expression of different MMPs (MMP-1, -2, -9 and -13) and TIMPs (TIMP-1 and -2) by reverse transcriptase-polymerase chain reaction (RT-PCR) in vulvar biopsies from patients with LS (n=11), classified according to Hewitt histological criteria and compared with clinically normal vulvar tissue (n=5), and the immunohistochemistry of MMP-2 and -9 and TIMP-1 and -2 distribution in the remodeling zone of LS (n=31) and in clinically normal vulvar tissue (n=28)., Results: Although no statistically significant difference between LS and normal skin groups at the mRNA level of MMP and TIMP transcripts was shown, an increase in the immunodistribution of MMP-2 and -9 and TIMP-1 and -2 in LS compared to normal vulvar skin was observed., Conclusions: These results suggest that these molecules could be related to the process of cutaneous collagen remodeling in LS pathology., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objective: To investigate the expression of endogenous hypoxia-related markers identified as being involved in vulvar squamous cell carcinoma (VSCC)., Methods: We performed immunohistochemical staining of hypoxia-inducible factor-1α(HIF-1α), glucose transporter-1 (GLUT-1), carbonic anhydrase 9 (CA-9) and vascular endothelial growth factor (VEGF), on tissue sections of 25 VSCC patients, 10 vulvar intraepithelial neoplasia (VIN) patients and 12 healthy controls., Results: HIF-1α expression was found in all sections, with no significant difference between controls, VIN and VSCC sections (all P<0.05). Glut-1 expression was found in 25% of control, 90% of VIN and 100% of VSCC sections. A significant difference between control and VIN or VSCC was observed (all P<0.05), while no difference was found between VIN and VSCC sections (P>0.05). CA-9 expression was negative in control sections, but it was found in 30% of VIN sections and 52% of VSCC sections with strong staining. Similarly, CA-9 expression also showed obvious differences between controls and VIN or VSCC sections (all P<0.05). However, there was no significant difference between VIN and VSCC (P>0.05). There were only 25% of control sections with weak VEGF expression, while strong staining was found in about 60% of VIN sections and 25% of VSCC sections (all P<0.05). In addition, a difference was also found between VIN and VSCC sections (P<0.05)., Conclusion: Expression of endogenous hypoxia markers (HIF-1α, GLUT-1, CA-9 and VEGF) might be involved in the malignant progression of VSCC.

Robust biological systems are expected to accumulate cryptic genetic variation that does not affect the system output in standard conditions yet may play an evolutionary role once phenotypically expressed under a strong perturbation. Genetic variation that is cryptic relative to a robust trait may accumulate neutrally as it does not change the phenotype, yet it could also evolve under selection if it affects traits related to fitness in addition to its cryptic effect. Cryptic variation affecting the vulval intercellular signaling network was previously uncovered among wild isolates of Caenorhabditis elegans. Using a quantitative genetic approach, we identify a non-synonymous polymorphism of the previously uncharacterized nath-10 gene that affects the vulval phenotype when the system is sensitized with different mutations, but not in wild-type strains. nath-10 is an essential protein acetyltransferase gene and the homolog of human NAT10. The nath-10 polymorphism also presents non-cryptic effects on life history traits. The nath-10 allele carried by the N2 reference strain leads to a subtle increase in the egg laying rate and in the total number of sperm, a trait affecting the trade-off between fertility and minimal generation time in hermaphrodite individuals. We show that this allele appeared during early laboratory culture of N2, which allowed us to test whether it may have evolved under selection in this novel environment. The derived allele indeed strongly outcompetes the ancestral allele in laboratory conditions. In conclusion, we identified the molecular nature of a cryptic genetic variation and characterized its evolutionary history. These results show that cryptic genetic variation does not necessarily accumulate neutrally at the whole-organism level, but may evolve through selection for pleiotropic effects that alter fitness. In addition, cultivation in the laboratory has led to adaptive evolution of the reference strain N2 to the laboratory environment, which may modify other phenotypes of interest., Competing Interests: The authors have declared that no competing interests exist.

