Transfer of polarity information via diffusion of Wnt ligands in C. elegans embryos.

Different signaling mechanisms concur to ensure robust tissue patterning and cell fate instruction during animal development. Most of these mechanisms rely on signaling proteins that are produced, transported, and detected. The spatiotemporal dynamics of signaling molecules are largely unknown, yet they determine signal activity's spatial range and time frame. Here, we use the Caenorhabditis elegans embryo to study how Wnt ligands, an evolutionarily conserved family of signaling proteins, dynamically organize to establish cell polarity in a developing tissue. We identify how Wnt ligands, produced in the posterior half of the embryos, spread extracellularly to transmit information to distant target cells in the anterior half. With quantitative live imaging and fluorescence correlation spectroscopy, we show that Wnt ligands diffuse through the embryo over a timescale shorter than the cell cycle, in the intercellular space, and outside the tissue below the eggshell. We extracted diffusion coefficients of Wnt ligands and their receptor Frizzled and characterized their co-localization. Integrating our different measurements and observations in a simple computational framework, we show how fast diffusion in the embryo can polarize individual cells through a time integration of the arrival of the ligands at the target cells. The polarity established at the tissue level by a posterior Wnt source can be transferred to the cellular level. Our results support a diffusion-based long-range Wnt signaling, which is consistent with the dynamics of developing processes.

Labelling of Active Transcription Sites with Argonaute NRDE-3-Image Active Transcription Sites in vivo in Caenorhabditis elegans.

Live labelling of active transcription sites is critical to our understanding of transcriptional dynamics. In the most widely used method, RNA sequence MS2 repeats are added to the transcript of interest, on which fluorescently tagged Major Coat Protein binds, and labels transcription sites and transcripts. Here we describe another strategy, using the Argonaute protein NRDE-3, repurposed as an RNA-programmable RNA binding protein. We label active transcription sites in C. elegans embryos and larvae, without editing the gene of interest. NRDE-3 is programmed by feeding nematodes with double-stranded RNA matching the target gene. This method does not require genome editing and is inexpensive and fast to apply to many different genes. Graphical abstract.

PRC1 chromatin factors strengthen the consistency of neuronal cell fate specification and maintenance in C. elegans.

In the nervous system, the specific identity of a neuron is established and maintained by terminal selector transcription factors that directly activate large batteries of terminal differentiation genes and positively regulate their own expression via feedback loops. However, how this is achieved in a reliable manner despite noise in gene expression, genetic variability or environmental perturbations remains poorly understood. We addressed this question using the AIY cholinergic interneurons of C. elegans, whose specification and differentiation network is well characterized. Via a genetic screen, we found that a loss of function of PRC1 chromatin factors induces a stochastic loss of AIY differentiated state in a small proportion of the population. PRC1 factors act directly in the AIY neuron and independently of PRC2 factors. By quantifying mRNA and protein levels of terminal selector transcription factors in single neurons, using smFISH and CRISPR tagging, we observed that, in PRC1 mutants, terminal selector expression is still initiated during embryonic development but the level is reduced, and expression is subsequently lost in a stochastic manner during maintenance phase in part of the population. We also observed variability in the level of expression of terminal selectors in wild type animals and, using correlation analysis, established that this noise comes from both intrinsic and extrinsic sources. Finally, we found that PRC1 factors increase the resistance of AIY neuron fate to environmental stress, and also secure the terminal differentiation of other neuron types. We propose that PRC1 factors contribute to the consistency of neuronal cell fate specification and maintenance by protecting neurons against noise and perturbations in their differentiation program.

Multiple neural bHLHs ensure the precision of a neuronal specification event in Caenorhabditis elegans.

