Cohen, Jennifer et al. (2019) International Worm Meeting "Structure and assembly of C. elegans' pre-cuticular apical extracellular matrix."

Cohen, Jennifer, Belfi, Alexandra, Birnbaum, Susanna, Sundaram, Meera

[International Worm Meeting, 2019]

A lipid and glycoprotein-rich apical extracellular matrix (aECM) lines and protects all exposed epithelial surfaces, including the skin and the internal walls of tubes, but the contents, assembly and functional properties of such matrices are not well understood. In C. elegans, an early pre-cuticular or "sheath" aECM lines developing (or molting) external epithelia such as the hypodermis, vulva, rectum, and excretory duct and pore. Somewhat different versions of this aECM are present in different tissues and stages, but all share many of the same types of proteins, including zona pellucida (ZP) domain proteins. Our studies of this early aECM showed that it shapes developing epithelia and patterns the subsequent cuticle: loss of individual matrix components can result in embryonic rupture, collapse of narrow excretory tubes, mis-shapen vulva tubes, molting defects, and/or cuticle alae defects. With many aECM proteins or regulators now identified, we can ask how each contributes to the layered aECM structure and test potential regulatory hierarchies for aECM layer secretion, assembly and disassembly. We've used CRISPR to tag some of these proteins with fluorescent markers, and we see beautiful and complex matrix patterns in different tissues. In the vulva lumen, different matrix factors assemble over 1? vs. 2? vulval cells, and there are dynamic changes in matrix organization as the lumen changes shape. For example, LET-653 is recruited specifically and transiently to the matrix over 1? vulva cells via a mechanism that requires the C-terminal portion of its ZP domain, proteolytic cleavage, and the Patched-related transmembrane protein PTR-4, which is found on the apical surfaces of 2? and 3? cells. In the hypodermis, various glycoproteins travel to or from the apical surface within different types of vesicles, and many accumulate significantly in lysosome-related organelles. Certain glycoproteins form stripes along the seam hypodermis that precede or coincide with alae formation, providing insights into the early steps of forming those specialized ridges. Together, our studies establish the worm pre-cuticular aECM as a model for probing structure/function relationships for conserved matrix protein families and for working out basic principles of aECM assembly.

Kanther, Michelle et al. (2013) International Worm Meeting "Identification of genes important for excretory system function and maintenance using Whole Genome Sequencing."

Sundaram, Meera, Parry, Jean, Cohen, Jennifer, Kanther, Michelle

[International Worm Meeting, 2013]

Tubes form the building blocks of organ development in invertebrates and vertebrates. Defects in tube development and maintenance are associated with several human diseases. The critical steps of tube development, including cell polarization and epithelial junction formation, have been well studied; however, the genes regulating these steps remain poorly defined. The C. elegans excretory system provides a simple and genetically tractable system for studying unicellular tube formation and maintenance. This tubular network is required for osmoregulation and its integrity is necessary for development and survival. It is composed of three tandem unicellular tubes: the canal, duct, and pore. These cells are connected by apicolateral junctions and form a continuous lumen that opens to the outside environment via the pore cell. Both the duct and pore cells form by a poorly-understood cell wrapping mechanism. The pore retains a characteristic autojunction, while the duct becomes a seamless tube. Defects in this system result in fluid accumulation and death of "rod-like" L1 larvae. To discover novel genes involved in the formation and maintenance of unicellular tubes, we performed an EMS mutagenesis screen. From our screen we isolated 85 recessive, rod-like lethal mutants. Phenotypic analysis of these mutants reveals three distinct classifications. Class A mutants initially display normal junctions within and between all excretory cells; mutants in this class include alleles of let-653, lpr-1, egg-6, let-4, and rdy-2,3,4. Class B mutants exhibit two pore cell autojunctions (similar to Ras loss-of-function), while Class C mutants lack a pore cell autojunction (similar to Ras gain-of-function or Notch loss-of-function). To identify the causative mutations underlying our observed Class A phenotypes, we used balancers to map 23 mutations to chromosomal regions. After testing known candidates by complementation, we performed SNP mapping and whole genome sequencing using a scheme adapted from Doitsidou et. al. (2010) and Minevich et. al. (2012). We are now testing which variants are causative and extending our approach to the other mutant classes. Note: Kanther & Cohen co-authors.

