Anchor Cell Research Articles

Phytoglobins regulate nitric oxide-dependent abscisic acid synthesis and ethylene-induced program cell death in developing maize somatic embryos.

During maize somatic embryogenesis, suppression of phytoglobins (Pgbs) reduced ABA levels leading to ethylene-induced programmed cell death in the developing embryos. These effects modulate embryonic yield depending on the cellular localization of specific phytoglobin gene expression. Suppression of Zea mays phytoglobins (ZmPgb1.1 or ZmPgb1.2) during somatic embryogenesis induces programmed cell death (PCD) by elevating nitric oxide (NO). While ZmPgb1.1 is expressed in many embryonic domains and its suppression results in embryo abortion, ZmPgb1.2 is expressed in the basal cells anchoring the embryos to the embryogenic tissue. Down-regulation of ZmPgb1.2 is required to induce PCD in these anchor cells allowing the embryos to develop further. Exogenous applications of ABA could reverse the effects caused by the suppression of either of the two ZmPgbs. A depletion of ABA, ascribed to a down-regulation of biosynthetic genes, was observed in those embryonic domains where the respective ZmPgbs were repressed. These effects were mediated by NO. Depletion in ABA content increased the transcription of genes participating in the synthesis and response of ethylene, as well as the accumulation of ethylene, which influenced embryogenesis. Somatic embryo number was reduced by high ethylene levels and increased with pharmacological treatments suppressing ethylene synthesis. The ethylene inhibition of embryogenesis was linked to the production of reactive oxygen species (ROS) and the execution of PCD. Integration of ABA and ethylene in the ZmPgb regulation of embryogenesis is proposed in a model where NO accumulates in ZmPgb-suppressing cells, decreasing the level of ABA. Abscisic acid inhibits ethylene biosynthesis and the NO-mediated depletion of ABA relieves this inhibition causing ethylene to accumulate. Elevated ethylene levels trigger production of ROS and induce PCD. Ethylene-induced PCD in the ZmPgb1.1-suppressing line [ZmPgb1.1 (A)] leads to embryo abortion, while PCD in the ZmPgb1.2-suppressing line [ZmPgb1.2 (A)] results in the elimination of the anchor cells and the successful development of the embryos.

Silk fibroin/collagen protein hybrid cell-encapsulating hydrogels with tunable gelation and improved physical and biological properties

Cell encapsulating hydrogels with tunable mechanical and biological properties are of special importance for cell delivery and tissue engineering. Silk fibroin and collagen, two typical important biological proteins, are considered potential as cell culture hydrogels. However, both have been used individually, with limited properties (e.g., collagen has poor mechanical properties and cell-mediated shrinkage, and silk fibroin from Bombyx mori (mulberry) lacks cell adhesion motifs). Therefore, the combination of them is considered to achieve improved mechanical and biological properties with respect to individual hydrogels. Here, we show that the cell-encapsulating hydrogels of mulberry silk fibroin / collagen are implementable over a wide range of compositions, enabled simply by combining the different gelation mechanisms. Not only the gelation reaction but also the structural characteristics, consequently, the mechanical properties and cellular behaviors are accelerated significantly by the silk fibroin / collagen hybrid hydrogel approach. Of note, the mechanical and biological properties are tunable to represent the combined merits of individual proteins. The shear storage modulus is tailored to range from 0.1 to 20 kPa along the iso-compositional line, which is considered to cover the matrix stiffness of soft-to-hard tissues. In particular, the silk fibroin / collagen hydrogels are highly elastic, exhibiting excellent resistance to permanent deformation under different modes of stress; without being collapsed or water-squeezed out (vs. not possible in individual proteins) – which results from the mechanical synergism of interpenetrating networks of both proteins. Furthermore, the role of collagen protein component in the hybrid hydrogels provides adhesive sites to cells, stimulating anchorage and spreading significantly with respect to mulberry silk fibroin gel, which lacks cell adhesion motifs. The silk fibroin / collagen hydrogels can encapsulate cells while preserving the viability and growth over a long 3D culture period. Our findings demonstrate that the silk / collagen hydrogels possess physical and biological properties tunable and significantly improved (vs. the individual protein gels), implying their potential uses for cell delivery and tissue engineering. Statement of SignificanceDevelopment of cell encapsulating hydrogels with excellent physical and biological properties is important for the cell delivery and cell-based tissue engineering. Here we communicate for the first time the novel protein composite hydrogels comprised of ‘Silk’ and ‘Collagen’ and report their outstanding physical, mechanical and biological properties that are not readily achievable with individual protein hydrogels. The properties include i) gelation accelerated over a wide range of compositions, ii) stiffness levels covering 0.1 kPa to 20 kPa that mimic those of soft-to-hard tissues, iii) excellent elastic behaviors under various stress modes (bending, twisting, stretching, and compression), iv) high resistance to cell-mediated gel contraction, v) rapid anchorage and spreading of cells, and vi) cell encapsulation ability with a long-term survivability. These results come from the synergism of individual proteins of alpha-helix and beta-sheet structured networks. We consider the current elastic cell-encapsulating hydrogels of silk-collagen can be potentially useful for the cell delivery and tissue engineering in a wide spectrum of soft-to-hard tissues.

