Single-animal, single-tube RNA extraction for quantitative analysis of transcripts in the tardigrade Hypsibius exemplaris.

The tardigrade Hypsibius exemplaris is an emerging model organism renowned for its ability to survive environmental extremes.1-3 To explore the molecular mechanisms and genetic basis of such extremotolerance, many studies rely on transcriptional profiling4, 5 and RNA interference (RNAi)6 to define molecular targets. Such studies require efficient, accurate, and robust RNA extraction methods; however, obtaining high-quality quantitative levels of RNA from H. exemplaris has been challenging6, 7. Possessing a layer of firm chitinous cuticle, tardigrade tissues are difficult to disrupt by chemical or mechanical means8. Here we present an efficient single-tardigrade, single-tube RNA extraction method (STST) that not only reliably isolates RNA from individual tardigrades but dramatically reduces the time required for extraction. We show that this RNA extraction method yields robust quantities of cDNA and can be used to amplify multiple transcripts by qRT-PCR. To validate the method, we use it to compare dynamic changes in expression of genes encoding two heat-shock-regulated proteins, Heat-Shock Protein 70 ß2 (HSP70ß2) and Heat-Shock Protein 90α (HSP90α) by quantifying their expression levels in heat-exposed and cold-exposed individuals using qRT-PCR across long-term and short-term heat stressors. Our method effectively complements existing bulk RNA extraction methods7, permitting rapid examination of individual tardigrade transcriptional data and quantification of phenotypic variations in expression profiles amongst individuals.

Regulation of defective mitochondrial DNA accumulation and transmission in C. elegans by the programmed cell death and aging pathways.

The heteroplasmic state of eukaryotic cells allows for cryptic accumulation of defective mitochondrial genomes (mtDNA). 'Purifying selection' mechanisms operate to remove such dysfunctional mtDNAs. We found that activators of programmed cell death (PCD), including the CED-3 and CSP-1 caspases, the BH3-only protein CED-13, and PCD corpse engulfment factors, are required in C. elegans to attenuate germline abundance of a 3.1-kb mtDNA deletion mutation, uaDf5, which is normally stably maintained in heteroplasmy with wildtype mtDNA. In contrast, removal of CED-4/Apaf1 or a mutation in the CED-4-interacting prodomain of CED-3, do not increase accumulation of the defective mtDNA, suggesting induction of a non-canonical germline PCD mechanism or non-apoptotic action of the CED-13/caspase axis. We also found that the abundance of germline mtDNAuaDf5 reproducibly increases with age of the mothers. This effect is transmitted to the offspring of mothers, with only partial intergenerational removal of the defective mtDNA. In mutants with elevated mtDNAuaDf5 levels, this removal is enhanced in older mothers, suggesting an age-dependent mechanism of mtDNA quality control. Indeed, we found that both steady-state and age-dependent accumulation rates of uaDf5 are markedly decreased in long-lived, and increased in short-lived, mutants. These findings reveal that regulators of both PCD and the aging program are required for germline mtDNA quality control and its intergenerational transmission.

Expression of the odd-2 Gene in C. elegans.

The odd-2 gene in C. elegans is an orthologue of the odd-skipped gene in Drosophila and the odd-skipped related genes in mammals. The mammalian genes have been shown to be expressed in a variety of tissues and cancers. It was previously reported that ODD-2 is expressed in the intestine and shows some expression outside of the intestine in the tail region. Using a partial ODD-2 ::GFP fusion, we hypothesize that the expression outside of the intestine may be in rectal gland cells, and we also report that ODD-2 may be expressed in the germline sheath cells.

Feedforward regulatory logic controls the specification-to-differentiation transition and terminal cell fate during Caenorhabditis elegans endoderm development.

The architecture of gene regulatory networks determines the specificity and fidelity of developmental outcomes. We report that the core regulatory circuitry for endoderm development in Caenorhabditis elegans operates through a transcriptional cascade consisting of six sequentially expressed GATA-type factors that act in a recursive series of interlocked feedforward modules. This structure results in sequential redundancy, in which removal of a single factor or multiple alternate factors in the cascade leads to a mild or no effect on gut development, whereas elimination of any two sequential factors invariably causes a strong phenotype. The phenotypic strength is successfully predicted with a computational model based on the timing and levels of transcriptional states. We found that one factor in the middle of the cascade, END-1, which straddles the distinct events of specification and differentiation, functions in both processes. Finally, we reveal roles for key GATA factors in establishing spatial regulatory state domains by repressing other fates, thereby defining boundaries in the digestive tract. Our findings provide a paradigm that could account for the genetic redundancy observed in many developmental regulatory systems.

Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae.

Gravity sensation is an important and relatively understudied process. Sensing gravity enables animals to navigate their surroundings and facilitates movement. Additionally, gravity sensation, which occurs in the mammalian inner ear, is closely related to hearing - thus, understanding this process has implications for auditory and vestibular research. Gravitaxis assays exist for some model organisms, including Drosophila. Single worms have previously been assayed for their orientation preference as they settle in solution. However, a reliable and robust assay for Caenorhabditis gravitaxis has not been described. The present protocol outlines a procedure for performing gravitaxis assays that can be used to test hundreds of Caenorhabditis dauers at a time. This large-scale, long-distance assay allows for detailed data collection, revealing phenotypes that may be missed on a standard plate-based assay. Dauer movement along the vertical axis is compared with horizontal controls to ensure that directional bias is due to gravity. Gravitactic preference can then be compared between strains or experimental conditions. This method can determine molecular, cellular, and environmental requirements for gravitaxis in worms.

Interstellar space biology via Project Starlight.

Our ability to explore the cosmos by direct contact has been limited to a small number of lunar and interplanetary missions. However, the NASA Starlight program points a path forward to send small, relativistic spacecraft far outside our solar system via standoff directed-energy propulsion. These miniaturized spacecraft are capable of robotic exploration but can also transport seeds and organisms, marking a profound change in our ability to both characterize and expand the reach of known life. Here we explore the biological and technological challenges of interstellar space biology, focusing on radiation-tolerant microorganisms capable of cryptobiosis. Additionally, we discuss planetary protection concerns and other ethical considerations of sending life to the stars.

An Autonomous Molecular Bioluminescent Reporter (AMBER) for Voltage Imaging in Freely Moving Animals.

Genetically encoded reporters have greatly increased our understanding of biology. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach, Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential is engineered. Unlike other bioluminescent systems, AMBER is a voltage-gated luciferase coupling the functionalities of the Ciona voltage-sensing domain (VSD) and bacterial luciferase, luxAB. When co-expressed with the luciferin-producing genes, AMBER reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods, it is shown that AMBER switches its enzymatic activity from an OFF to an ON state as a function of the membrane potential. Upon depolarization, AMBER switches from a low to a high enzymatic activity state, showing a several-fold increase in the bioluminescence output (ΔL/L). AMBER in the pharyngeal muscles and mechanosensory touch neurons of Caenorhabditis elegans is expressed. Using the compressed sensing approach, the electropharingeogram of the C. elegans pharynx is reconstructed, validating the sensor in vivo. Thus, AMBER represents the first fully genetically encoded bioluminescent reporter without requiring exogenous luciferin addition.

Stressful development: integrating endoderm development, stress, and longevity.

In addition to performing digestion and nutrient absorption, the intestine serves as one of the first barriers to the external environment, crucial for protecting the host from environmental toxins, pathogenic invaders, and other stress inducers. The gene regulatory network (GRN) governing embryonic development of the endoderm and subsequent differentiation and maintenance of the intestine has been well-documented in C. elegans. A key regulatory input that initiates activation of the embryonic GRN for endoderm and mesoderm in this animal is the maternally provided SKN-1 transcription factor, an ortholog of the vertebrate Nrf1 and 2, which, like C. elegans SKN-1, perform conserved regulatory roles in mediating a variety of stress responses across metazoan phylogeny. Other key regulatory factors in early gut development also participate in stress response as well as in innate immunity and aging and longevity. In this review, we discuss the intersection between genetic nodes that mediate endoderm/intestine differentiation and regulation of stress and homeostasis. We also consider how direct signaling from the intestine to the germline, in some cases involving SKN-1, facilitates heritable epigenetic changes, allowing transmission of adaptive stress responses across multiple generations. These connections between regulation of endoderm/intestine development and stress response mechanisms suggest that varying selective pressure exerted on the stress response pathways may influence the architecture of the endoderm GRN, thereby leading to genetic and epigenetic variation in early embryonic GRN regulatory events.

Natural cryptic variation in epigenetic modulation of an embryonic gene regulatory network.

