Repulsive guidance molecule acts in axon branching in Caenorhabditis elegans

Repulsive guidance molecules (RGMs) are evolutionarily conserved proteins implicated in repulsive axon guidance. Here we report the function of the Caenorhabditis elegans ortholog DRAG-1 in axon branching. The axons of hermaphrodite-specific neurons (HSNs) extend dorsal branches at the region abutting the vulval muscles. The drag-1 mutants exhibited defects in HSN axon branching in addition to a small body size phenotype. DRAG-1 expression in the hypodermal cells was required for the branching of the axons. Although DRAG-1 is normally expressed in the ventral hypodermis excepting the vulval region, its ectopic expression in vulval precursor cells was sufficient to induce the branching. The C-terminal glycosylphosphatidylinositol anchor of DRAG-1 was important for its function, suggesting that DRAG-1 should be anchored to the cell surface. Genetic analyses suggested that the membrane receptor UNC-40 acts in the same pathway with DRAG-1 in HSN branching. We propose that DRAG-1 expressed in the ventral hypodermis signals via the UNC-40 receptor expressed in HSNs to elicit branching activity of HSN axons.

Repulsive Guidance Molecule Acts in Axon Branching in Caenorhabditis elegans

Repulsive guidance molecules (RGMs) are evolutionarily conserved proteins implicated in repulsive axon guidance. Here we report the function of the Caenorhabditis elegans ortholog DRAG-1 in axon branching. The axons of hermaphrodite-specific neurons (HSNs) extend dorsal branches at the region abutting the vulval muscles. The drag-1 mutants exhibited defects in HSN axon branching in addition to a small body size phenotype. DRAG-1 expression in the hypodermal cells was required for the branching of the axons. Although DRAG-1 is normally expressed in the ventral hypodermis excepting the vulval region, its ectopic expression in vulval precursor cells was sufficient to induce the branching. The C-terminal glycosylphosphatidylinositol anchor of DRAG-1 was important for its function, suggesting that DRAG-1 should be anchored to the cell surface. Genetic analyses suggested that the membrane receptor UNC-40 acts in the same pathway with DRAG-1 in HSN branching. We propose that DRAG-1 expressed in the ventral hypodermis signals via the UNC-40 receptor expressed in HSNs to elicit branching activity of HSN axons.

Nuclear translocation of tagged endogenous MPK-1/ERK denotes a subset of activation events in C. elegans development

The extracellular signal-regulated kinase (ERK) MAP kinase is utilized downstream of Ras>Raf>MEK signaling to control activation of a wide array of targets. Activation of ERK is elevated in Ras-driven tumors and RASopathies, and is thus a target for pharmacological inhibition. Regulatory mechanisms of ERK activation have been studied extensively in vitro and in cultured cells but little in living animals. We tagged the C. elegans ERK-encoding gene, mpk-1. MPK-1 is ubiquitously expressed with elevated expression in certain contexts. We detected cytosol-to-nuclear translocation of MPK-1 in maturing oocytes and hence validated nuclear translocation as a reporter of some activation events. During patterning of vulval precursor cells, MPK-1 is necessary and sufficient for the central cell, P6.p, to assume 1˚ fate. Yet MPK-1 translocates to the nuclei of all six VPCs in a temporal and concentration gradient centered on P6.p. This observation contrasts with previous results using the ERK-nKTR reporter of substrate activation, raising questions about mechanisms and indicators of MPK-1 activation. This system and reagent promise to provide critical insights into regulation of MPK-1 activation within a complex intercellular signaling network.

Nets-within-nets for modeling emergent patterns in ontogenetic processes

ABSTRACTOntogenesis is the development of an organism from its earliest stage to maturity, including homeostasis maintenance throughout adulthood despite environmental perturbations. Almost all cells of a multicellular organism share the same genomic information. Nevertheless, phenotypic diversity and complex supra-cellular architectures emerge at every level, starting from tissues and organs. This is possible thanks to a robust and dynamic interplay of regulative mechanisms.To study ontogenesis, it is necessary to consider different levels of regulation, both genetic and epigenetic. Each cell undergoes a specific path across a landscape of possible regulative states affecting both its structure and its functions during development. This paper proposes using the Nets-Within-Nets formalism, which combines Petri Nets’ simplicity with the capability to represent and simulate the interplay between different layers of regulation connected by non-trivial and context-dependent hierarchical relations.In particular, this work introduces a modeling strategy based on Nets-Within-Nets that can model several critical processes involved in ontogenesis. Moreover, it presents a case study focusing on the first phase of Vulval Precursor Cells specification in C. Elegans. The case study shows that the proposed model can simulate the emergent morphogenetic pattern corresponding to the observed developmental outcome of that phase, in both the physiological case and different mutations. The model presented in the results section is available online at https://github.com/sysbio-polito/NWN_CElegans_VPC_model/

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

ABSTRACTActivation 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.

