Nutritional vitamin B12 regulates RAS/MAPK-mediated cell fate decisions through one-carbon metabolism.

Vitamin B12 is an essential nutritional co-factor for the folate and methionine cycles, which together constitute one-carbon metabolism. Here, we show that dietary uptake of vitamin B12 modulates cell fate decisions controlled by the conserved RAS/MAPK signaling pathway in C. elegans. A bacterial diet rich in vitamin B12 increases vulval induction, germ cell apoptosis and oocyte differentiation. These effects are mediated by different one-carbon metabolites in a tissue-specific manner. Vitamin B12 enhances via the choline/phosphatidylcholine metabolism vulval induction by down-regulating fat biosynthesis genes and increasing H3K4 tri-methylation, which results in increased expression of RAS/MAPK target genes. Furthermore, the nucleoside metabolism and H3K4 tri-methylation positively regulate germ cell apoptosis and oocyte production. Using mammalian cells carrying different activated KRAS and BRAF alleles, we show that the effects of methionine on RAS/MAPK-regulated phenotype are conserved in mammals. Our findings suggest that the vitamin B12-dependent one-carbon metabolism is a limiting factor for diverse RAS/MAPK-induced cellular responses.

Functional distinction in oncogenic Ras variant activity in Caenorhabditis elegans.

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.

Cell cycle perturbation uncouples mitotic progression and invasive behavior in a post-mitotic cell.

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 (p21CIP1/p27KIP1) 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 (p21CIP1/p27KIP1), LIN-35 (pRb/p107/p130), FZR-1 (Cdh1/Hct1), and LIN-23 (ß-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.

TP53 mutation and human papilloma virus status as independent prognostic factors in a Norwegian cohort of vulva squamous cell carcinoma.

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 p16INK4A (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 p16INK4A 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. p16INK4A 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 p16INK4A 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.

Prolonging somatic cell proliferation through constitutive hox gene expression in C. elegans.

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.

Genetic characterization of C. elegans TMED genes.

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, ß, 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.

Positive p16 Immunostaining Is an Independent Prognostic Variable for Disease-free Survival and Overall Survival in Patients With Squamous Cell Carcinoma of the Vulva Treated With Radical Surgery and Inguinofemoral Lymphadenectomy: An Italian Single Center Retrospective Study.

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.

Reevaluating the relationship between EGL-43 (EVI1) and LIN-12 (Notch) during C. elegans anchor cell invasion.

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.

Discovery of nonautonomous modulators of activated Ras.

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.

Identifying the Caenorhabditis elegans vulval transcriptome.

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.

HPV-independent, p53-wild-type vulvar intraepithelial neoplasia: a review of nomenclature and the journey to characterize verruciform and acanthotic precursor lesions of the vulva.

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.

Influences of HLH-2 stability on anchor cell fate specification during Caenorhabditis elegans gonadogenesis.

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 ß cells-have the potential to be the sole anchor cell. The ß 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 ß 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 ß 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 ß cells that correlates with known differences in POP-1/TCF levels and anchor cell potential between α and ß cells. Together, our results indicate that hlh-2 activity is regulated at multiple levels to restrict the anchor cell fate to a single cell.

A DNA replication-independent function of pre-replication complex genes during cell invasion in C. elegans.

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.

Prereplication complex proteins get caught moonlighting.

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.

Reciprocal EGFR signaling in the anchor cell ensures precise inter-organ connection during Caenorhabditis elegans vulval morphogenesis.

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.

Genetic analysis of Caenorhabditis elegans pry-1/Axin suppressors identifies genes involved in reproductive structure development, stress responses, and aging.

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.

Ras, Ral, and Rap1 in C. elegans.

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.

Golgi localization of the LIN-2/7/10 complex points to a role in basolateral secretion of LET-23 EGFR in the Caenorhabditiselegans vulval precursor cells.

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 Caenorhabditiselegans, 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.

LIN-10 can promote LET-23 EGFR signaling and trafficking independently of LIN-2 and LIN-7.

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.

Negative feedback by conserved kinases patterns the degradation of Caenorhabditiselegans Raf in vulval fate patterning.

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.