Birnbaum, Susanna et al. (2021) International Worm Meeting "Characterizing a Matrix Protease important for epithelial tissue shaping in C. elegans"

Sundaram, Meera, Cohen, Jennifer, Birnbaum, Susanna

[International Worm Meeting, 2021]

The apical extracellular matrix (aECM) is a lipid-and glycoprotein-rich protective layer that lines epithelial surfaces exposed to the environment. Despite its importance in tissue shaping and protection, the components and organization of aECM are not well understood. C. eleganshas a pre-cuticular aECM that lines and shapes developing epithelia such as the hypodermis, vulva, rectum, and excretory duct and pore tubes. Our lab has identified proteins in the C. eleganspre-cuticular aECM, many of which share domains with mammalian matrix proteins. We are working towards characterizing these aECM components to understand function and hierarchy within the matrix. Proteases in the matrix often regulate assembly, disassembly and proper function of aECM components; we are interested in identifying proteases that cleave some of our matrix components. One candidate is BLI-4, a relative of the mammalian proprotein convertase subtilisin/kexin (PCSK) family. We generated bli-4 knock-out mutants using CRISPR and found its phenotypes were very similar to those of some of our aECM mutants. We are conducting further experiments to investigate endogenous BLI-4 expression and which BLI-4 isoform(s) is necessary for pre-cuticle development/embryonic viability. Understanding the role of BLI-4 will contribute to a greater knowledge of basic aECM structure and assembly.

Forman-Rubinsky, Rachel et al. (2017) International Worm Meeting "Lipocalins are required for apical extracellular matrix organization."

Sundaram, Meera, Forman-Rubinsky, Rachel, Cohen, Jennifer

[International Worm Meeting, 2017]

Biological tubes are lined by a poorly understood apical (luminal) extracellular matrix (aECM) or "glycocalyx" consisting of lipids, carbohydrates and glycoproteins. This aECM influences tube shape and integrity, and its disruption or loss can cause organ failure and disease. Very narrow tubes, such as capillaries and lung alveoli, appear particularly susceptible to aECM-related defects. We are using C. elegans to study the specific composition and structure of aECM and its tube-protecting functions. C. elegans external epithelia develop in the presence of a glycocalyx-like aECM that later matures to form the cuticle. Several components of the early glycocalyx are required for integrity of the narrow excretory duct and pore tubes. Others are required to inflate the larger vulva tube. By screening for mutants with excretory duct and pore defects, and then visualizing the discovered proteins in the vulva (where they are also present), we are beginning to learn about the multi-layered organization and dynamics of the protective glycocalyx. EMS mutagenesis screens for lethal mutants with duct or pore cell abnormalities identified two lipocalins; lpr-1 and lpr-3. Lipocalins are a family of functionally diverse, cup-shaped secreted proteins that bind and transport various lipophilic molecules. In humans, lipocalins are often found in luminal or aECM compartments such as blood plasma, urine or tear film, and they are used as biomarkers for detecting tissue damage. LPR-1 and LPR-3 are apically secreted into the glycocalyx, but they have different patterns of localization. LPR-1 localizes diffusely and can function tissue non-autonomously. LPR-3 localizes specifically to a matrix layer near the apical membrane, adjacent to the glycoprotein LET-653. In addition to the excretory phenotypes, both lpr mutants have other aECM related phenotypes such as defects in alae, cuticle permeability, or molting. We conclude that these lipocalins are required for aECM organization and its tube-protecting functions. Current studies are testing relationships between lipocalins and other aECM glycoproteins, and investigating whether lipocalins transport or sequester aECM lipids.

Gill, Hasreet et al. (2015) International Worm Meeting "Critical roles for the apical extracellular matrix in C. elegans excretory duct and pore tube development."

