The Rac pathway prevents cell fragmentation in a nonprotrusively migrating leader cell during C. elegans gonad organogenesis.

The C. elegans hermaphrodite distal tip cell (DTC) leads gonadogenesis. Loss-of-function mutations in a C. elegans ortholog of the Rac1 GTPase (ced-10) and its GEF complex (ced-5/DOCK180, ced-2/CrkII, ced-12/ELMO) cause gonad migration defects related to directional sensing; we discovered an additional defect class of gonad bifurcation in these mutants. Using genetic approaches, tissue-specific and whole-body RNAi, and in vivo imaging of endogenously tagged proteins and marked cells, we find that loss of Rac1 or its regulators causes the DTC to fragment as it migrates. Both products of fragmentation-the now-smaller DTC and the membranous patch of cellular material-localize important stem cell niche signaling (LAG-2 ligand) and migration (INA-1/integrin subunit alpha) factors to their membranes, but only one retains the DTC nucleus and therefore the ability to maintain gene expression over time. The enucleate patch can lead a bifurcating branch off the gonad arm that grows through germ cell proliferation. Germ cells in this branch differentiate as the patch loses LAG-2 expression. While the nucleus is surprisingly dispensable for aspects of leader cell function, it is required for stem cell niche activity long term. Prior work found that Rac1-/-;Rac2-/- mouse erythrocytes fragment; in this context, our new findings support the conclusion that maintaining a cohesive but deformable cell is a conserved function of this important cytoskeletal regulator.

Getting there in one piece: The Rac pathway prevents cell fragmentation in a nonprotrusively migrating leader cell during organogenesis.

The C. elegans hermaphrodite distal tip cell (DTC) leads gonadogenesis. Loss-of-function mutations in a C. elegans ortholog of the Rac1 GTPase (ced-10) and its GEF complex (ced-5/DOCK180, ced-2/CrkII, ced-12/ELMO) cause gonad migration defects related to directional sensing; we discovered an additional defect class of gonad bifurcation in these mutants. Using genetic approaches, tissue-specific and whole-body RNAi, and in vivo imaging of endogenously tagged proteins and marked cells, we find that loss of Rac1 or its regulators causes the DTC to fragment as it migrates. Both products of fragmentation-the now-smaller DTC and the membranous patch of cellular material-localize important stem cell niche signaling (LAG-2/DSL ligand) and migration (INA-1/integrin subunit alpha) factors to their membranes, but only one retains the DTC nucleus and therefore the ability to maintain gene expression over time. The enucleate patch can lead a bifurcating branch off the gonad arm that grows through germ cell proliferation. Germ cells in this branch differentiate as the patch loses LAG-2 expression. While the nucleus is surprisingly dispensable for aspects of leader cell function, it is required for stem cell niche activity long-term. Prior work found that Rac1-/-;Rac2-/- mouse erythrocytes fragment; in this context, our new findings support the conclusion that maintaining a cohesive but deformable cell is a conserved function of this important cytoskeletal regulator.

The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics.

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. This study reveals phenotypic consequences of whole-animal tetraploidy that make C. elegans an excellent model for ploidy differences.

The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics.

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. Overall, this study reveals the phenotypic consequences of whole-animal tetraploidy in C. elegans.

The C. elegans gonadal sheath Sh1 cells extend asymmetrically over a differentiating germ cell population in the proliferative zone.

The Caenorhabditis elegans adult hermaphrodite germline is surrounded by a thin tube formed by somatic sheath cells that support germ cells as they mature from the stem-like mitotic state through meiosis, gametogenesis, and ovulation. Recently, we discovered that the distal Sh1 sheath cells associate with mitotic germ cells as they exit the niche Gordon et al., 2020. Here, we report that these sheath-associated germ cells differentiate first in animals with temperature-sensitive mutations affecting germ cell state, and stem-like germ cells are maintained distal to the Sh1 boundary. We analyze several markers of the distal sheath, which is best visualized with endogenously tagged membrane proteins, as overexpressed fluorescent proteins fail to localize to distal membrane processes and can cause gonad morphology defects. However, such reagents with highly variable expression can be used to determine the relative positions of the two Sh1 cells, one of which often extends further distal than the other.

Stem cell niche exit in C. elegans via orientation and segregation of daughter cells by a cryptic cell outside the niche.