Neural bHLH transcription factors play a key role in the early steps of neuronal specification in many animals. We have previously observed that the Achaete-Scute HLH-3, the Olig HLH-16 and their binding partner the E-protein HLH-2 activate the terminal differentiation program of a specific class of cholinergic neurons, AIY, in Caenorhabditis elegans. Here we identify a role for a fourth bHLH, the Neurogenin NGN-1, in this process, raising the question of why so many neural bHLHs are required for a single neuronal specification event. Using quantitative imaging we show that the combined action of different bHLHs is needed to activate the correct level of expression of the terminal selector transcription factors TTX-3 and CEH-10 that subsequently initiate and maintain the expression of a large battery of terminal differentiation genes. Surprisingly, the different bHLHs have an antagonistic effect on another target, the proapoptotic BH3-only factor EGL-1, normally not expressed in AIY and otherwise detrimental for its specification. We propose that the use of multiple neural bHLHs allows robust neuronal specification while, at the same time, preventing spurious activation of deleterious genes.

Imaging of native transcription and transcriptional dynamics in vivo using a tagged Argonaute protein.

A flexible method to image unmodified transcripts and transcription in vivo would be a valuable tool to understand the regulation and dynamics of transcription. Here, we present a novel approach to follow native transcription, with fluorescence microscopy, in live C. elegans. By using the fluorescently tagged Argonaute protein NRDE-3, programmed by exposure to defined dsRNA to bind to nascent transcripts of the gene of interest, we demonstrate transcript labelling of multiple genes, at the transcription site and in the cytoplasm. This flexible approach does not require genetic manipulation, and can be easily scaled up by relying on whole-genome dsRNA libraries. We apply this method to image the transcriptional dynamics of the heat-shock inducible gene hsp-4 (a member of the hsp70 family), as well as two transcription factors: ttx-3 (a LHX2/9 orthologue) in embryos, and hlh-1 (a MyoD orthologue) in larvae, respectively involved in neuronal and muscle development.

Neuronal specification in C. elegans: combining lineage inheritance with intercellular signaling.

The nervous system is composed of a high diversity of neuronal types. How this diversity is generated during development is a key question in neurobiology. Addressing this question is one of the reasons that led Sydney Brenner to develop the nematode C. elegans as a model organism. While there was initially a debate on whether the neuronal specification follows a 'European' model (determined by ancestry) or an 'American' model (determined by intercellular communication), several decades of research have established that the truth lies somewhere in between. Neurons are specified by the combination of transcription factors inherited from the ancestor cells and signaling between neighboring cells (especially Wnt and Notch signaling). This converges to the activation in newly generated postmitotic neurons of a specific set of terminal selector transcription factors that initiate and maintain the differentiation of the neuron. In this review, we also discuss the evolution of these specification mechanisms in other nematodes and beyond.

Wnt ligands regulate the asymmetric divisions of neuronal progenitors in C. elegans embryos.

Wnt/ß-catenin signalling has been implicated in the terminal asymmetric divisions of neuronal progenitors in vertebrates and invertebrates. However, the role of Wnt ligands in this process remains poorly characterized. Here, we used the terminal divisions of the embryonic neuronal progenitors in C. elegans to characterize the role of Wnt ligands during this process, focusing on a lineage that produces the cholinergic interneuron AIY. We observed that, during interphase, the neuronal progenitor is elongated along the anteroposterior axis, then divides along its major axis, generating an anterior and a posterior daughter with different fates. Using time-controlled perturbations, we show that three Wnt ligands, which are transcribed at higher levels at the posterior of the embryo, regulate the orientation of the neuronal progenitor and its asymmetric division. We also identify a role for a Wnt receptor (MOM-5) and a cortical transducer APC (APR-1), which are, respectively, enriched at the posterior and anterior poles of the neuronal progenitor. Our study establishes a role for Wnt ligands in the regulation of the shape and terminal asymmetric divisions of neuronal progenitors, and identifies downstream components.

Zic Genes in Nematodes: A Role in Nervous System Development and Wnt Signaling.