Forman-Rubinsky, Rachel, Gill, Hasreet, Sundaram, Meera, Cohen, Jennifer, Pu, Pu

[International Worm Meeting, 2015]

Polarized tubular epithelia are vital components of most organ systems. The apical (or lumenal) and basal domains in tube cells are demarcated by specialized apical junctions that link adjacent cells. These domains also associate with distinct extracellular matrix (ECM) environments. While it has been well established that basal ECM proteins, such as laminins and collagens, facilitate proper cell shaping, promote tissue integrity, and influence cell-cell signaling, contributions of apical ECM proteins to tubulogenesis and signaling have only recently begun to be elucidated. Importantly, defects in the apical ECM have been shown to be major contributors to diseases affecting tubular epithelia, such as chronic kidney disease and cardiovascular disease.Here, we describe broad insights into the role of the apical ECM in morphogenesis and maintenance of the C. elegans excretory duct and pore tubes. The mature duct and pore are narrow, cuticle-lined unicellular tubes that connect the excretory canal cell to the outside environment. Development and shaping of these cells during embryogenesis occurs in the context of a poorly understood pre-cuticular apical ECM. Multiple aspects of duct tube development also depend on LIN-3/EGF signaling from the canal cell, which is connected to the duct via an apical junction and lumen.In EMS mutagenesis screens for lethal mutants with duct or pore abnormalities, we identified many transmembrane or secreted proteins, including the extracellular leucine-rich repeat only (eLLRon) proteins LET-4 and EGG-6, the lipocalins LPR-1 and LPR-3, the tetraspan protein RDY-2, the nidogen and zonadhesion-related protein DEX-1, and the Zona Pellucida domain and mucin-related protein LET-653. Many of these proteins localize to apical epithelial domains and affect apical ECM organization. A combination of live imaging and transmission electron microscopy (TEM) revealed that all of these gene products are required to maintain duct lumen continuity during morphogenesis, when the duct undergoes elongation and narrowing; most are also required to maintain the shape and/or integrity of apical junctions within and between the tube cells. Epistasis experiments suggest at least two separate groups that act in parallel, where the defects in one group are suppressible by hyperactivation of EGF-Ras-ERK signaling. Together, these results challenge the paradigm of a passive apical ECM, and suggest instead that, like the basal ECM, the apical ECM plays important roles in epithelial cell shaping, maintenance and signaling.

Denham, J. et al. (2017) International Worm Meeting "An integrated neuro-mechanical model of C. elegans forward locomotion."

Ranner, T., Denham, J., Cohen, N.

[International Worm Meeting, 2017]

Behavioural responses in C. elegans can be observed through changes in locomotive patterns. It is therefore important to consider the role of the physics in computational models of the neural circuit for motor behaviours. We present a dynamical systems model of C. elegans forward locomotion in which the neural circuit is divided into a series of repeating identical units, coupled via posterior stretch receptor feedback. Each unit includes cholinergic, bistable B-type motor neurons (modelled after bistable RMD neurons, Mellem et al. 2008, Boyle et al. 2012), implicit GABAergic D-type motor neurons (Boyle et al. 2012) and nonlinear viscoelastic muscle forcing along the body (Boyle et. al. 2012). The neural model incorporates proprioceptive feedback as the mechanism for producing sustained oscillations. Integrating the neural model with a recent continuum mechanical body model (Cohen and Ranner 2017) provides this feedback and is also the method for incorporating environmental drag from the surrounding fluid, closing the neuronal-environmental loop. Biophysically realistic parameters are used to obtain sustained travelling waves in muscle activation which respond to changes in environmental viscoelasticity. To explore the pattern generation mechanism, we present results from bifurcation analysis performed in the isolated neural framework and in the fully integrated neuro-mechanical model. We show how these results are modulated by changes in the external drag and internal material properties of the passive and active body. References [1] Boyle JH, Berri S, Cohen N: Gait modulation in c. elegans: an integrated neuro-mechanical model. Frontiers in computational neuroscience 2012, 6:10. [2] Mellem JE, Brockie PJ, Madsen DM, Maricq AV: Action potentials contribute to neuronal signaling in C. elegans, Nature Neuroscience 2008, 11:865-867 [3] Cohen N, Ranner T: A new computational method for a model of C. elegans biomechanics: Insights into elasticity and locomotion performance, arXiv:1702.04988, 20