Anogenital malignancy has a significant association with high-risk mucosal alpha-human papillomaviruses (alpha-PV), particularly HPV 16 and 18 whereas extragenital SCC has been linked to the presence of cutaneous beta and gamma-HPV types. Vulval skin may be colonised by both mucosal and cutaneous (beta-, mu-, nu- and gamma-) PV types, but there are few systematic studies investigating their presence and their relative contributions to vulval malignancy. Dysregulation of AKT, a serine/threonine kinase, plays a significant role in several cancers. Mucosal HPV types can increase AKT phosphorylation and activity whereas cutaneous HPV types down-regulate AKT1 expression, probably to weaken the cornified envelope to promote viral release. We assessed the presence of mucosal and cutaneous HPV in vulval malignancy and its relationship to AKT1 expression in order to establish the corresponding HPV and AKT1 profile of normal vulval skin, vulval intraepithelial neoplasia (VIN) and vulval squamous cell carcinoma (vSCC). We show that HPV16 is the principle HPV type present in VIN, there were few detectable beta types present and AKT1 loss was not associated with the presence of these cutaneous HPV. We show that HPV16 early gene expression reduced AKT1 expression in transgenic mouse epidermis. AKT1 loss in our VIN cohort correlated with presence of high copy number, episomal HPV16. Maintained AKT1 expression correlated with low copy number, an increased frequency of integration and increased HPV16E7 expression, a finding we replicated in another untyped cohort of vSCC. Since expression of E7 reflects tumour progression, these findings suggest that AKT1 loss associated with episomal HPV16 may have positive prognostic implications in vulval malignancy.

Vulval development in Caenorhabditis elegans serves as an excellent model to examine the crosstalk between different conserved signaling pathways that are deregulated in human cancer. The concerted action of the RAS/MAPK, NOTCH, and WNT pathways determines an invariant pattern of cell fates in three vulval precursor cells. We have discovered a novel form of crosstalk between components of the Insulin and the RAS/MAPK pathways. The insulin receptor DAF-2 stimulates, while DAF-18 PTEN inhibits, RAS/MAPK signaling in the vulval precursor cells. Surprisingly, the inhibitory activity of DAF-18 PTEN on the RAS/MAPK pathway is partially independent of its PIP(3) lipid phosphatase activity and does not involve further downstream components of the insulin pathway, such as AKT and DAF-16 FOXO. Genetic and biochemical analyses indicate that DAF-18 negatively regulates vulval induction by inhibiting MAPK activation. Thus, mutations in the PTEN tumor suppressor gene may result in the simultaneous hyper-activation of two oncogenic signaling pathways., Competing Interests: The authors have declared that no competing interests exist.

The Rab7 GTPase regulates late endosome trafficking of the Epidermal Growth Factor Receptor (EGFR) to the lysosome for degradation. However, less is known about how Rab7 activity, functioning late in the endocytic pathway, affects EGFR signaling. Here we used Caenorhabditis elegans vulva cell fate induction, a paradigm for genetic analysis of EGFR/Receptor Tyrosine Kinase (RTK) signaling, to assess the genetic requirements for rab-7. Using a rab-7 deletion mutant, we demonstrate that rab-7 antagonizes LET-23 EGFR signaling to a similar extent, but in a distinct manner, as previously described negative regulators such as sli-1 c-Cbl. Epistasis analysis places rab-7 upstream of or in parallel to lin-3 EGF and let-23 EGFR. However, expression of gfp::rab-7 in the Vulva Presursor Cells (VPCs) is sufficient to rescue the rab-7(-) VPC induction phenotypes indicating that RAB-7 functions in the signal receiving cell. We show that components of the Endosomal Sorting Complex Required for Transport (ESCRT)-0, and -I, complexes, hgrs-1 Hrs, and vps-28, also antagonize signaling, suggesting that LET-23 EGFR likely transits through Multivesicular Bodies (MVBs) en route to the lysosome. Consistent with RAB-7 regulating LET-23 EGFR trafficking, rab-7 mutants have increased number of LET-23::GFP-positive endosomes. Our data imply that Rab7, by mediating EGFR trafficking and degradation, plays an important role in downregulation of EGFR signaling. Failure to downregulate EGFR signaling contributes to oncogenesis, and thus Rab7 could possess tumor suppressor activity in humans.