Stem cells reside in and rely upon their niche to maintain stemness but must balance self-renewal with the production of daughters that leave the niche to differentiate. We discovered a mechanism of stem cell niche exit in the canonical C. elegans distal tip cell (DTC) germ stem cell niche mediated by previously unobserved, thin, membranous protrusions of the adjacent somatic gonad cell pair (Sh1). A disproportionate number of germ cell divisions were observed at the DTC-Sh1 interface. Stem-like and differentiating cell fates segregated across this boundary. Spindles polarized, pairs of daughter cells oriented between the DTC and Sh1, and Sh1 grew over the Sh1-facing daughter. Impeding Sh1 growth by RNAi to cofilin and Arp2/3 perturbed the DTC-Sh1 interface, reduced germ cell proliferation, and shifted a differentiation marker. Because Sh1 membrane protrusions eluded detection for decades, it is possible that similar structures actively regulate niche exit in other systems.

Stem cells have the rare ability to divide and specialize into the many different types of cells necessary for an organism to survive. For instance, germ stem cells can multiply to produce precursor cells that go on to become eggs or sperm needed for reproduction. When a stem cell divides, the daughter cells can either remain 'naïve', or start to specialize into a given cell type. In many cases, this decision is strongly influenced by the properties of the environment that surrounds the stem cell. However, in the microscopic worm Caenorhabditis elegans, how the daughters of germ stem cells specialize was thought to be a random process, with nearby cells equally likely to specialize or remain naïve. In this animal, germ stem cells reside in tube-shaped structures called gonads, which are enclosed by a large 'distal tip' cell. In addition, cells known as Sh1 surround the gonad. Here, Gordon et al. tracked dividing germ stem cells in the gonads of live worms. This revealed that both the distal tip cell and Sh1 cells have finger-like extensions that form contacts with the germ stem cells. The fate of dividing germ stem cells is shaped by these interactions. If they touch only the distal tip cell, they remain in a naïve state. However, if they contact both the distal tip cell and an Sh1 cell, one daughter of the stem cell becomes an egg precursor ­ with the daughter closest to the distal tip cell staying naïve. In fact, germ stem cells that are prevented from contacting Sh1 cells divide less often. Many other types of stem cells, for example in human skin, are believed to make the decision to remain naïve or undergo specialization randomly. The results from Gordon et al. could provide a roadmap to discover hidden layers of control in other organisms, some of which may be potentially relevant in health and disease.

Recent Advances in the Genetic, Anatomical, and Environmental Regulation of the C. elegans Germ Line Progenitor Zone.

The C. elegans germ line and its gonadal support cells are well studied from a developmental genetics standpoint and have revealed many foundational principles of stem cell niche biology. Among these are the observations that a niche-like cell supports a self-renewing stem cell population with multipotential, differentiating daughter cells. While genetic features that distinguish stem-like cells from their differentiating progeny have been defined, the mechanisms that structure these populations in the germ line have yet to be explained. The spatial restriction of Notch activation has emerged as an important genetic principle acting in the distal germ line. Synthesizing recent findings, I present a model in which the germ stem cell population of the C. elegans adult hermaphrodite can be recognized as two distinct anatomical and genetic populations. This review describes the recent progress that has been made in characterizing the undifferentiated germ cells and gonad anatomy, and presents open questions in the field and new directions for research to pursue.

MANF deletion abrogates early larval Caenorhabditis elegans stress response to tunicamycin and Pseudomonas aeruginosa.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is the only human neurotrophic factor with an evolutionarily-conserved C. elegans homolog, Y54G2A.23 or manf-1. MANF is a small, soluble, endoplasmic-reticulum (ER)-resident protein that is secreted upon ER stress and promotes survival of target cells such as neurons. However, the role of MANF in ER stress and its mechanism of cellular protection are not clear and the function of MANF in C. elegans is only beginning to emerge. In this study, we show that depletion of C. elegans manf-1 causes a slight decrease in lifespan and brood size; furthermore, combined depletion of manf-1 and the IRE-1/XBP-1 ER stress/UPR pathway resulted in sterile animals that did not produce viable progeny. We demonstrate upregulation of markers of ER stress in L1 larval nematodes, as measured by hsp-3 and hsp-4 transcription, upon depletion of manf-1 by RNAi or mutation; however, there was no difference in tunicamycin-induced expression of hsp-3 and hsp-4 between wild-type and MANF-deficient worms. Surprisingly, larval growth arrest observed in wild-type nematodes reared on tunicamycin is completely prevented in the manf-1 (tm3603) mutant. Transcriptional microarray analysis revealed that manf-1 mutant L1 larvae exhibit a novel modulation of innate immunity genes in response to tunicamycin. The hypothesis that manf-1 negatively regulates the innate immunity pathway is supported by our finding that the development of manf-1 mutant larvae compared to wild-type larvae is not inhibited by growth on P. aeruginosa. Together, our data represent the first characterization of C. elegans MANF as a key modulator of organismal ER stress and immunity.

