Alex Hajnal et al. (2002) European Worm Meeting "The C. elegans MAP kinase phosphatase LIP-1 is required for the G2/M cell cycle arrest in developing oocytes"

ALEX HAJNAL, Thomas Berset

[European Worm Meeting, 2002]

In the C. elegans hermaphrodite germ line, spatially restricted MAP kinase signaling controls the meiotic cell cycle. First, the MAP kinase signal is necessary for the germ cells to progress through pachytene of meiotic prophase I (Church et al.1995). As the germ cells exit pachytene and enter diplotene/diakinesis, the MAP kinase is inactivated and the developing oocytes arrest in diakinesis (G2/M arrest). During oocyte maturation, a signal from the sperm reactivates the MAP kinase to promote M-phase entry (Miller et al. 2000). We have previously reported that the dual- specificity phosphatase LIP-1 negatively regulates MAP kinase signaling during vulval induction (Berset et al. 2000). Here, we show that LIP-1 dephosphorylates the MAP kinase in germ cells that exit pachytene in order to maintain the MAP kinase in an inactive state in the developing oocytes that arrest in diakinesis. Germ cells lacking LIP-1 fail to arrest the cell cycle at the G2/M boundary and enter a mitotic cell cycle without fertilization. LIP-1 thus coordinates meiotic cell cycle progression and maturation with ovulation and fertilization.

Itay, Nakdimon et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Cross talk between the Ras-MAPK and Insulin pathways during vulval development"

Hajnal, Alex, Itay, Nakdimon

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The development of the vulva, the egg-laying organ of the hermaphrodite, serves as an excellent model to study how inter- and intracellular signals control cell fate specification and pattern formation during organogenesis. Previous work from our lab showed that a InsR/daf-2 reduction-of-function (rf) mutation reduces the Multivulva (Muv) phenotype of a let-60 gain-of-function (gf) allele (Battu et al. 2003). Further epistasis analysis showed that although loss of PI3K/age-1 function (lf) only partially suppresses the let-60(gf) Muv phenotype, PTEN/daf-18(lf) significantly increased the number of VPCs induced in a let-60(gf) background independently of AKT-1/-2 and DAF-16 function. Moreover, a mutated DAF-18(G174E) rescue construct that can dephosphorylate proteins but not lipids only showed a weak rescue, suggesting that elevated P IP3 levels enhance RAS/MAPK-induced vulval development and that an alternative PI3K might function besides AGE-1. daf-18(lf) partially suppressed the Vulvaless (Vul) phenotype of mutants in other RAS/MAPK pathway components such as lin-2(lf), let-23(rf), sos-1(lf) and lin-45(rf), but failed to suppress mpk-1(lf) mutants. These results suggest that elevated PIP3 levels in daf-18(lf) activate the RAS/MAPK pathway downstream of or in parallel with SOS-1 and upstream of or at the level of MPK-1. A rescuing translational DAF-18::GFP reporter showed strong membrane expression in all induced VPCs, the uterus, the spermatheca and the ventral nerve cord. In conclusion, we have shown that PIP3 levels, which are regulated by Insulin signaling and DAF-18, modulate RAS/MAPK signaling in the VPCs.

Kehrli, Debora et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Cell specific mRNA pull-down using the FLPase system"