The Caenorhabditis elegans class A and B synthetic multivulva (synMuv) genes redundantly antagonize an EGF/Ras pathway to prevent ectopic vulval induction. We identify a class A synMuv mutation in the promoter of the lin-3 EGF gene, establishing that lin-3 is the key biological target of the class A synMuv genes in vulval development and that the repressive activities of the class A and B synMuv pathways are integrated at the level of lin-3 expression. Using FISH with single mRNA molecule resolution, we find that lin-3 EGF expression is tightly restricted to only a few tissues in wild-type animals, including the germline. In synMuv double mutants, lin-3 EGF is ectopically expressed at low levels throughout the animal. Our findings reveal that the widespread ectopic expression of a growth factor mRNA at concentrations much lower than that in the normal domain of expression can abnormally activate the Ras pathway and alter cell fates. These results suggest hypotheses for the mechanistic basis of the functional redundancy between the tumor-suppressor-like class A and B synMuv genes: the class A synMuv genes either directly or indirectly specifically repress ectopic lin-3 expression; while the class B synMuv genes might function similarly, but alternatively might act to repress lin-3 as a consequence of their role in preventing cells from adopting a germline-like fate. Analogous genes in mammals might function as tumor suppressors by preventing broad ectopic expression of EGF-like ligands., Competing Interests: The authors have declared that no competing interests exist.

The C. elegans cell lineage is overall invariant. One rare instance of variability concerns P3.p, the most anterior vulva precursor cell, which may either fuse with the epidermis without dividing, or remain competent to form vulval tissue and divide. Here we examine the evolutionary properties of this stochastic variation in P3.p fate. In the Caenorhabditis genus, high P3.p competence is ancestral and reduction in P3.p competence and division frequency occurred in C. sp. 14 and in a clade of nine species. Within this clade, the frequency of P3.p division further varies within and among species, being intermediate in C. elegans and low in C. briggsae. P3.p fate frequency is sensitive to random mutation accumulation, suggesting that this trait may evolve rapidly because of its sensitivity to mutational impact. P3.p fate depends on LIN-39/Hox5 expression and we find that the peak of LIN-39/Hox5 protein level is displaced posteriorly in C. briggsae compared to C. elegans. However, P3.p fate specification is most sensitive to the dose of EGL-20 and CWN-1, two Wnts that are secreted in a long-range gradient from the posterior end of C. elegans larvae (accompanying article). A half-dose of either of these Wnts is sufficient to affect division frequency in C. elegans N2 to levels similar to those in C. briggsae. Symmetrically, we show that an increase in Wnt dose rescues anterior competence in C. briggsae. We propose that evolutionary variation in the concentration or interpretation of the long-range Wnt gradient may be involved in the rapid evolution of P3.p fate in Caenorhabditis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cell competence is a key developmental property. The Caenorhabditis elegans vulval competence group consists of P(3-8).p, six cells aligned along the antero-posterior axis in a wide central body region. The six cells are not equal in their competence: 1) P3.p quits the competence group in half of the individuals; 2) the posterior cells P7.p and P8.p are less competent than central vulval precursor cells. Competence to adopt a vulval fate is controlled by expression of the HOM-C gene lin-39, and maintained through Wnt signals that are secreted from the tail in a long-range gradient. Here we quantify the LIN-39 protein profile in vulval precursor cells of early L2 stage larvae, prior to P3.p fusion and inductive signaling. We show that LIN-39 levels are very low in P3.p and P4.p, peak in P5.p and progressively decrease until P8.p. This unexpectedly centered profile arises independently from the gonad. Posterior Wnt signaling reduces LIN-39 level in the posterior cells by activating the next-posterior HOM-C gene, mab-5. On the anterior side, P3.p and P4.p competence and division are sensitive to the already low LIN-39 and Wnt doses; most dramatically, each of the cwn-1/Wnt and egl-20/Wnt genes show haplo-insufficience for P3.p fate. In contrast to previous results, we find that these Wnts maintain P3.p and P4.p competence without affecting their LIN-39 level. The centered vulval competence profile is thus under the control of the posterior Wnts and of cross-regulation of three HOM-C genes and prepatterns the later induction of vulval fates., (Copyright © 2011 Elsevier Inc. All rights reserved.)