Transcription factors of the Zic family play important roles during animal development, and their misregulation has been implicated in several human diseases. Zic proteins are present in nematodes, and their function has been mostly studied in the model organism C. elegans. C. elegans possesses only one Zic family member, REF-2. Functional studies have shown that this factor plays a key role during the development of the nervous system, epidermis, and excretory system. In addition, they have revealed that the C. elegans Zic protein acts as an atypical mediator of the Wnt/ß-catenin pathway. In other animals including vertebrates, Zic factors are also regulators of nervous system development and modulators of Wnt signaling, suggesting that these are evolutionary ancient functions of Zic proteins.

Zic-Proteins Are Repressors of Dopaminergic Forebrain Fate in Mice and C. elegans.

In the postnatal forebrain regionalized neural stem cells along the ventricular walls produce olfactory bulb (OB) interneurons with varying neurotransmitter phenotypes and positions. To understand the molecular basis of this region-specific variability we analyzed gene expression in the postnatal dorsal and lateral lineages in mice of both sexes from stem cells to neurons. We show that both lineages maintain transcription factor signatures of their embryonic site of origin, the pallium and subpallium. However, additional factors, including Zic1 and Zic2, are postnatally expressed in the dorsal stem cell compartment and maintained in the lineage that generates calretinin-positive GABAergic neurons for the OB. Functionally, we show that Zic1 and Zic2 induce the generation of calretinin-positive neurons while suppressing dopaminergic fate in the postnatal dorsal lineage. We investigated the evolutionary conservation of the dopaminergic repressor function of Zic proteins and show that it is already present in C. elegansSIGNIFICANCE STATEMENT The vertebrate brain generates thousands of different neuron types. In this work we investigate the molecular mechanisms underlying this variability. Using a genomics approach we identify the transcription factor signatures of defined neural stem cells and neuron populations. Based thereon we show that two related transcription factors, Zic1 and Zic2, are essential to control the balance between two defined neuron types in the postnatal brain. We show that this mechanism is conserved in evolutionary very distant species.

ß-catenin-driven binary cell fate decisions in animal development.

The Wnt/ß-catenin pathway plays key roles during animal development. In several species, ß-catenin is used in a reiterative manner to regulate cell fate diversification between daughter cells following division. This binary cell fate specification mechanism has been observed in animals that belong to very diverse phyla: the nematode Caenorhabditis elegans, the annelid Platynereis, and the ascidian Ciona. It may also play a role in the regulation of several stem cell lineages in vertebrates. While the molecular mechanism behind this binary cell fate switch is not fully understood, it appears that both secreted Wnt ligands and asymmetric cortical factors contribute to the generation of the difference in nuclear ß-catenin levels between daughter cells. ß-Catenin then cooperates with lineage specific transcription factors to induce the expression of novel sets of transcription factors at each round of divisions, thereby diversifying cell fate. For further resources related to this article, please visit the WIREs website.

Atypical Transcriptional Activation by TCF via a Zic Transcription Factor in C. elegans Neuronal Precursors.

Transcription factors of the TCF family are key mediators of the Wnt/ß-catenin pathway. TCF usually activates transcription on cis-regulatory elements containing TCF binding sites when the pathway is active and represses transcription when the pathway is inactive. However, some direct targets display an opposite regulation (activated by TCF in the absence of Wnt), but the mechanism behind this atypical regulation remains poorly characterized. Here, we use the cis-regulatory region of an opposite target gene, ttx-3, to dissect the mechanism of this atypical regulation. Using a combination of genetic, molecular, and biochemical experiments, we establish that, in the absence of Wnt pathway activation, TCF activates ttx-3 expression via a Zic binding site by forming a complex with a Zic transcription factor. This mechanism is later reinforced by specific bHLH factors. This study reveals an atypical mode of action for TCF that may apply to other binary decisions mediated by Wnt signaling.

How targets select activation or repression in response to Wnt.