Hajnal, Alex, Kehrli, Debora

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

During vulval development, the highly conserved EGFR/RAS/MAPK, DELTA/NOTCH and Wnt pathways are activated. Mutations in components of these pathways lead to diseases in humans such as cancer or Alzheimer. The EGFR/RAS/MAPK pathway is activated in P6.p to specify the primary (1) cell fate. The secondary (2) cell fate is induced in P5.p and P7.p via the lateral DELTA/NOTCH signal from P6.p. NOTCH signaling in P5.p and P7.p blocks the activation of the EGFR/RAS/MAPK pathway and promotes the expression of 2 specific genes. The LIN-1 and LIN-31 transcription factors are phosphorylated in P6.p by the MAP kinase MPK-1, leading to the expression of 1 fate-specific genes. However, only a few LIN-1 and LIN-31 target genes are currently known. Moreover, no downstream targets of the DELTA/NOTCH pathway that specify the 2 cell fate in P5.p and P7.p are known. We are attempting to systematically identify targets of the EGFR/RAS/MAPK pathway in the 1 vulval cells by performing a lineage-specific mRNA pull-down. For this purpose, we use the method developed by Roy et al. (2002) expressing an epitope-tagged poly(A)-binding protein (PAB-1) in the 1 cells. To further increase the specificity of the method, we have used the FLP/FRT recombination system (Davis et al., 2008) with one 1 lineage-specific promoter driving the FLPase and another 1 lineage promoter driving PAB-1 cDNA preceded by the FRT-Stop cassette. An analogous approach will be used to isolate 2 cell-specific mRNAs. Our progress will be presented at the meeting.

Walser, Michael et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A Mass Spectrometry-Based Approach to Identify New Interaction Partners of the Tyrosine Phosphatase DEP-1"

Walser, Michael, Hajnal, Alex

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

During hermaphrodite vulval development six vulval precursor cells (VPCs, P3.p P8.p) are competent to generate vulval tissue and adopt an invariant pattern of three cell fates. The anchor cell secretes the LIN-3 EGF growth factor that activates in P6.p the EGFR/RAS/MAPK pathway to specify the primary (1) cell fate. Subsequently, a lateral signal from P6.p inhibits the 1 and induces the secondary (2) cell fate in the neighboring VPCs P5.p and P7.p via the LIN-12 Notch pathway. Several negative regulators of the EGFR/RAS/MAPK signaling pathway, such as LIP-1 and the density enhanced phosphatase DEP-1 are up-regulated in P5.p and P7.p to inhibit the primary fate specification in these VPCs. DEP-1, which is a member of the class III receptor protein tyrosine phosphatase family (den Hertog 1999), negatively regulates LET-23 EGFR signaling (Berset et al. 2005). Human Dep-1 is often mutated in colon, breast, skin, and lung carcinomas (Ruivenkamp et al. 2002). Despite its importance as a tumor suppressor in various epithelial tissues, the physiological functions of mammalian Dep-1 are still poorly understood. Furthermore, the available data are based almost exclusively on in vitro experiments. Therefore the role of Dep-1 in cell fate specification, pattern formation during normal development, tumorigenesis, as well as its physiological substrates have still to be elucidated. In order to identify new interaction partners of DEP-1 in C. elegans, we are performing a mass spectrometry based approach. For the co-purification of DEP-1 together with its physiological binding partners we are using HA-Strep-III-tagged versions of wild-type DEP-1 and a substrate trapping mutant in the catalytic phosphatase domain. After tandem affinity purification of the tagged fusion proteins, the interacting partners can be identified by mass spectrometry and further analyzed in vivo and in vitro . Progress of these approaches will be presented at the meeting.

Nakdimon, Itay et al. (2009) International Worm Meeting "Cross-talk between the RAS/MAPK and PI3K/PTEN signaling pathways during vulval development."

Nakdimon, Itay, Hajnal, Alex

[International Worm Meeting, 2009]