lag-2 encodes a ligand for LIN-12/Notch and is a component of the lateral signal that activates LIN-12/Notch during Caenorhabditis elegans vulval precursor cell (VPC) fate patterning. lag-2 is specifically transcribed in one VPC, named P6.p, in response to activation of EGFR/Ras/MAPK by the inductive signal that initiates vulval development. Here, we show that a critical molecular event linking inductive and lateral signaling is the relief of VPC-wide lag-2 repression in P6.p. We find that the lag-2 promoter contains an element, VPCrep, which mediates repression in all VPCs when the inductive signal is absent, and another promoter element, VPCact, which is required for activation when repression is relieved by the inductive signal. We show that repression through VPCrep is mediated by the Elk1 ortholog LIN-1, and that the level and subcellular accumulation of a functional LIN-1::GFP protein is similar in all six VPCs before and after vulval induction, suggesting that relief of LIN-1-mediated repression in P6.p is likely due to the known MAPK-dependent phosphorylation of LIN-1. We also provide evidence that the factor(s) acting through VPCact is present in all VPCs but is not modulated by the inductive signal, and that transcription of lag-2 requires the Hth/Meis ortholog UNC-62 and the Mediator complex component SUR-2. Relief of repression of lag-2 in P6.p offers a plausible mechanistic basis for spatial restriction of lag-2 in generating the precise spatial pattern of VPC fates.

The invasive and fully antigenic trophoblast of the chorionic girdle portion of the equine fetal membranes has the capacity to survive and differentiate after transplantation to ectopic sites. The objectives of this study were to determine i) the survival time of ectopically transplanted allogeneic trophoblast cells in non-pregnant recipient mares, ii) whether equine chorionic gonadotropin (eCG) can be delivered systemically by transplanted chorionic girdle cells, and iii) whether eCG delivered by the transplanted cells is biologically active and can suppress behavioral signs associated with estrus. Ectopically transplanted chorionic girdle survived for up to 105 days with a mean lifespan of 75 days (95% confidence interval 55-94) and secreted sufficient eCG for the hormone to be measurable in the recipients' circulation. Immunohistochemical labeling of serial biopsies of the transplant sites and measurement of eCG profiles demonstrated that graft survival was similar to the lifespan of equine endometrial cups in normal horse pregnancy. The eCG secreted by the transplanted cells induced corpora lutea formation and sustained systemic progesterone levels in the recipient mares, effects that are also observed during pregnancy. This in turn caused suppression of estrus behavior in the recipients for up to 3 months. Thus, ectopically transplanted equine trophoblast provides an unusual example of sustained viability and function of an immunogenic transplant in a recipient with an intact immune system. This model highlights the importance of innate immunoregulatory capabilities of invasive trophoblast cells and describes a new method to deliver sustained circulating concentrations of eCG in non-pregnant mares., Competing Interests: Declarations of interest There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Objective: To assess whether primary and secondary vestibulodynia represent different pathologic pathways., Methods: This was an analysis of archived vestibulectomy specimens from 88 premenopausal women with vestibulodynia (2002-2008). Patient records were reviewed to classify the type of vestibulodynia, duration of symptoms, and hormone status. Histologic sections were stained for hematoxylin and eosin to grade inflammation, S100 to highlight nerves, CD117 for mast cells, estrogen receptor α, and progesterone receptor. Differences between primary and secondary vestibulodynia were tested by t tests, chi-square analysis, and linear and logistic regression., Results: Primary vestibulodynia showed significant neural hypertrophy and hyperplasia (P=.02, adjusted odds ratio [OR] 3.01, 95% confidence interval [CI] 1.2-7.6) and increased progesterone receptor nuclear immunostaining (P=.004, adjusted OR 3.94, CI 1.6-9.9) compared with secondary vestibulodynia. Estrogen receptor α expression was also greater in primary vestibulodynia when symptom diagnosis was less than 5 years (P=.004, adjusted OR 5.53 CI 1.71-17.91)., Conclusion: Primary and secondary vestibulodynia have significantly different histologic features, suggesting that they may have separate mechanistic pathways. Clinically, this may mean the discovery of distinct conditions.