Long-term C. elegans immobilization enables high resolution developmental studies in vivo.

Live-imaging of C. elegans is essential for the study of conserved cellular pathways (e.g. EGFR/Wnt signaling) and morphogenesis in vivo. However, the usefulness of live imaging as a research tool has been severely limited by the need to immobilize worms prior to and during imaging. Conventionally, immobilization is achieved by employing both physical and chemical interventions. These are known to significantly affect many physiological processes, and thus limit our understanding of dynamic developmental processes. Herein we present a novel, easy-to-use microfluidic platform for the long-term immobilization of viable, normally developing C. elegans, compatible with image acquisition at high resolution, thereby overcoming the limitations associated with conventional worm immobilization. The capabilities of the platform are demonstrated through the continuous assessment of anchor cell (AC) invasion and distal tip cell (DTC) migration in larval C. elegans and germ cell apoptosis in adult C. elegans in vivo for the first time.

Cell Invasion In Vivo via Rapid Exocytosis of a Transient Lysosome-Derived Membrane Domain

Invasive cells use small invadopodia to breach basement membrane (BM), a dense matrix that encases tissues. Following the breach, a large protrusion forms to clear a path for tissue entry by poorly understood mechanisms. Using RNAi screening for defects in Caenorhabditis elegans anchor cell (AC) invasion, we found that UNC-6(netrin)/UNC-40(DCC) signaling at the BM breach site directs exocytosis of lysosomes using the exocyst and SNARE SNAP-29 to form a large protrusion that invades vulval tissue. Live-cell imaging revealed that the protrusion is enriched in the matrix metalloprotease ZMP-1 and transiently expands AC volume by more than 20%, displacing surrounding BM and vulval epithelium. Photobleaching and genetic perturbations showed that the BM receptor dystroglycan forms a membrane diffusion barrier at the neck of the protrusion, which enables protrusion growth. Together these studies define a netrin-dependent pathway that builds an invasive protrusion, an isolated lysosome-derived membrane structure specialized to breach tissue barriers.

Recent progress of divertor simulation research using the GAMMA 10/PDX tandem mirror

This paper describes the recent progress in divertor simulation research using the GAMMA 10/PDX tandem mirror towards the development of divertors in fusion reactors. During a plasma flow generation experiment in the end cell of the GAMMA 10/PDX, ICRF heating in the anchor cell successfully extended the particle flux up to 3.3 × 1023 m2 s−1. Superimposing the short pulse of the ECH also attained a maximum heat flux of ~30 MW m−2. We have succeeded in achieving and characterizing the detachment of the high-temperature plasma, which is equivalent to the SOL plasma of tokamaks, by using the divertor simulation experimental module (D-module) in the GAMMA 10/PDX end cell, in spite of using a linear device with a short magnetic field line connection length. Various gases (Ar, Xe, Ne and N2) are examined to evaluate the effect of radiation cooling against the plasma flow at the MW m−2 level in the divertor simulation region and the following results are obtained: (i) Xe gas was most effective in the reduction of heat and particle fluxes (1%, 3%, respectively) and has a stronger effect on electron cooling (down to ~1.6 eV) in the used gas species. (ii) Ne gas was less effective. On the other hand, (iii) N2 gas showed more favorable effects than Ar in the lower pressure range. These results will contribute to the progress in detached plasma operation and in clarifying the radiation cooling mechanism towards the development of future divertors.