Jennifer Pilipiuk et al. (2006) European Worm Meeting "Analysis of Epithelial Genes during Postembryonic Development of C. elegans"

Olaf Bossinger, Jennifer Pilipiuk, Gisela Helbig

[European Worm Meeting, 2006]

Jennifer Pilipiuk, Gisela Helbig and Olaf Bossinger. We wish to understand how epithelial polarity and tissue integrity is maintained. Here, we consider the role of DLG-1 (Discs large), LET-413 (Scribble), ERM-1 (Ezrin-Radixin-Moesin), and the catenin-cadherin complex (HMP-1HMP-2HMR-1) during postembryonic development of C. elegans. In the course of embryogenesis these genes play an important role in the establishment and maintenance of epithelial polarity, the formation of the lumen, and cell-cell adhesion. In contrast, during larval and adult development only newly established epithelia (e.g. the spermatheka or the vulva) show severe defects after bacterial RNAi against DLG-1, LET-413 and ERM-1, while the depletion of the catenin-cadherin complex seems not to cause visible defects. Nevertheless, how polarity of already established epithelia (e.g. the intestine or the hypodermis) is maintained during postembryonic development remains elusive. In the case of DLG-1, our results suggest that a small amount of protein is sufficient, but cannot fall below a certain threshold without causing defects. Intestinal and hypodermal polarity during larval and adult development of C. elegans might also depend on other, so far unidentified proteins. A detailed analysis of DLG-1, LET-413 and ERM-1 phenotypes during postembryonic epithelial development in C. elegans will be presented and discussed.

Stefano Berri et al. (2008) European Worm Meeting "The role of the body in determining C. elegans locomotion waveforms: experiment and theory."

Ian A Hope, Stefano Berri, Netta Cohen, Jordan H Boyle

[European Worm Meeting, 2008]

C. elegans locomotion is typically studied on agar, where forward crawling. is well described by a characteristic waveform. Models of the neural and. neuromuscular control of locomotion have been restricted to modeling such. crawling behavior. Specifically, Niebur and Erdos (1991) suggest that the. sinusoidal track, or groove, left by a worm on an agar surface is. functionally significant.. The importance of the groove in determining the shape of the body may. resolve an important difference between the two existing classes of models. of forward locomotion. In the model of Niebur and Erdos (1991), the worm. relies on a central pattern generator (CPG) in the head to determine the. frequency of undulations and effectively create a groove for the rest of. the body to follow through. The body shape is therefore determined by the. path of the head. In contrast, the Bryden and Cohen model (2004, 2008) does. not include the constraints of a groove, and so the shape of the body is. determined by the muscle activation pattern all along the worm. To. determine to what extent the entire body is used by the worm to determine. its waveform during crawling behavior, we set out to investigate whether. the worm can determine its own waveform in the absence of a groove.. We compared the locomotion on agar with that of worms on a smooth, solid. (nitrocellulose) surface, which precludes the possible formation of a. groove.. Results indicate the following:. (i) The worm is capable of generating and propagating a waveform very. similar to that of forward crawling on agar.. (ii) Nonetheless, no forward motion results on the solid surface.. (iii) Using locomotion data analysis, we are able to estimate the. properties of the groove on agar and to confirm the absence of similar. forces on a solid surface.. (iv) Using a model adapted from Niebur and Erdos (1991) (Boyle, Bryden and. Cohen 2007), we successfully replicate the results in an environment. mimicking a groove. However, for insufficiently strong grooves (in. particular matching the strength of the groove to our observations of. behavior on agar) the mechanism breaks down, as the muscle activation. pattern alone is not sufficient to fully determine the body shape.. (v) Simulations of model worms with imposed waveforms and undulation. frequencies on a solid surface successfully explain the observed. experimental results.. Our work suggests that the crawling waveform of the worm does not require a. groove, and therefore that the body shape cannot be purely determined by. the motion of the head and the properties of the environment. In. particular, a minimal muscle activation pattern that generates thrust. within a groove is not sufficient to explain the behavior of the worm on a. solid surface. The worm must recruit muscles along the body to determine. the shape of the body in time.. Our conclusions may have important implications for further modeling of. neural control of locomotion in general and for predictions about the. distribution of postulated stretch receptors along ventral cord. motoneurons, in particular.