The restricted expression of epidermal growth factor (EGF) family ligands is important for proper development and for preventing cancerous growth in mammals. In Caenorhabditis elegans, the class A and B synthetic multivulva (synMuv) genes redundantly repress expression of lin-3 EGF to negatively regulate Ras-mediated vulval development. The class B synMuv genes encode proteins homologous to components of the NuRD and Myb-MuvB/dREAM transcriptional repressor complexes, indicating that they likely silence lin-3 EGF through chromatin remodeling. The two class A synMuv genes cloned thus far, lin-8 and lin-15A, both encode novel proteins. The LIN-8 protein is nuclear. We have characterized the class A synMuv gene lin-56 and found it to encode a novel protein that shares a THAP-like C(2)CH motif with LIN-15A. Both the LIN-56 and LIN-15A proteins localize to nuclei. Wild-type levels of LIN-56 require LIN-15A, and wild-type levels and/or localization of LIN-15A requires LIN-56. Furthermore, LIN-56 and LIN-15A interact in the yeast two-hybrid system. We propose that LIN-56 and LIN-15A associate in a nuclear complex that inhibits vulval specification by repressing lin-3 EGF expression., (© 2011 by the Genetics Society of America)

The C. elegans vulva is patterned by epidermal growth factor (EGF) activation of Ras to control 1° fate, and 1° fate induces antagonistic Notch-dependent 2° fate. Furthermore, a spatial EGF gradient, in addition to inducing 1° fate, directly contributes to 2° fate via an unknown pathway. We find that in addition to its canonical effector, Raf, vulval Ras utilizes an exchange factor for the Ral small GTPase (RalGEF), such that Ras-RalGEF-Ral antagonizes Ras-Raf pro-1° fate activity. Consistent with its restricted expression pattern, Ral participates in EGF pro-2° activity. Thus, we have delineated a Ras effector-switching mechanism whereby position within the morphogen gradient dictates that Ras effector usage is switched to RalGEF from Raf to promote 2° instead of 1° fate. Our observations define the utility of Ras effector switching during normal development and may provide a possible mechanistic basis for cell and cancer-type differences in effector dependency and activation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background/objectives: The basement membrane zone (BMZ) is an anatomically defined region present in all types of skin and mucosa, linking the epithelium to the mesenchyme with a complex structure to provide adhesion. Altered antigenic expression of the BMZ is implicated in interface dermatoses, and the BMZ is targeted by autoantibodies in subepidermal immunobullous dermatoses. This study aims to compare the antigenic expression of the BMZ and the dermal extracellular matrix in female genital skin and mucosa and amnion, with non-reproductive skin and mucosa., Methods: An indirect immunofluorescence technique was used to compare the antigenic expression of hemidesmosome, lamina lucida, anchoring filaments, lamina densa, anchoring fibrils and extracellular matrix in samples of non-reproductive skin (three), oral mucosa (three), vulval skin (two), vagina (three) and amnion (four)., Results: Antigenic expression was similar in the stratified epithelium of reproductive and non-reproductive skin and mucosa, but differed in the simple cuboidal epithelium of amnion, which had reduced expression of dermal-associated antigens., Conclusions: The BMZ and dermal extracellular matrix of vagina and vulva are very similar to those of non-reproductive skin and mucosa despite their various functions, but differs from amnion. Their antigenic expression does not fully account for the anatomical distribution of immunobullous and interface dermatoses., (© 2010 The Authors. Australasian Journal of Dermatology © 2010 The Australasian College of Dermatologists.)