Gene regulatory networks (GRNs) that direct animal embryogenesis must respond to varying environmental and physiological conditions to ensure robust construction of organ systems. While GRNs are evolutionarily modified by natural genomic variation, the roles of epigenetic processes in shaping plasticity of GRN architecture are not well understood. The endoderm GRN in Caenorhabditis elegans is initiated by the maternally supplied SKN-1/Nrf2 bZIP transcription factor; however, the requirement for SKN-1 in endoderm specification varies widely among distinct C. elegans wild isotypes, owing to rapid developmental system drift driven by accumulation of cryptic genetic variants. We report here that heritable epigenetic factors that are stimulated by transient developmental diapause also underlie cryptic variation in the requirement for SKN-1 in endoderm development. This epigenetic memory is inherited from the maternal germline, apparently through a nuclear, rather than cytoplasmic, signal, resulting in a parent-of-origin effect (POE), in which the phenotype of the progeny resembles that of the maternal founder. The occurrence and persistence of POE varies between different parental pairs, perduring for at least 10 generations in one pair. This long-perduring POE requires piwi-interacting RNA (piRNA) function and the germline nuclear RNA interference (RNAi) pathway, as well as MET-2 and SET-32, which direct histone H3K9 trimethylation and drive heritable epigenetic modification. Such nongenetic cryptic variation may provide a resource of additional phenotypic diversity through which adaptation may facilitate evolutionary changes and shape developmental regulatory systems.

Intertwined Functions of Separase and Caspase in Cell Division and Programmed Cell Death.

Timely sister chromatid separation, promoted by separase, is essential for faithful chromosome segregation. Separase is a member of the CD clan of cysteine proteases, which also includes the pro-apoptotic enzymes known as caspases. We report a role for the C. elegans separase SEP-1, primarily known for its essential activity in cell division and cortical granule exocytosis, in developmentally programmed cell death when the predominant pro-apoptotic caspase CED-3 is compromised. Loss of SEP-1 results in extra surviving cells in a weak ced-3(-) mutant, and suppresses the embryonic lethality of a mutant defective for the apoptotic suppressor ced-9/Bcl-2 implicating SEP-1 in execution of apoptosis. We also report apparent non-apoptotic roles for CED-3 in promoting germ cell proliferation, meiotic chromosome disjunction, egg shell formation, and the normal rate of embryonic development. Moreover, loss of the soma-specific (CSP-3) and germline-specific (CSP-2) caspase inhibitors result in CED-3-dependent suppression of embryonic lethality and meiotic chromosome non-disjunction respectively, when separase function is compromised. Thus, while caspases and separases have evolved different substrate specificities associated with their specialized functions in apoptosis and cell division respectively, they appear to have retained the residual ability to participate in both processes, supporting the view that co-option of components in cell division may have led to the innovation of programmed cell suicide early in metazoan evolution.

Evolution and Developmental System Drift in the Endoderm Gene Regulatory Network of Caenorhabditis and Other Nematodes.

Developmental gene regulatory networks (GRNs) underpin metazoan embryogenesis and have undergone substantial modification to generate the tremendous variety of animal forms present on Earth today. The nematode Caenorhabditis elegans has been a central model for advancing many important discoveries in fundamental mechanistic biology and, more recently, has provided a strong base from which to explore the evolutionary diversification of GRN architecture and developmental processes in other species. In this short review, we will focus on evolutionary diversification of the GRN for the most ancient of the embryonic germ layers, the endoderm. Early embryogenesis diverges considerably across the phylum Nematoda. Notably, while some species deploy regulative development, more derived species, such as C. elegans, exhibit largely mosaic modes of embryogenesis. Despite the relatively similar morphology of the nematode gut across species, widespread variation has been observed in the signaling inputs that initiate the endoderm GRN, an exemplar of developmental system drift (DSD). We will explore how genetic variation in the endoderm GRN helps to drive DSD at both inter- and intraspecies levels, thereby resulting in a robust developmental system. Comparative studies using divergent nematodes promise to unveil the genetic mechanisms controlling developmental plasticity and provide a paradigm for the principles governing evolutionary modification of an embryonic GRN.

Improving Academic Performance, Belonging, and Retention through Increasing Structure of an Introductory Biology Course.

Integration of active-learning approaches into increased-structure postsecondary classrooms significantly improves student academic outcomes. We describe here two parallel sections of Introductory Biology that shared learning objectives and content but varied in course structure. The large-enrollment traditional course consisted of four 50-minute lectures coupled with minimal active-learning techniques, while an increased-structure intervention course integrated multiple active-learning approaches, had limited enrollment, and comprised three 50-minute lectures combined with a fourth peer-led team-learning discussion section. Additionally, the intervention course employed weekly review quizzes and multiple in-class formative assessments. The academic impact of these two course formats was evaluated by use of common exam questions, final grade, and student retention. We showed that academic achievement and retention of participants enrolled in the intervention course was significantly improved when compared with the traditional section. Further, we explored whether promoting in-class student-student/student-instructor interactions and peer-led discussion sections fostered a greater sense of belonging. At the end of the course, participants in the intervention course reported greater perceptions of classroom belonging. Therefore, this study begins to characterize the importance of combining pedagogical methods that promote both academic success and belonging to effectively improve retention in science, technology, engineering, and mathematics majors.