The development of the Caenorhabditis elegans vulva, the egg-laying organ of the hermaphrodite, serves as an excellent model to study how inter- and intracellular signals control cell fate specification and pattern formation during organogenesis. We are interested in the crosstalk between the different signaling pathways controlling vulval fate specification. One well-characterized example for signaling crosstalk is the antagonism between the RAS/MAPK and Notch pathways. Here, we examined a possible connection between the phosphoinositide-3 kinase (PI-3K)/PTEN and the RAS/MAPK signaling pathways during vulval development. It has been previously shown in mammalian cells that RAS can directly activate PI3K, while AKT, a downstream target of PI3K, phosphorylates and thereby inhibits the activity of RAF, the downstream target of RAS. To examine a possible role of the Insulin/PI3K pathway during vulval development, we combined mutations that either decrease or increase the activity of the canonical insulin pathway with mutations that alter the RAS/MAPK signaling intensity. daf-18 , the homologue of the mammalian tumor suppressor PTEN, encodes a lipid phosphatase that catalyzes the dephosphorylation of phophatydylinositol-3,-4,-5-trisphosphate (PIP3), thereby antagonizing the role of the AGE-1 PI-3 Kinase. A loss-of-function (lf) mutation in daf-18 blocks entry into the dauer stage and reduces life span due to hyperactivation of the PI-3K pathway. Our results suggest that DAF-18 also inhibits LET-60 RAS signaling during vulval development. Loss of daf-18 function increases the number of Vulva Precursor Cells (VPCs) induced in a let-60(gf) background and suppresses the vulvaless phenotype of different RAS pathway mutants. Our results also indicate that this effect is, at least in part, independent of the insulin pathway. Loss-of-function mutations in age-1 or daf-16, which are core components of the insulin pathway, do not change the number of VPCs induced in a let-60(gf) background. Furthermore, daf-18(lf) not only affects the number of VPCs induced, but also their fates, since an increase in the expression level of the primary cell fate marker egl-17::cfp was observed in a daf-18(lf) mutant. Mammalian PTEN is reported to have both lipid as well as protein phosphatase activities. The fact that part of the effect of daf-18 on vulval development is independent of the Insulin pathway suggests that a protein phosphatase activity of DAF-18 might play a role in the negative regulation of RAS/MAPK signaling. Experiments that distinguish between the two DAF-18 activities will be presented at the meeting.

Walser, Michael et al. (2009) International Worm Meeting "A Mass Spectrometry-Based Approach to Identify New Interaction Partners of the Tyrosine Phosphatase DEP-1."

Hajnal, Alex, Walser, Michael

[International Worm Meeting, 2009]

During hermaphrodite vulval development six vulval precursor cells (VPCs, P3.p - P8.p) are competent to generate vulval tissue and adopt an invariant pattern of three cell fates. The anchor cell secretes the LIN-3 EGF growth factor that activates in P6.p the EGFR/RAS/MAPK pathway to specify the primary (1 deg ) cell fate. Subsequently, a lateral signal from P6.p inhibits the 1 deg and induces the secondary (2 deg ) cell fate in the neighboring VPCs P5.p and P7.p via the LIN-12 Notch pathway. Several negative regulators of the EGFR/RAS/MAPK signaling pathway, such as LIP-1 and the density enhanced phosphatase DEP-1 are up-regulated in P5.p and P7.p to inhibit the primary fate specification in these VPCs. DEP-1, which is a member of the class III receptor protein tyrosine phosphatase family (den Hertog 1999), negatively regulates LET-23 EGFR signaling (Berset et al. 2005). Human Dep-1 is often mutated in colon, breast, skin, and lung carcinomas (Ruivenkamp et al. 2002). Despite its importance as a tumor suppressor in various epithelial tissues, the physiological functions of mammalian Dep-1 are still poorly understood. Furthermore, the available data are based almost exclusively on in vitro experiments. Therefore the role of Dep-1 in cell fate specification, pattern formation during normal development, tumorigenesis, as well as its physiological substrates have still to be elucidated. In order to identify new interaction partners of DEP-1 in C. elegans, we are performing mass spectrometry based approaches. For the co-purification of DEP-1 together with its physiological binding partners we are using HA-Strep-III- and GST-tagged versions of wild-type DEP-1 and a substrate trapping mutant in the catalytic phosphatase domain. After affinity purification of the tagged fusion proteins, the interacting partners can then be identified by mass spectrometry. Progress of these approaches will be presented at the meeting.

Deng, Ting et al. (2017) International Worm Meeting "Cell cycle-independent role of the pre-replication complex during anchor cell invasion."