Insulated Switches: Dual-Function Protein RalGEFRGL-1 Promotes Developmental Fidelity

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

The Signaling Network Controlling C. elegans Vulval Cell Fate Patterning

EGF, emitted by the Anchor Cell, patterns six equipotent C. elegans vulval precursor cells to assume a precise array of three cell fates with high fidelity. A group of core and modulatory signaling cascades forms a signaling network that demonstrates plasticity during the transition from naïve to terminally differentiated cells. In this review, we summarize the history of classical developmental manipulations and molecular genetics experiments that led to our understanding of the signals governing this process, and discuss principles of signal transduction and developmental biology that have emerged from these studies.

Developmental fidelity imposed by the RGL-1 “Balanced Switch” mediating opposing signals

The six C. elegans vulval precursor cells (VPCs) are induced to form the 30-30-20-Γ-20-3° pattern of cell fates with high fidelity. In response to EGF signal, the LET-60/Ras-LIN-45/Raf-MEK-2/MEK-MPK-1/ERK canonical MAP kinase cascade is necessary to induce 1° fate and synthesis of DSL ligands. In turn, LIN-12/Notch signal is necessary to induce neighboring cells to become 2°. We previously showed that, in response to lower dose of EGF signal, the modulatory LET-60/Ras-RGL-1/RalGEF-RAL-1/Ral signal promotes 2° fate in support of LIN-12. In this study we identify two key differences between RGL-1 and RAL-1 functions. First, deletion of RGL-1 confers no overt developmental defects, while previous studies showed RAL-1 to be essential for viability and fertility. From this observation we hypothesize that the developmentally essential functions of RAL-1 are independent of upstream activation. Second, RGL-1 plays opposing and genetically separable roles in VPC fate patterning. RGL-1 promotes 2° fate via canonical GEF-dependent activation of RAL-1 and 1° fate via a non-canonical GEF-independent activity. Our genetic epistasis experiments are consistent with RGL-1 functioning in the modulatory 1°-promoting AGE-1/PI3-Kinase-PDK-1-AKT-1 cascade. Additionally, animals without RGL-1 experience 15-fold higher rates of VPC patterning errors compared to the wild type. Yet VPC patterning in RGL-1 deletion mutants is not more sensitive to environmental perturbations. We propose that RGL-1 functions as a “Balanced Switch” that orchestrates opposing 1°- and 2°-promoting modulatory cascades to decrease inappropriate fate decisions. We speculate that such switches are broadly conserved but mostly masked by paralog redundancy or essential genes.

Description and developmental biology of the predatory diplogastrid Acrostichus nudicapitatus (Steiner, 1914) Massey, 1962 (Nematoda: Rhabditida)

SummaryAcrostichus nudicapitatus (Steiner, 1914) Massey, 1962 is redescribed and illustrated along with observations on its developmental biology. Most morphometrics of the present population agree well with those of A. nudicapitatus (Steiner, 1914) Massey, 1962. The largely oviparous females of A. nudicapitatus lay eggs in single-celled or two-celled stage, 1.5 – 2 h after fertilization. In cultured females, the uterine tract was observed to accommodate occasionally as many as 4 – 6 eggs. The eggs are smooth-shelled, oval in shape measuring 45 – 48 x 23 – 26 μm in dimension. The pole of entry of sperm marks the posterior end of the developing embryo. The embryonation time has been recorded to be 20 – 25 h at 25 ± 2 °C. The first moult occurs inside the egg and the juvenile hatches as second stage juvenile. The gonad development follows the trends found in most rhabditids, however, three prime cells of the 12 vulval precursor cells have been observed to be involved in vulva formation.