Baran, Renee A. et al. (2009) International Worm Meeting "zyg-8 Function in C. elegans Motor Neuron Development."

Baran, Renee A., Shirazi, Farah, Tang, Garland

[International Worm Meeting, 2009]

Mutations in alpha-tubulin, doublecortin, and LIS1 block neuron migration and cause lissencephaly in humans. Doublecortin/DCX and doublecortin-like proteins bind microtubules and function to promote microtubule assembly and stability while LIS1 mediates interactions with the dynein motor complex. Doublecortin proteins have also been shown to localize to neurite endings and influence axon outgrowth and dendrite arborization (Friocourt et al., 2003; Deuel et al., 2006; Cohen et al., 2008). zyg-8, the sole C. elegans member of the doublecortin family, encodes a doublecortin-like protein required for positioning the mitotic spindle during embryonic divisions (Gonzcy et al., 2001). We utilized conditional zyg-8 mutants to study the role of zyg-8 in later stages of neuronal development. zyg-8(b235ts) mutants were shifted to the nonpermissive temperature as late embryos or early L1 larvae prior to synapse formation, and the GABAergic motor neurons of mutant adults were analysed for expression of the synaptic vesicle marker SNB-1::GFP, active zone marker UNC-10::RFP, and an axon-restricted microtubule plus-end binding protein. zyg-8 mutants exhibited defects in synapse morphology similar to gain-of-function tba-1 mutants (R. Baran & Y. Jin). SNB-1::GFP puncta were irregular in size and spacing and GFP was mislocalized in the commissures. Synapses were sometimes missing from the most distal regions of axons along the dorsal nerve cord, a phenotype also observed in tba-1(ju89) mutants. These results suggest that zyg-8 may play a significant role in larval and adult motor neurons in maintaining axon and synapse integrity.

Sanders, Tom et al. (2015) International Worm Meeting "Nonlinear sensory integration in C. elegans: a computational model."

Cohen, Netta, Ghosh, D.D., Sanders, Tom, Nitabach, M.N.

[International Worm Meeting, 2015]

Animal survival depends on a combination of often conflicting demands such as foraging and evading of dangers. To navigate effectively in such unknown and changing conditions, animals must continuously integrate over a variety of sensory cues, and adapt their decision making strategy in a context dependent manner. Here, we examine the neural control of a sensory integration task in the nematode C. elegans. The task involves an ASH-triggered aversive response to high osmolarity fructose and an AWA-triggered attractive response to diacetyl [1]. In the assay, worms are placed in the center of a ring of fructose; two drops of diacetyl are located outside the ring. We present a computational model, consisting of point worms, situated in a virtual arena that closely mimics this experimental assay, and endowed with a sensory motor pathway of two sensory neurons, a neural integration pathway and two motor programs (pirouettes and steering). A monoamine (PDF-2 and tyramine) modulation circuit involving RIM and ASH is overlaid on the synaptic circuit, in line with molecular data [1]. Model parameters were constrained by behavioral data for wild type and mutant animals for a range of stimulus concentrations. Based on our simulation results, we reject a null hypothesis of a linear sensory integration mechanism in RIM and present results that are consistent with the data for a sensory "coincidence detector" like process in RIM.[1] Ghosh, D.D., Sanders, T., Hong, S., Chase, D.L., Cohen, N., Koelle, M.R., and Nitabach, M.N. "Neuroendocrine reinforcement of a dynamic multisensory decision." International C. elegans meeting.