Extensive intraspecies cryptic variation in an ancient embryonic gene regulatory network.

Innovations in metazoan development arise from evolutionary modification of gene regulatory networks (GRNs). We report widespread cryptic variation in the requirement for two key regulatory inputs, SKN-1/Nrf2 and MOM-2/Wnt, into the C. elegans endoderm GRN. While some natural isolates show a nearly absolute requirement for these two regulators, in others, most embryos differentiate endoderm in their absence. GWAS and analysis of recombinant inbred lines reveal multiple genetic regions underlying this broad phenotypic variation. We observe a reciprocal trend, in which genomic variants, or knockdown of endoderm regulatory genes, that result in a high SKN-1 requirement often show low MOM-2/Wnt requirement and vice-versa, suggesting that cryptic variation in the endoderm GRN may be tuned by opposing requirements for these two key regulatory inputs. These findings reveal that while the downstream components in the endoderm GRN are common across metazoan phylogeny, initiating regulatory inputs are remarkably plastic even within a single species.

The multipotency-to-commitment transition in Caenorhabditis elegans-implications for reprogramming from cells to organs.

In animal embryos, cells transition from a multipotential state, with the capacity to adopt multiple fates, into an irreversible, committed state of differentiation. This multipotency-to-commitment transition (MCT) is evident from experiments in which cell fate is reprogrammed by transcription factors for cell type-specific differentiation, as has been observed extensively in Caenorhabditis elegans. Although factors that direct differentiation into each of the three germ layer types cannot generally reprogram cells after the MCT in this animal, transcription factors for endoderm development are able to do so in multiple differentiated cell types. In one case, these factors can redirect the development of an entire organ in the process of "transorganogenesis". Natural transdifferentiation also occurs in a small number of differentiated cells during normal C. elegans development. We review these reprogramming and transdifferentiation events, highlighting the cellular and developmental contexts in which they occur, and discuss common themes underlying direct cell lineage reprogramming. Although certain aspects may be unique to the model system, growing evidence suggests that some mechanisms are evolutionarily conserved and may shed light on cellular plasticity and disease in humans.

Quantitating transcription factor redundancy: The relative roles of the ELT-2 and ELT-7 GATA factors in the C. elegans endoderm.

The two GATA transcription factors ELT-2 and ELT-7 function in the differentiation of the C. elegans intestine. ELT-2 loss causes lethality. ELT-7 loss causes no obvious phenotype but enhances the elt-2(-) intestinal phenotype. Thus, ELT-2 and ELT-7 appear partially redundant, with ELT-2 being more influential. To investigate the different regulatory roles of ELT-2 and ELT-7, we compared the transcriptional profiles of pure populations of wild-type, elt-2(-), elt-7(-), and elt-7(-); elt-2(-) double mutant L1-stage larvae. Consistent with the mutant phenotypes, loss of ELT-2 had a>25 fold greater influence on the number of significantly altered transcripts compared to the loss of ELT-7; nonetheless, the levels of numerous transcripts changed upon loss of ELT-7 in the elt-2(-) background. The quantitative responses of individual genes revealed a more complicated behaviour than simple redundancy/partial redundancy. In particular, genes expressed only in the intestine showed three distinguishable classes of response in the different mutant backgrounds. One class of genes responded as if ELT-2 is the major transcriptional activator and ELT-7 provides variable compensatory input. For a second class, transcript levels increased upon loss of ELT-2 but decreased upon further loss of ELT-7, suggesting that ELT-7 actually overcompensates for the loss of ELT-2. For a third class, transcript levels also increased upon loss of ELT-2 but remained elevated upon further loss of ELT-7, suggesting overcompensation by some other intestinal transcription factor(s). In spite of its minor loss-of-function phenotype and its limited sequence similarity to ELT-2, ELT-7 expressed under control of the elt-2 promoter is able to rescue elt-2(-) lethality. Indeed, appropriately expressed ELT-7, like appropriately expressed ELT-2, is able to replace all other core GATA factors in the C. elegans endodermal pathway. Overall, this study focuses attention on the quantitative intricacies behind apparent redundancy or partial redundancy of two related transcription factors.

Heterotaxy in Caenorhabditis: widespread natural variation in left-right arrangement of the major organs.