The Invading Anchor Cell Induces Lateral Membrane Constriction during Vulval Lumen Morphogenesis in C. elegans

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

Computing linear systems on metric graphs

The linear system |D| of a divisor D on a metric graph has the structure of a cell complex. We introduce the anchor divisors and anchor cells in it – they serve as the landmarks for us to compute the f-vector of the complex and find all cells in the complex. A linear system can also be identified as a tropical convex hull of rational functions. We compute its extremal generators using the landmarks. We apply these methods to some examples – namely the canonical linear systems of some small trivalent graphs.

Microtubule Motors in Establishment of Epithelial Cell Polarity

Epithelial cells play a key role in insuring physiological homeostasis by acting as a barrier between the outside environment and internal organs. They are also responsible for the vectorial transport of ions and fluid essential to the function of many organs. To accomplish these tasks, epithelial cells must generate an asymmetrically organized plasma membrane comprised of structurally and functionally distinct apical and basolateral membranes. Adherent and occluding junctions, respectively, anchor cells within a layer and prevent lateral diffusion of proteins in the outer leaflet of the plasma membrane and restrict passage of proteins and solutes through intercellular spaces. At a fundamental level, the establishment and maintenance of epithelial polarity requires that signals initiated at cell-substratum and cell-cell adhesions are transmitted appropriately and dynamically to the cytoskeleton, to the membrane-trafficking machinery, and to the regulation of occluding and adherent junctions. Rigorous descriptive and mechanistic studies published over the last 50 years have provided great detail to our understanding of epithelial polarization. Yet still, critical early steps in morphogenesis are not yet fully appreciated. In this review, we discuss how cytoskeletal motor proteins, primarily kinesins, contribute to coordinated modification of microtubule and actin arrays, formation and remodeling of cell adhesions to targeted membrane trafficking, and to initiating the formation and expansion of an apical lumen.

Canalization of C. elegans Vulva Induction against Anatomical Variability

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

The extracellular metalloprotease AdamTS-A anchors neural lineages in place within and preserves the architecture of the central nervous system.

The extracellular matrix (ECM) regulates cell migration and sculpts organ shape. AdamTS proteins are extracellular metalloproteases known to modify ECM proteins and promote cell migration, but demonstrated roles for AdamTS proteins in regulating CNS structure and ensuring cell lineages remain fixed in place have not been uncovered. Using forward genetic approaches in Drosophila, we find that reduction of AdamTS-A function induces both the mass exodus of neural lineages out of the CNS and drastic perturbations to CNS structure. Expressed and active in surface glia, AdamTS-A acts in parallel to perlecan and in opposition to viking/collagen IV and βPS-integrin to keep CNS lineages rooted in place and to preserve the structural integrity of the CNS. viking/collagen IV and βPS-integrin are known to promote tissue stiffness and oppose the function of perlecan, which reduces tissue stiffness. Our work supports a model in which AdamTS-A anchors cells in place and preserves CNS architecture by reducing tissue stiffness.

Defining the hierarchical organisation of collagen VI microfibrils at nanometre to micrometre length scales.

Cartilage is a connective tissue rich in extracellular matrix molecules and is tough and compressive to cushion the bones of joints. However, in adults cartilage is poorly repaired after injury and so this is an important target for tissue engineering. Many connective tissues contain collagen VI, which forms microfibrils and networks but we understand very little about these assemblies or the tissue structures they form. Therefore, we have use complementary imaging techniques to image collagen VI microfibrils from the nano-scale to the micro-scale in order to understand the structure and the assemblies it forms. These findings will help to inform the future design of scaffolds to mimic connective tissues in regenerative medicine applications.

Amino acid duplication in the coiled-coil structure of collagen XVII alters its maturation and trimerization causing mild junctional epidermolysis bullosa.