Deng, Ting, Hajnal, Alex, Lattmann, Evelyn

[International Worm Meeting, 2017]

Anchor-cell (AC) invasion is serving as an excellent model of study fundamental aspects of cell invasion, a process occurring during normal development as well as tumor metastasis. By screening the C. elegans orthologs of genes up-regulated in invasive human melanoma cells, we have identified five pre-replication complex (pre-RC) components, mcm-7, cdc-6, cdt-1, orc-2 and orc-5, as regulators of AC invasion. The pre-RC is required for the licensing of replication origins during the G1 phase. Since the invading AC never replicates its DNA and remains arrested in G1, the similar penetrance of AC-specific and systemic mcm-7 RNAi suggest a cell-autonomous and cell cycle-independent function of MCM-7. An endogenous GFP::MCM-7 reporter shows a transient nuclear expression pattern in the AC prior to invasion, suggesting a function of the pre-RC in inducing the epithelial to mesenchymal transition of the AC. Accordingly, MCM-7 regulates expression of metalloproteinase zmp-1 in parallel with FOS-1A. To further study the function of the pre-RC during AC invasion, we have inserted FRT sites into the mcm-7 locus, allowing us to perform a conditional mcm-7 knock-out. We propose that components of the pre-RC have adopted a cell-cycle independent function in the AC to regulate cell invasion.

Farooqui, Sarfarazhussain et al. (2009) International Worm Meeting "The ROCK homolog LET-502 is temporally and spatially regulated by LIN-12 Notch and LIN-1 ETS during vulval morphogenesis."

Rimann, Ivo, Farooqui, Sarfarazhussain, Hajnal, Alex

[International Worm Meeting, 2009]

Cell fate specification and morphogenesis are two important aspects of vulval development. After the vulval precursor cells (VPCs) have acquired their fates, the VPCs divide thrice and their descendants undergo migration, elongation and fusion at the vulval midline to form seven toroidal rings stacked one over the other in an invariant fashion (Ranjana Sharma-Kishore et. al, 1999). Recently, genetic approaches have revealed that two parallel pathways regulate vulval morphogenesis. The first pathway requires the SMP-1 Semaphorin ligand, the PLX-1 Plexin receptor and the CED-10 Rac small GTPase, while the second pathway utilizes the MIG-2 Rac small GTPase and its activator UNC-73 GEF (Dalpe et al. 2005; Ranjana S Kishore et al. 2002). Here, we show a role for the ROCK homolog LET-502 during vulval morphogenesis. Maternally rescued let-502(ok1283) animals display defects in vulval toroid formation, with evidence of abnormal cell migration and elongation. The expression of the vulval fate markers was not affected in the let-502(ok1283) animals, suggesting that let-502 does not affect VPC fate specification. Genetic epistasis reveals redundancy of LET-502 with both the SMP-1/PLX-1/CED-10 and UNC-73/MIG-2 signaling pathways. During vulval morphogenesis, let-502 is specifically expressed in the secondary vulval cells. Analysis of the let-502 promoter reveals spatial and temporal regulation during morphogenesis by LIN-12 Notch signaling and the transcription factor LIN-1 ETS. Accordingly, lin-12 notch loss-of-function mutants display defects in toroid formation similar to the defects observed in let-502(ok1283) mutants. Hence, Notch signaling may regulate vulval morphogenesis through the activation of let-502 transcription.

Schmid, Tobias et al. (2011) International Worm Meeting "The Panacea Consortium: Identification of new Modulators of EGFR/RAS/MAPK and WNT signaling during vulval development."