In metazoans, the Wnt signaling pathway plays a key role in the regulation of binary decisions during development. During this process different sets of target genes are activated in cells where the Wnt pathway is active (classic target genes) versus cells where the pathway is inactive (opposite target genes). While the mechanism of transcriptional activation is well understood for classic target genes, how opposite target genes are activated in the absence of Wnt remains poorly characterized. Here we discuss how the key transcriptional mediator of the Wnt pathway, the TCF family member POP-1, regulates opposite target genes during C. elegans development. We examine recent findings suggesting that the direction of the transcriptional output (activation or repression) can be determined by the way TCF is recruited and physically interacts with its target gene.

Setting up a simple light sheet microscope for in toto imaging of C. elegans development.

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Notch-dependent induction of left/right asymmetry in C. elegans interneurons and motoneurons.

Although nervous systems are largely bilaterally symmetric on a structural level, they display striking degrees of functional left/right (L/R) asymmetry. In Caenorhabditis elegans, two structurally symmetric pairs of sensory neurons, ASE and AWC, display two distinctly controlled types of functional L/R asymmetries (stereotyped versus stochastic asymmetry). Beyond these two cases, the extent of neuronal asymmetry in the C. elegans nervous system was unclear. Here, we report that the Beta3/Olig-type bHLH transcription factor hlh-16 is L/R asymmetrically expressed in several distinct, otherwise bilaterally symmetric interneuron and motoneuron pairs that are part of a known navigation circuit. We find that hlh-16 asymmetry is generated during gastrulation by an asymmetric LAG-2/Delta signal originating from the mesoderm that promotes hlh-16 expression in neurons on the left side through direct binding of the Notch effector LAG-1/Su(H)/CBF to a cis-regulatory element in the hlh-16 locus. Removal of hlh-16 reveals an unanticipated asymmetry in the ability of the axons of the AIY interneurons to extend into the nerve ring, with the left AIY axon requiring elevated hlh-16 expression for correct extension. Our study suggests that the extent of molecular L/R asymmetry in the C. elegans nervous system is broader than previously anticipated, establishes a novel signaling mechanism that crosses germ layers to diversify bilaterally symmetric neuronal lineages, and reveals L/R asymmetric control of axonal outgrowth of bilaterally symmetric neurons.

Lineage programming: navigating through transient regulatory states via binary decisions.

Lineage-based mechanisms are widely used to generate cell type diversity in both vertebrates and invertebrates. For the past few decades, the nematode Caenorhabditis elegans has served as a primary model system to study this process because of its fixed and well-characterized cell lineage. Recent studies conducted at the level of single cells and individual cis-regulatory elements suggest a general model by which cellular diversity is generated in this organism. During its developmental history a cell passes through multiple transient regulatory states characterized by the expression of specific sets of transcription factors. The transition from one state to another is driven by a general binary decision mechanism acting at each successive division in a reiterative manner and ending up with the activation of the terminal differentiation program upon terminal division. A similar cell fate specification system seems to play a role in generating cellular diversity in the nervous system of more complex organisms such as Drosophila and vertebrates.

Analysis of multiple ethyl methanesulfonate-mutagenized Caenorhabditis elegans strains by whole-genome sequencing.

Whole-genome sequencing (WGS) of organisms displaying a specific mutant phenotype is a powerful approach to identify the genetic determinants of a plethora of biological processes. We have previously validated the feasibility of this approach by identifying a point-mutated locus responsible for a specific phenotype, observed in an ethyl methanesulfonate (EMS)-mutagenized Caenorhabditis elegans strain. Here we describe the genome-wide mutational profile of 17 EMS-mutagenized genomes as assessed with a bioinformatic pipeline, called MAQGene. Surprisingly, we find that while outcrossing mutagenized strains does reduce the total number of mutations, a striking mutational load is still observed even in outcrossed strains. Such genetic complexity has to be taken into account when establishing a causative relationship between genotype and phenotype. Even though unintentional, the 17 sequenced strains described here provide a resource of allelic variants in almost 1000 genes, including 62 premature stop codons, which represent candidate knockout alleles that will be of further use for the C. elegans community to study gene function.