Although the arrangement of internal organs in most metazoans is profoundly left-right (L/R) asymmetric with a predominant handedness, rare individuals show full (mirror-symmetric) or partial (heterotaxy) reversals. While the nematode Caenorhabditis elegans is known for its highly determinate development, including stereotyped L/R organ handedness, we found that L/R asymmetry of the major organs, the gut and gonad, varies among natural isolates of the species in both males and hermaphrodites. In hermaphrodites, heterotaxy can involve one or both bilaterally asymmetric gonad arms. Male heterotaxy is probably not attributable to relaxed selection in this hermaphroditic species, as it is also seen in gonochoristic Caenorhabditis species. Heterotaxy increases in many isolates at elevated temperature, with one showing a pregastrulation temperature-sensitive period, suggesting a very early embryonic or germline effect on this much later developmental outcome. A genome-wide association study of 100 isolates showed that male heterotaxy is associated with three genomic regions. Analysis of recombinant inbred lines suggests that a small number of loci are responsible for the observed variation. These findings reveal that heterotaxy is a widely varying quantitative trait in an animal with an otherwise highly stereotyped anatomy, demonstrating unexpected plasticity in an L/R arrangement of the major organs even in a simple animal.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

Transorganogenesis and transdifferentiation in C. elegans are dependent on differentiated cell identity.

The differentiated cell identities and structure of fully formed organs are generally stable after their development. In contrast, we report here that development of the C. elegans proximal somatic gonad (hermaphrodite uterus and spermathecae, and male vas deferens) can be redirected into intestine-like organs by brief expression of the ELT-7 GATA transcription factor. This process converts one developing organ into another and can hence be considered "transorganogenesis." We show that, following pulsed ELT-7 expression, cells of the uterus activate and maintain intestine-specific gene expression and are transformed at the ultrastructural level to form an epithelial tube resembling the normal intestine formed during embryogenesis. Ubiquitous ELT-7 expression activates intestinal markers in many different cell types but only cells in the somatic gonad and pharynx appear to become fully reprogrammed. We found that ectopic expression of other endoderm-promoting transcription factors, but not muscle- or ectoderm- promoting transcription factors, redirects the fate of these organs, suggesting that pharyngeal and somatic gonad cells are specifically competent to adopt intestine identity. Although the intestine, pharynx, and somatic gonad are derived from distant cell lineages, they all express the PHA-4/FoxA transcription factor. While we found that post-embryonic PHA-4 is not necessary for pharynx or uterus reprogramming and PHA-4 is not sufficient in combination with ELT-7 to induce reprogramming in other cells types, knock down of PHA-4 during embryogenesis, which abolishes normal pharynx differentiation, prevents pharyngeal precursors from being reprogrammed into intestine. These results suggest that differentiated cell identity determines susceptibility to transdifferentiation and highlight the importance of cellular context in controlling competency for reprogramming.

NEGotiating Cell Identity through Regulated Cytoplasmic Polyadenylation.

In this issue of Developmental Cell, Elewa et al. (2015) show that combinatorial action of RNA binding proteins modulates poly(A) tail length of maternal mRNAs, leading to asymmetric expression of a cell fate determinant in early C. elegans embryos. Genome-wide profiling suggests this mechanism may be widely used to establish cell identities.

Natural reversal of left-right gut/gonad asymmetry in C. elegans males is independent of embryonic chirality.

Anatomical left-right (L/R) asymmetry in C. elegans is established in the four-cell embryo as a result of anteroposterior skewing of transverse mitotic spindles with a defined handedness. This event creates a chiral embryo and ultimately an adult body plan with fixed L/R positioning of internal organs and components of the nervous system. While this "dextral" configuration is invariant in hermaphrodites, it can be reversed by physical manipulation of the early embryo or by mutations that interfere with mitotic spindle orientation, which leads to viable, mirror-reversed (sinistral) animals. During normal development of the C. elegans male, the gonad develops on the right of the midline, with the gut bilaterally apposed on the left. However, we found that in males of the laboratory N2 strain and Hawaiian ("Hw") wild isolate, the gut/gonad asymmetry is frequently reversed in a temperature-dependent manner, independent of normal embryonic chirality. We also observed sporadic errors in gonad migration occurring naturally during early larval stages of these and other wild strains; however, the incidence of such errors does not correlate with the frequency of L/R gut/gonad reversals in these strains. Analysis of N2/Hw hybrids and recombinant inbred advanced intercross lines (RIAILs) indicate that the L/R organ reversals are likely to result from recessively acting variations in multiple genes. Thus, unlike the highly reproducible L/R asymmetries of most structures in hermaphrodites, the L/R asymmetry of the male C. elegans body plan is less rigidly determined and subject to natural variation that is influenced by a multiplicity of genes.