The function and stability of collagens depend on the accurate triple helix formation of three distinct polypeptide chains. Disruption of this triple-helical structure can result in connective-tissue disorders. Triple helix formation is thought to depend on three-stranded coiled-coil oligomerization sites within non-collagenous domains. However, only little is known about the physiological relevance of these coiled-coil structures. Transmembrane collagen XVII, also known as 180 kDa bullous pemphigoid antigen provides mechanical stability through the anchorage of epithelial cells to the basement membrane. Mutations in the collagen XVII gene, COL17A1, cause junctional epidermolysis bullosa (JEB), characterized by chronic trauma-induced skin blistering. Here we exploited a novel naturally occurring COL17A1 mutation, leading to an in-frame lysine duplication within the coiled-coil structure of the juxtamembranous NC16A domain of collagen XVII, which resulted in a mild phenotype of JEB due to reduced membrane-anchored collagen XVII molecules. This mutation causes structural changes in the mutant molecule and interferes with its maturation. The destabilized coiled-coil structure of the mutant collagen XVII unmasks a furin cleavage site that results in excessive and non-physiological ectodomain shedding during its maturation. Furthermore, it decreases its triple-helical stability due to defective coiled-coil oligomerization, which makes it highly susceptible to proteolytic degradation. As a consequence of altered maturation and decreased stability of collagen XVII trimers, reduced collagen XVII is incorporated into the cell membrane, resulting in compromised dermal-epidermal adhesion. Taken together, using this genetic model, we provide the first proof that alteration of the coiled-coil structure destabilizes oligomerization and impairs physiological shedding of collagen XVII in vivo.

Abstract A18: G1 cell cycle arrest is required for invasive behavior

Abstract The ability for a cell to breach the surrounding basement membrane and adopt an invasive phenotype is a critical, but poorly understood step in the progression of cancer. Like many aspects of cancer cell behavior, the genetic programs that regulate invasive behavior are likely shared with normal cell biological processes, as invasion occurs during embryonic development and immune surveillance. Due to difficulties of studying this dynamic behavior in vivo, elucidating the genetic and epigenetic controls of cell invasive activity has been difficult. Our laboratory uses a unique in vivo model to examine cell invasion that combines functional genomic and genetic tools with single-cell high resolution visual analyses. We examine anchor cell (AC) invasion into the vulval epithelium during the larval development of the model roundworm, C. elegans. Our data functionally links G1 cell cycle arrest to acquisition of invasive behavior. Previously we have identified that a single transcription factor, the conserved nuclear hormone receptor nhr-67 (NR2E1/TLX) is required in the AC to prevent the invasive AC from entering the cell cycle. NHR-67 maintains the AC in G1 cell cycle arrest, in part through upregulation of the cyclin-dependent kinase inhibitor cki-1 (p21CIP1/p27Kip1). Loss of nhr-67 results in non-invasive mitotic ACs that fail to express matrix metalloproteinases (MMPs) and actin regulators or form invadopodia, F-actin rich membrane-localized protrusions that are required for invasion. Strikingly, AC invasion can be rescued through induction of G1 arrest, preventing cell division and promoting differentiation. Downstream of G1 arrest, the AC requires the activity of the conserved histone deacetylase HDA-1, a key regulator of cell differentiation, to regulate the expression of pro-invasive genes and localize invadopodia. Through loss-of-function RNA interference (RNAi) screening, we have identified new cell cycle regulatory components (lin-35/RB, skr-2/SKP1, cdc-14/CDC14A, cul-1/ Cullin-1 and cul-4/ Cullin-4B) and epigenetic modifiers (let-418/Mi-2/CHD3 and Swi/snf-components) that function with cki-1 and hda-1, respectively, to maintain G1 cell cycle arrest and differentiate the invasive phenotype. Together our results suggest that the acquisition of the invasive phenotype is a post-mitotic differentiated state, which may help explain the paradoxical reports that the invasive fronts of many metastatic cancers are non-proliferative. Citation Format: Abraham Q. Kohrman, Mana Chandhok, Wan Zhang, David Q. Matus. G1 cell cycle arrest is required for invasive behavior. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A18.