Linking asymmetric cell division to the terminal differentiation program of postmitotic neurons in C. elegans.

How asymmetric divisions are connected to the terminal differentiation program of neuronal subtypes is poorly understood. In C. elegans, two homeodomain transcription factors, TTX-3 (a LHX2/9 ortholog) and CEH-10 (a CHX10 ortholog), directly activate a large battery of terminal differentiation genes in the cholinergic interneuron AIY. We establish here a transcriptional cascade linking asymmetric division to this differentiation program. A transient lineage-specific input formed by the Zic factor REF-2 and the bHLH factor HLH-2 directly activates ttx-3 expression in the AIY mother. During the terminal division of the AIY mother, an asymmetric Wnt/beta-catenin pathway cooperates with TTX-3 to directly restrict ceh-10 expression to only one of the two daughter cells. TTX-3 and CEH-10 automaintain their expression, thereby locking in the differentiation state. Our study establishes how transient lineage and asymmetric division inputs are integrated and suggests that the Wnt/beta-catenin pathway is widely used to control the identity of neuronal lineages.

A combinatorial code of maternal GATA, Ets and beta-catenin-TCF transcription factors specifies and patterns the early ascidian ectoderm.

Our understanding of the maternal factors that initiate early chordate development, and of their direct zygotic targets, is still fragmentary. A molecular cascade is emerging for the mesendoderm, but less is known about the ectoderm, giving rise to epidermis and nervous tissue. Our cis-regulatory analysis surprisingly places the maternal transcription factor Ci-GATAa (GATA4/5/6) at the top of the ectodermal regulatory network in ascidians. Initially distributed throughout the embryo, Ci-GATAa activity is progressively repressed in vegetal territories by accumulating maternal beta-catenin. Once restricted to the animal hemisphere, Ci-GATAa directly activates two types of zygotic ectodermal genes. First, Ci-fog is activated from the 8-cell stage throughout the ectoderm, then Ci-otx is turned on from the 32-cell stage in neural precursors only. Whereas the enhancers of both genes contain critical and interchangeable GATA sites, their distinct patterns of activation stem from the additional presence of two Ets sites in the Ci-otx enhancer. Initially characterized as activating elements in the neural lineages, these Ets sites additionally act as repressors in non-neural lineages, and restrict GATA-mediated activation of Ci-otx. We thus identify a precise combinatorial code of maternal factors responsible for zygotic onset of a chordate ectodermal genetic program.

A quantitative approach to the study of cell shapes and interactions during early chordate embryogenesis.

BACKGROUND: The prospects of deciphering the genetic program underlying embryonic development were recently boosted by the generation of large sets of precisely organized quantitative molecular data. In contrast, although the precise arrangement, interactions, and shapes of cells are crucial for the fulfilment of this program, their description remains coarse and qualitative. To bridge this gap, we developed a generic software, 3D Virtual Embryo, to quantify the geometry and interactions of cells in interactive three-dimensional embryo models. We applied this approach to early ascidian embryos, chosen because of their simplicity and their phylogenetic proximity to vertebrates. RESULTS: We generated a collection of 19 interactive ascidian embryos between the 2- and 44-cell stages. We characterized the evolution with time, and in different cell lineages, of the volume of cells and of eight mathematical descriptors of their geometry, and we measured the surface of contact between neighboring blastomeres. These analyses first revealed that early embryonic blastomeres adopt a surprising variety of shapes, which appeared to be under strict and dynamic developmental control. Second, we found novel asymmetric cell divisions in the posterior vegetal lineages, which gave birth to sister cells with different fates. Third, during neural induction, differences in the area of contact between individual competent animal cells and inducing vegetal blastomeres appeared important to select the induced cells. CONCLUSIONS: In addition to novel insight into both cell-autonomous and inductive processes controlling early ascidian development, we establish a generic conceptual framework for the quantitative analysis of embryo geometry that can be applied to other model organisms.