Ectopic Germ Cells Can Induce Niche-like Enwrapment by Neighboring Body Wall Muscle.

Niche cell enwrapment of stem cells and their differentiating progeny is common and provides a specialized signaling and protective environment. Elucidating the mechanisms underlying enwrapment behavior has important basic and clinical significance in not only understanding how niches are formed and maintained but also how they can be engineered and how they are misregulated in human pathologies, such as cancer. Previous work in C. elegans found that, when germ cells, which are enwrapped by somatic gonadal niche cells, are freed into the body cavity, they embed into other tissues. We investigated this phenomenon using live-cell imaging and discovered that ectopic germ cells preferentially induce body-wall muscle to extend cellular processes that enwrap the germ cells, the extent of which was strikingly similar to the distal tip cell (DTC)-germ stem cell niche. Enwrapment was specific for escaped germ cells, and genetic analysis revealed it did not depend on pathways that control cell death and engulfment or muscle arm extension. Instead, using a large-scale RNAi screen and GFP knockin strains, we discovered that the enwrapping behavior of muscle relied upon the same suite of cell-cell adhesion molecules that functioned in the endogenous niche: the C. elegans E-cadherin HMR-1, its intracellular associates α-catenin (HMP-1) and ß-catenin (HMP-2), and the L1CAM protein SAX-7. This ectopic niche-like behavior resembles the seed-and-soil model of cancer metastasis and offers a new model to understand factors regulating ectopic niche formation.

Swimming Exercise and Transient Food Deprivation in Caenorhabditis elegans Promote Mitochondrial Maintenance and Protect Against Chemical-Induced Mitotoxicity.

Exercise and caloric restriction improve health, including reducing risk of cardiovascular disease, neurological disease, and cancer. However, molecular mechanisms underlying these protections are poorly understood, partly due to the cost and time investment of mammalian long-term diet and exercise intervention studies. We subjected Caenorhabditis elegans nematodes to a 6-day, twice daily swimming exercise regimen, during which time the animals also experienced brief, transient food deprivation. Accordingly, we included a non-exercise group with the same transient food deprivation, a non-exercise control with ad libitum access to food, and a group that exercised in food-containing medium. Following these regimens, we assessed mitochondrial health and sensitivity to mitochondrial toxicants. Exercise protected against age-related decline in mitochondrial morphology in body-wall muscle. Food deprivation increased organismal basal respiration; however, exercise was the sole intervention that increased spare respiratory capacity and proton leak. We observed increased lifespan in exercised animals compared to both control and transiently food-deprived nematodes. Finally, exercised animals (and to a lesser extent, transiently food-deprived animals) were markedly protected against lethality from acute exposures to the mitotoxicants rotenone and arsenic. Thus, swimming exercise and brief food deprivation provide effective intervention in C. elegans, protecting from age-associated mitochondrial decline and providing resistance to mitotoxicant exposures.

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.

Identification of regulators of germ stem cell enwrapment by its niche in C. elegans.

Many stem cell niches contain support cells that increase contact with stem cells by enwrapping them in cellular processes. One example is the germ stem cell niche in C. elegans, which is composed of a single niche cell termed the distal tip cell (DTC) that extends cellular processes, constructing an elaborate plexus that enwraps germ stem cells. To identify genes required for plexus formation and to explore the function of this specialized enwrapping behavior, a series of targeted and tissue-specific RNAi screens were performed. Here we identify genes that promote stem cell enwrapment by the DTC plexus, including a set that specifically functions within the DTC, such as the chromatin modifier lin-40/MTA1, and others that act within the germline, such as the 14-3-3 signaling protein par-5. Analysis of genes that function within the germline to mediate plexus development reveal that they are required for expansion of the germ progenitor zone, supporting the emerging idea that germ stem cells signal to the niche to stimulate enwrapping behavior. Examination of wild-type animals with asymmetric plexus formation and animals with reduced DTC plexus elaboration via loss of two candidates including lin-40 indicate that cellular enwrapment promotes GLP-1/Notch signaling and germ stem cell fate. Together, our work identifies novel regulators of cellular enwrapment and suggests that reciprocal signaling between the DTC niche and the germ stem cells promotes enwrapment behavior and stem cell fate.