Boundary cells restrict dystroglycan trafficking to control basement membrane sliding during tissue remodeling.

Epithelial cells and their underlying basement membranes (BMs) slide along each other to renew epithelia, shape organs, and enlarge BM openings. How BM sliding is controlled, however, is poorly understood. Using genetic and live cell imaging approaches during uterine-vulval attachment in C. elegans, we have discovered that the invasive uterine anchor cell activates Notch signaling in neighboring uterine cells at the boundary of the BM gap through which it invades to promote BM sliding. Through an RNAi screen, we found that Notch activation upregulates expression of ctg-1, which encodes a Sec14-GOLD protein, a member of the Sec14 phosphatidylinositol-transfer protein superfamily that is implicated in vesicle trafficking. Through photobleaching, targeted knockdown, and cell-specific rescue, our results suggest that CTG-1 restricts BM adhesion receptor DGN-1 (dystroglycan) trafficking to the cell-BM interface, which promotes BM sliding. Together, these studies reveal a new morphogenetic signaling pathway that controls BM sliding to remodel tissues.

Redundancy and Molecular Evolution: The Rapid Induction of Bone Formation by the Mammalian Transforming Growth Factor-β3 Isoform.

The soluble osteogenic molecular signals of the transforming growth factor-β (TGF-β) supergene family are the molecular bases of the induction of bone formation and postnatal bone tissue morphogenesis with translation into clinical contexts. The mammalian TGF-β3 isoform, a pleiotropic member of the family, controls a vast array of biological processes including the induction of bone formation. Recombinant hTGF-β3 induces substantial bone formation when implanted with either collagenous bone matrices or coral-derived macroporous bioreactors in the rectus abdominis muscle of the non-human primate Papio ursinus. In marked contrast, the three mammalian TGF-βs do not initiate the induction of bone formation in rodents and lagomorphs. The induction of bone by hTGF-β3/preloaded bioreactors is orchestrated by inducing fibrin-fibronectin rings that structurally organize tissue patterning and morphogenesis within the macroporous spaces. Induced advancing extracellular matrix rings provide the structural anchorage for hyper chromatic cells, interpreted as differentiating osteoblasts re-programmed by hTGF-β3 from invading myoblastic and/or pericytic differentiated cells. Runx2 and Osteocalcin expression are significantly up-regulated correlating to multiple invading cells differentiating into the osteoblastic phenotype. Bioreactors pre-loaded with recombinant human Noggin (hNoggin), a BMPs antagonist, show down-regulation of BMP-2 and other profiled osteogenic proteins' genes resulting in minimal bone formation. Coral-derived macroporous constructs preloaded with binary applications of hTGF-β3 and hNoggin also show down-regulation of BMP-2 with the induction of limited bone formation. The induction of bone formation by hTGF-β3 is via the BMPs pathway and it is thus blocked by hNoggin. Our systematic studies in P. ursinus with translational hTGF-β3 in large cranio-mandibulo-facial defects in humans are now requesting the re-evaluation of “Bone: formation by autoinduction” in primate models including humans.

Evolution of New cis-Regulatory Motifs Required for Cell-Specific Gene Expression in Caenorhabditis.

Patterning of C. elegans vulval cell fates relies on inductive signaling. In this induction event, a single cell, the gonadal anchor cell, secretes LIN-3/EGF and induces three out of six competent precursor cells to acquire a vulval fate. We previously showed that this developmental system is robust to a four-fold variation in lin-3/EGF genetic dose. Here using single-molecule FISH, we find that the mean level of expression of lin-3 in the anchor cell is remarkably conserved. No change in lin-3 expression level could be detected among C. elegans wild isolates and only a low level of change—less than 30%—in the Caenorhabditis genus and in Oscheius tipulae. In C. elegans, lin-3 expression in the anchor cell is known to require three transcription factor binding sites, specifically two E-boxes and a nuclear-hormone-receptor (NHR) binding site. Mutation of any of these three elements in C. elegans results in a dramatic decrease in lin-3 expression. Yet only a single E-box is found in the Drosophilae supergroup of Caenorhabditis species, including C. angaria, while the NHR-binding site likely only evolved at the base of the Elegans group. We find that a transgene from C. angaria bearing a single E-box is sufficient for normal expression in C. elegans. Even a short 58 bp cis-regulatory fragment from C. angaria with this single E-box is able to replace the three transcription factor binding sites at the endogenous C. elegans lin-3 locus, resulting in the wild-type expression level. Thus, regulatory evolution occurring in cis within a 58 bp lin-3 fragment, results in a strict requirement for the NHR binding site and a second E-box in C. elegans. This single-cell, single-molecule, quantitative and functional evo-devo study demonstrates that conserved expression levels can hide extensive change in cis-regulatory site requirements and highlights the evolution of new cis-regulatory elements required for cell-specific gene expression.