The molecular pathogenesis of human papilloma virus-unrelated vulvar squamous cell carcinoma is not well known. Whether malignant progression of lichen sclerosus and differentiated vulvar intraepithelial neoplasia to vulvar squamous cell carcinoma could be accompanied by altered DNA content has not been studied extensively. DNA content in isolated nuclei of microdissected normal vulvar epithelium (n = 2), lichen sclerosus (n = 9), differentiated vulvar intraepithelial neoplasia (n = 13), and squamous cell carcinoma (n = 17) from 22 patients was measured via DNA image cytometry. For additional analysis, 6 differentiated vulvar intraepithelial neoplasia lesions were selected, bringing the number of patients to 28. p53 expression was determined by immunohistochemistry on consecutive tissue sections. Thirty-eight percent (5/13) of differentiated vulvar intraepithelial neoplasia lesions and 65% (11/17) of squamous cell carcinomas were DNA aneuploid or tetraploid. In lesions that contained differentiated vulvar intraepithelial neoplasia and adjacent squamous cell carcinoma, the ploidy status of differentiated vulvar intraepithelial neoplasia did not exceed that of squamous cell carcinoma. We observed a strong correlation between high p53 expression and DNA aneuploidy. This relation was also present at the level of a single nucleus, measured by sequential image cytometry of p53 immunohistochemistry followed by DNA image cytometry on formalin-fixed tissue sections. Similarly, we found p53-positive nonproliferating cells with increased DNA content in the superficial compartment of 6 additional solitary differentiated vulvar intraepithelial neoplasia lesions that were not associated with squamous cell carcinoma, indicating ascending aneuploid cells from the basal compartment. DNA ploidy measurements suggest that differentiated vulvar intraepithelial neoplasia has a higher malignant potential than lichen sclerosus and thus is a more likely precursor of squamous cell carcinoma. Furthermore, high p53 expression correlates with increased DNA content and aneuploidy; but it requires further research to unveil a possible causal relation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Ste20 kinases constitute a large family of serine/threonine kinases with a plethora of biological functions. Members of the GCK-VI subfamily have been identified as important regulators of osmohomeostasis across species functioning upstream of ion channels. Although the expression of the two highly similar mammalian GCK-VI kinases is eminent in a wide variety of tissues, which includes also the testis, their potential roles in development remain elusive. Caenorhabditis elegans contains a single ancestral ortholog termed GCK-3. Here, we report a comprehensive analysis of gck-3 function and demonstrate its requirement for several developmental processes independent of ion homeostasis, i.e., larval progression, vulva, and germ line formation. Consistent with a wide range of gck-3 function we find that endogenous GCK-3 is expressed ubiquitously. The serine/threonine kinase activity of GCK-3, but not its presumed C-terminal substrate interaction domain, is essential for gck-3 gene function. Although expressed in female germ cells, we find GCK-3 progressively accumulating during spermatogenesis where it promotes the first meiotic cell division and facilitates faithful chromosome segregation. In particular, we find that different levels of gck-3 activity appear to be important for various aspects of germ line development. Taken together, our findings suggest that members of the GCK-VI kinase subfamily may act as key regulators of many developmental processes and that this newly described role in meiotic progression might be conserved and an important part of sexual reproduction., (Copyright 2010 Elsevier Inc. All rights reserved.)