Phylum-Level Conservation of Regulatory Information in Nematodes despite Extensive Non-coding Sequence Divergence.

Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs (unc-47, unc-25, mec-3 and elt-2) from distantly-related nematode species. These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements.

Coevolution within and between regulatory loci can preserve promoter function despite evolutionary rate acceleration.

Phenotypes that appear to be conserved could be maintained not only by strong purifying selection on the underlying genetic systems, but also by stabilizing selection acting via compensatory mutations with balanced effects. Such coevolution has been invoked to explain experimental results, but has rarely been the focus of study. Conserved expression driven by the unc-47 promoters of Caenorhabditis elegans and C. briggsae persists despite divergence within a cis-regulatory element and between this element and the trans-regulatory environment. Compensatory changes in cis and trans are revealed when these promoters are used to drive expression in the other species. Functional changes in the C. briggsae promoter, which has experienced accelerated sequence evolution, did not lead to alteration of gene expression in its endogenous environment. Coevolution among promoter elements suggests that complex epistatic interactions within cis-regulatory elements may facilitate their divergence. Our results offer a detailed picture of regulatory evolution in which subtle, lineage-specific, and compensatory modifications of interacting cis and trans regulators together maintain conserved gene expression patterns.

Tempo and mode in evolution of transcriptional regulation.

Perennial questions of evolutionary biology can be applied to gene regulatory systems using the abundance of experimental data addressing gene regulation in a comparative context. What is the tempo (frequency, rate) and mode (way, mechanism) of transcriptional regulatory evolution? Here we synthesize the results of 230 experiments performed on insects and nematodes in which regulatory DNA from one species was used to drive gene expression in another species. General principles of regulatory evolution emerge. Gene regulatory evolution is widespread and accumulates with genetic divergence in both insects and nematodes. Divergence in cis is more common than divergence in trans. Coevolution between cis and trans shows a particular increase over greater evolutionary timespans, especially in sex-specific gene regulation. Despite these generalities, the evolution of gene regulation is gene- and taxon-specific. The congruence of these conclusions with evidence from other types of experiments suggests that general principles are discoverable, and a unified view of the tempo and mode of regulatory evolution may be achievable.

Distinct functional constraints partition sequence conservation in a cis-regulatory element.

Different functional constraints contribute to different evolutionary rates across genomes. To understand why some sequences evolve faster than others in a single cis-regulatory locus, we investigated function and evolutionary dynamics of the promoter of the Caenorhabditis elegans unc-47 gene. We found that this promoter consists of two distinct domains. The proximal promoter is conserved and is largely sufficient to direct appropriate spatial expression. The distal promoter displays little if any conservation between several closely related nematodes. Despite this divergence, sequences from all species confer robustness of expression, arguing that this function does not require substantial sequence conservation. We showed that even unrelated sequences have the ability to promote robust expression. A prominent feature shared by all of these robustness-promoting sequences is an AT-enriched nucleotide composition consistent with nucleosome depletion. Because general sequence composition can be maintained despite sequence turnover, our results explain how different functional constraints can lead to vastly disparate rates of sequence divergence within a promoter.

Developmental expression of COE across the Metazoa supports a conserved role in neuronal cell-type specification and mesodermal development.

The transcription factor COE (collier/olfactory-1/early B cell factor) is an unusual basic helix-loop-helix transcription factor as it lacks a basic domain and is maintained as a single copy gene in the genomes of all currently analysed non-vertebrate Metazoan genomes. Given the unique features of the COE gene, its proposed ancestral role in the specification of chemosensory neurons and the wealth of functional data from vertebrates and Drosophila, the evolutionary history of the COE gene can be readily investigated. We have examined the ways in which COE expression has diversified among the Metazoa by analysing its expression from representatives of four disparate invertebrate phyla: Ctenophora (Mnemiopsis leidyi); Mollusca (Haliotis asinina); Annelida (Capitella teleta and Chaetopterus) and Echinodermata (Strongylocentrotus purpuratus). In addition, we have studied COE function with knockdown experiments in S. purpuratus, which indicate that COE is likely to be involved in repressing serotonergic cell fate in the apical ganglion of dipleurula larvae. These analyses suggest that COE has played an important role in the evolution of ectodermally derived tissues (likely primarily nervous tissues) and mesodermally derived tissues. Our results provide a broad evolutionary foundation from which further studies aimed at the functional characterisation and evolution of COE can be investigated.