The C. elegans hox gene lin-39 controls cell cycle progression during vulval development

Cell fate specification during organogenesis is usually followed by a phase of cell proliferation to produce the required number of differentiated cells. The Caenorhabditis elegans vulva is an excellent model to study how cell fate specification and cell proliferation are coordinated. The six vulval precursor cells (VPCs) are born at the first larval stage, but they arrest in the G1 phase of the cell cycle until the beginning of the third larval stage, when their fates are specified and the three proximal VPCs proliferate to generate 22 vulval cells. An epidermal growth factor (EGF) signal from the gonadal anchor cell combined with lateral DELTA/NOTCH signaling between the VPCs determine the primary (1°) and secondary (2°) fates, respectively. The hox gene lin-39 plays a key role in integrating these spatial patterning signals and in maintaining the VPCs as polarized epithelial cells. Using a fusion-defective eff-1(lf) mutation to keep the VPCs polarized, we find that VPCs lacking lin-39 can neither activate lateral NOTCH signaling nor proliferate. LIN-39 promotes cell cycle progression through two distinct mechanisms. First, LIN-39 maintains the VPCs competent to proliferate by inducing cdk-4 cdk and cye-1 cyclinE expression via a non-canonical HOX binding motif. Second, LIN-39 activates in the adjacent VPCs the NOTCH signaling pathway, which promotes VPC proliferation independently of LIN-39. The hox gene lin-39 is therefore a central node in a regulatory network coordinating VPC differentiation and proliferation.

Abstract 919: A genome-wide screen in C. elegans identifies cell non-autonomous suppressors of let-60/RAS mediated oncogenic over-proliferation

Abstract Coordinated proliferative signals from the mesenchymal cells play a crucial role in the regulation of proliferation of epithelial cells during normal development, wound healing and several other normal physiological conditions. However, when epithelial cells acquire a set of malignant mutations, they respond differently to these extrinsic proliferative signals leading to enhanced abnormal proliferation of mutant epithelial cells and hence tumor growth. Despite mounting evidence that stromal cells influence the growth of tumors and cancer progression, it is unclear which specific genes in these mesenchymal cells regulate the molecular signals that promote the over-proliferation of the adjacent mutant cells. The complexity of several cell types and their interactions in vivo in the cancer mouse models and human tumor samples limits our ability to identify mesenchymal genes important in this process. Thus, we took a cross species approach to use C. elegans vulval development as a model to better understand the impact of mesenchymal (mesodermal) cells on the proliferation of the epithelial (epidermal) cells. This model includes well-described signaling interactions between mesodermal (anchor cell, gonad, and muscle) cells and epidermal (vulva precursor cells, VPCs) cells. In this system, three of the six equipotent VPCs normally acquire vulva fates (termed 1° and 2°) upon receiving coordinated EGF and Wnt signals from the mesodermal cells, and lateral Notch signaling between the VPCs. Then, they divide 3 times to produce a symmetric vulva comprised of 22 daughter cells. Abnormal activation of the EGF pathway, such as by introducing gain-of-function (gf) mutations in let-60/Ras, leads to the promotion of vulval fates in the remaining three VPCs, which then divide inappropriately resulting in development of multiple vulval protrusions (Muv phenotype).To identify genes that function in mesodermal cells to inhibit the proliferation of Ras-activated epidermal cells, we genetically engineered C. elegans to develop a strain that possesses let-60(gf)/Ras mediated over-proliferation (Muv phenotype) and RNA interference (RNAi) competence specific to the mesodermal tissues. This strain was subjected to a genome-wide RNAi screen. Our screen identified 47 genes that, upon RNAi knock-down of a gene in mesodermal cells, suppress over-proliferation of let-60(gf)/Ras epithelial cells in a genotype-specific manner. Notably, these genes encode chromatin remodeling proteins, ribosomal proteins, proteins involved in vesicle transport, and metabolic factors, rather than secreted molecules. Importantly, candidate genes emerged from this screen are significantly enriched for the conserved genes from C. elegans to humans. These results form an initial understanding of molecular factors in mesenchymal cells that impact oncogenic cell proliferation. Citation Format: Komal Rambani, Raleigh Kladney, Serena Chang, Markus Harrigan, James Dowdle, Xing Tang, Huayang Liu, Elizabeth Brunner, Helen Chamberlin, Gustavo Leone. A genome-wide screen in C. elegans identifies cell non-autonomous suppressors of let-60/RAS mediated oncogenic over-proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 919.