Mammalian hyaluronidases degrade hyaluronan and some structurally related glycosaminoglycans. We generated a deletion mutant in the Caenorhabditis elegans orthologue of mammalian hyaluronidase, hya-1. Mutant animals are viable and grossly normal, but exhibit defects in vulval morphogenesis and egg-laying and showed increased staining with alcian blue, consistent with an accumulation of glycosaminoglycan. A hya-1::GFP reporter was expressed in a restricted pattern in somatic tissues of the animal with strongest expression in the intestine, the PLM sensory neurons and the vulva. Total protein extracts from wild-type animals exhibited chondroitin-degrading but not hyaluronan-degrading activity. Chondroitinase activities were observed at both neutral and acidic pH conditions while both neutral and acidic activities were absent in extracts from hya-1 mutant strains. We also evaluated the function of oga-1, which encodes the C. elegans orthologue of MGEA-5, a protein with hyaluronan-degrading activity in vitro. oga-1 is expressed in muscles, vulval cells and the scavenger-like coelomocytes. An oga-1 mutant strain exhibited egg-laying and vulval defects similar to those of hya-1; chondroitinase activity was unaffected in this mutant., (Copyright © 2010 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Background: Extramammary Paget's disease (EMPD) is a rare neoplasm of apocrine gland-bearing skin. It is known that over-expression of survivin and human telomerase reverse transcriptase (hTERT) correlates with malignancies. We investigated the expression of hTERT and survivin by Paget's cells and their role in the tissue invasion and recurrence of EMPD., Method: Forty-two patients were enrolled into the study. Expression of survivin and hTERT were analyzed by immunohistochemistry and in situ hybridization techniques. The variables including the expression level of survivin and hTERT, gender, age, lesion location, invasion level and number of surgeries were statistically analyzed using Fisher's exact test., Results: Survivin was positively stained in 18 of 22 cases (81.8%), and hTERT in 18 of 29 cases (62.1%). Significantly higher level of survivin expression was detected in patients with multiple surgeries than those with single one (p = 0.0458). Expression of hTERT was significantly higher in the patients with micro-invasive and invasive lesions than those with non-invasive lesions (p = 0.0478)., Conclusions: Over-expression of survivin and hTERT correlated strongly with recurrence and local invasion of EMPD lesions. EMPD has male gender predominance in Oriental population.

The specification and patterning of vulval precursor cells (VPCs) in Caenorhabditiselegans is achieved using a conserved EGFR/RAS signaling pathway that is activated by the ligand lin-3/EGF, which is secreted by the neighboring somatic gonad. Previous work has demonstrated that the expression of lin-3 must be tightly regulated to ensure that only three of six equivalent VPCs are induced to differentiate into the mature vulva. Here, we have identified a novel regulator of EGFR/RAS signaling, let-765/nsh-1, that functions upstream of the pathway to promote vulval induction. let-765 encodes a conserved DExD/H box helicase protein and is the C. elegans ortholog of Drosophila strawberry notch. By investigating genetic interactions between let-765 and RAS pathway genes as well as with synthetic multivulva (synMuv) genes, we have demonstrated that let-765 positively regulates the RAS pathway and antagonizes synMuv activity at the level of lin-3/EGF. In support of these proposals, we found that LET-765 is required for producing wild-type levels of lin-3 mRNA. Mutations in let-765 result in pleiotropic phenotypes that imply its function must be required in multiple developmental processes and, together with data presented here, suggest that LET-765 promotes the expression of diverse targets, potentially through interactions with transcriptional activator or repressor complexes., (Copyright 2010 Elsevier Inc. All rights reserved.)

Vulvar cancer contributes to about 5% of all gynaecological cancers. Galectin-1, a member of an ubiquitous expressed beta-galactoside-binding family that comprises over 140 members to date, has been shown to be involved in many physiological and pathological processes, such as tumour progression, by promoting cancer cell invasion and metastasis, in apoptosis, embryogenesis and immunobiology. As the result of these findings, galectin-1 has been described as a potential marker for tumour progression in some malignancies. In this study, the expression pattern of galectin-1 was determined in 73 formalin-fixed, paraffin-embedded vulvar tissues by a standard immunohistochemical method: 12 benign vulvar specimen, 41 vulvar intraepithelial lesions (VIN), according to their differentiation were subdivided into VIN I, II and III and 20 invasive squamous cell carcinomas (ISCC). The immunohistochemical analyses showed that the intensity of galectin-1 expression on stromal cells next to the neoplastic cells steadily increased according to the pathological grade: benign vulvar tissue