Schmid, Tobias, Largey, Fabienne, Hajnal, Alex

[International Worm Meeting, 2011]

Complex human diseases such as cancer are responsible for the death of more than 50% of the adult human population. They arise from interactions between different signaling networks and the environment. Previous successful studies were mainly focused on the identification of genes involved in the development of rare monogenic diseases. Nevertheless, investigations of the more common complex, polygenic diseases are required. The genetic background has a considerable effect on the pathogenesis of many complex diseases, but further investigation is needed to decipher the underlying genetic risk factors. Cancer-related signaling networks can be investigated in the C. elegans vulva with the aim to expand the knowledge about complex signaling pathways. The development of the C. elegans vulva depends on the interplay between the EGFR/RAS/MAPK, the Notch and the Wnt signaling pathways, all of which play a central role in the development of cancer in humans. As part of the Panacea Consortium, we are addressing the question of how the genetic background influences the output of these signaling networks. We therefore crossed mutants in the RAS, Notch and WNT pathways originally isolated in the N2 Bristol background into CB4856 Hawaii and selected for suppressors or enhancers of the mutant phenotypes to establish recombinant inbred (RIL) lines. A RIL set containing the let-60(n1046) allele in a mixed CB4856/N2 background exhibits a strongly enhanced Multivulva (Muv) phenotype when compared to let-60(n1046) in the "pure" N2 background. We have identified by QTL mapping genomic regions that contribute to the enhanced Muv phenotype and are investigating candidate genes in these regions for their role in EGFR/RAS/MAPK signaling. Another RIL set generated with the bar-1(ga80) allele using the same approach exhibits severe larval lethality and an enhancement of the bar-1(ga80) vulval and gonad migration phenotypes when compared to the N2 background. The quantification of these phenotypes is underway and will be combined with the genotype data to predict regions containing modifiers of the WNT pathway.

Maxeiner, Sabrina et al. (2013) International Worm Meeting "Animal to Animal Variability During Vulval Cell Fate Specification."

Maxeiner, Sabrina, Hajnal, Alex, Roiz, Daniel

[International Worm Meeting, 2013]

Cell fate determination and differentiation depend on the precise temporal and spatial regulation of intercellular signaling pathways (Euling et al. 1996). During vulval development, the anchor cell in the gonad induces the primary (1 deg ) vulval cell fate by secreting an epidermal growth factor LIN-3 that activates the EGFR/RAS/MAPK signaling pathway in the nearest vulval precursor cell (VPC) P6.p (Sternberg 2005). A lateral signal from P6.p then induces the secondary (2 deg ) cell fate in P5.p and P7.p by activating the LIN-12 NOTCH pathway. The distal VPCs (P3.p, P4.p and P8.p) receiving low levels of inductive and lateral signals adopt the tertiary (3 deg ) fate. In particular, the connection between cell cycle progression and the NOTCH pathway ensures that 1 deg , 2 deg and 3 deg cell fates are specified in a temporally defined manner (Nusser-Stein et al. 2012). Although the vulval cell fate patterning exhibits an invariable outcome, the progression of fate acquisition by individual VPCs occurs with a certain degree of variability (Barkoulas et al. 2013). Defective coupling of signaling and cell cycle progression as it occurs in heterochronic mutants leads to mistakes in the timing of VPC division and cell fate determination (Ambros et al. 1984). To investigate the variability of EGFR/RAS/MAPK and LIN-12 NOTCH signaling during vulval fate specification quantitatively, we are establishing branched DNA single molecule fluorescence in situ hybridization (bDNA FISH) in C. elegans. bDNA FISH exhibits higher sensitivity and robustness compared to oligo DNA single molecule FISH (smFISH) allowing quantification at single RNA molecule resolution with improved signal-to-noise-ratio (Player at al. 2001, Raj et al. 2008). We are applying bDNA FISH to measure the dynamics and variability of 1 deg and 2 deg cell fate markers during VPC fate specification in L2 larvae. We assume that early during the cell fate acquisition process before the establishment of a stable fate pattern, the VPCs exhibit a certain degree of randomness in EGFR/RAS/MAPK and LIN-12 NOTCH signaling strength. To test this hypothesis, we are quantifying VPC fate specification in heterochronic mutants, where VPC induction is precocious (lin-28(lf)) or retarded (lin-14(gf)).