Anchor cell signaling and vulval precursor cell positioning establish a reproducible spatial context during C. elegans vulval induction

How cells coordinate their spatial positioning through intercellular signaling events is poorly understood. Here we address this topic using Caenorhabditis elegans vulval patterning during which hypodermal vulval precursor cells (VPCs) adopt distinct cell fates determined by their relative positions to the gonadal anchor cell (AC). LIN-3/EGF signaling by the AC induces the central VPC, P6.p, to adopt a 1° vulval fate. Exact alignment of AC and VPCs is thus critical for correct fate patterning, yet, as we show here, the initial AC-VPC positioning is both highly variable and asymmetric among individuals, with AC and P6.p only becoming aligned at the early L3 stage. Cell ablations and mutant analysis indicate that VPCs, most prominently 1° cells, move towards the AC. We identify AC-released LIN-3/EGF as a major attractive signal, which therefore plays a dual role in vulval patterning (cell alignment and fate induction). Additionally, compromising Wnt pathway components also induces AC-VPC alignment errors, with loss of posterior Wnt signaling increasing stochastic vulval centering on P5.p. Our results illustrate how intercellular signaling reduces initial spatial variability in cell positioning to generate reproducible interactions across tissues.

Adhesive organ regeneration in Macrostomum lignano.

BackgroundFlatworms possess pluripotent stem cells that can give rise to all cell types, which allows them to restore lost body parts after injury or amputation. This makes flatworms excellent model systems for studying regeneration. In this study, we present the adhesive organs of a marine flatworm as a simple model system for organ regeneration. Macrostomum lignano has approximately 130 adhesive organs at the ventral side of its tail plate. One adhesive organ consists of three interacting cells: one adhesive gland cell, one releasing gland cell, and one modified epidermal cell, called an anchor cell. However, no specific markers for these cell types were available to study the regeneration of adhesive organs.ResultsWe tested 15 commercially available lectins for their ability to label adhesive organs and found one lectin (peanut agglutinin) to be specific to adhesive gland cells. We visualized the morphology of regenerating adhesive organs using lectin- and antibody staining as well as transmission electron microscopy. Our findings indicate that the two gland cells differentiate earlier than the connected anchor cells. Using EdU/lectin staining of partially amputated adhesive organs, we showed that their regeneration can proceed in two ways. First, adhesive gland cell bodies are able to survive partial amputation and reconnect with newly formed anchor cells. Second, adhesive gland cell bodies are cleared away, and the entire adhesive organ is build anew.ConclusionOur results provide the first insights into adhesive organ regeneration and describe ten new markers for differentiated cells and tissues in M. lignano. The position of adhesive organ cells within the blastema and their chronological differentiation have been shown for the first time. M. lignano can regenerate adhesive organs de novo but also replace individual anchor cells in an injured organ. Our findings contribute to a better understanding of organogenesis in flatworms and enable further molecular investigations of cell-fate decisions during regeneration.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0121-1) contains supplementary material, which is available to authorized users.