Control of successive unequal cell divisions by neural cell fate regulators determines embryonic neuroblast cell size.

Asymmetric cell divisions often generate daughter cells of unequal size in addition to different fates. In some contexts, daughter cell size asymmetry is thought to be a key input to specific binary cell fate decisions. An alternative possibility is that unequal division is a mechanism by which a variety of cells of different sizes are generated during embryonic development. We show here that two unequal cell divisions precede neuroblast formation in the C lineage of Caenorhabditis elegans. The equalisation of these divisions in a pig-1/MELK mutant background has little effect on neuroblast specification. Instead, we demonstrate that let-19/MDT13 is a regulator of the proneural basic helix-loop-helix transcription factor hlh-14/ASCL1 and find that both are required to concomitantly regulate the acquisition of neuroblast identity and neuroblast cell size. Thus, embryonic neuroblast cell size in this lineage is progressively regulated in parallel with identity by key neural cell fate regulators. We propose that key cell fate determinants have a previously unappreciated function in regulating unequal cleavage, and therefore cell size, of the progenitor cells whose daughter cell fates they then go on to specify.

Cell-specific effects of the sole C. elegans Daughterless/E protein homolog, HLH-2, on nervous system development.

Are there common mechanisms of neurogenesis used throughout an entire nervous system? We explored to what extent canonical proneural class I/II bHLH complexes are responsible for neurogenesis throughout the entire Caenorhabditis elegans nervous system. Distinct, lineage-specific proneural class II bHLH factors are generally thought to operate via interaction with a common, class I bHLH subunit, encoded by Daughterless in flies, the E proteins in vertebrates and HLH-2 in C. elegans. To eliminate function of all proneuronal class I/II bHLH complexes, we therefore genetically removed maternal and zygotic hlh-2 gene activity. We observed broad effects on neurogenesis, but still detected normal neurogenesis in many distinct neuron-producing lineages of the central and peripheral nervous system. Moreover, we found that hlh-2 selectively affects some aspects of neuron differentiation while leaving others unaffected. Although our studies confirm the function of proneuronal class I/II bHLH complexes in many different lineages throughout a nervous system, we conclude that their function is not universal, but rather restricted by lineage, cell type and components of differentiation programs affected.

Increasing Complexity of the N-Glycome During Caenorhabditis Development.

Caenorhabditis elegans is a frequently employed genetic model organism and has been the object of a wide range of developmental, genetic, proteomic, and glycomic studies. Here, using an off-line MALDI-TOF-MS approach, we have analyzed the N-glycans of mixed embryos and liquid- or plate-grown L4 larvae. Of the over 200 different annotatable N-glycan structures, variations between the stages as well as the mode of cultivation were observed. While the embryonal N-glycome appears less complicated overall, the liquid- and plate-grown larvae differ especially in terms of methylation of bisecting fucose, α-galactosylation of mannose, and di-ß-galactosylation of core α1,6-fucose. Furthermore, we analyzed the O-glycans by LC-electrospray ionization-MS following ß-elimination; especially the embryonal O-glycomes included a set of phosphorylcholine-modified structures, previously not shown to exist in nematodes. However, the set of glycan structures cannot be clearly correlated with levels of glycosyltransferase transcripts in developmental RNA-Seq datasets, but there is an indication for coordinated expression of clusters of potential glycosylation-relevant genes. Thus, there are still questions to be answered in terms of how and why a simple nematode synthesizes such a diverse glycome.

Piecemeal regulation of convergent neuronal lineages by bHLH transcription factors in Caenorhabditis elegans.

Cells of the same type can be generated by distinct cellular lineages that originate in different parts of the developing embryo ('lineage convergence'). Several Caenorhabditis elegans neuron classes composed of left/right or radially symmetric class members display such lineage convergence. We show here that the C. elegans Atonal homolog lin-32 is differentially expressed in neuronal lineages that give rise to left/right or radially symmetric class members. Loss of lin-32 results in the selective loss of the expression of pan-neuronal markers and terminal selector-type transcription factors that confer neuron class-specific features. Another basic helix-loop-helix (bHLH) gene, the Achaete-Scute homolog hlh-14, is expressed in a mirror image pattern relative to lin-32 and is required to induce neuronal identity and terminal selector expression on the contralateral side of the animal. These findings demonstrate that distinct lineage histories converge via different bHLH factors at the level of induction of terminal selector identity determinants, which thus serve as integrators of distinct lineage histories. We also describe neuron-to-neuron identity transformations in lin-32 mutants, which we propose to also be the result of misregulation of terminal selector gene expression.

Disruption of the Caenorhabditis elegans Integrator complex triggers a non-conventional transcriptional mechanism beyond snRNA genes.

Gene expression is generally regulated by recruitment of transcription factors and RNA polymerase II (RNAP II) to specific sequences in the gene promoter region. The Integrator complex mediates processing of small nuclear RNAs (snRNAs) as well as the initiation and release of paused RNAP II at specific genes in response to growth factors. Here we show that in C. elegans, disruption of the Integrator complex leads to transcription of genes located downstream of the snRNA loci via a non-conventional transcription mechanism based on the lack of processing of the snRNAs. RNAP II read-through generates long chimeric RNAs containing snRNA, the intergenic region and the mature mRNA of the downstream gene located in sense. These chimeric sn-mRNAs remain as untranslated long non-coding RNAs, in the case of U1- and U2-derived sn-mRNAs, but can be translated to proteins in the case of SL-derived sn-mRNAs. The transcriptional effect caused by disruption of the Integrator complex is not restricted to genes located downstream of the snRNA loci but also affects key regulators of signal transduction such as kinases and phosphatases. Our findings highlight that these transcriptional alterations may be behind the correlation between mutations in the Integrator complex and tumor transformation.

Reduction of mRNA export unmasks different tissue sensitivities to low mRNA levels during Caenorhabditis elegans development.

Animal development requires the execution of specific transcriptional programs in different sets of cells to build tissues and functional organs. Transcripts are exported from the nucleus to the cytoplasm where they are translated into proteins that, ultimately, carry out the cellular functions. Here we show that in Caenorhabditis elegans, reduction of mRNA export strongly affects epithelial morphogenesis and germline proliferation while other tissues remain relatively unaffected. Epithelialization and gamete formation demand a large number of transcripts in the cytoplasm for the duration of these processes. In addition, our findings highlight the existence of a regulatory feedback mechanism that activates gene expression in response to low levels of cytoplasmic mRNA. We expand the genetic characterization of nuclear export factor NXF-1 to other members of the mRNA export pathway to model mRNA export and recycling of NXF-1 back to the nucleus. Our model explains how mutations in genes involved in general processes, such as mRNA export, may result in tissue-specific developmental phenotypes.

Neurotransmitter-mediated activity spatially controls neuronal migration in the zebrafish cerebellum.

Neuronal migration during embryonic development contributes to functional brain circuitry. Many neurons migrate in morphologically distinct stages that coincide with differentiation, requiring tight spatial regulation. It had been proposed that neurotransmitter-mediated activity could exert this control. Here, we demonstrate that intracellular calcium transients occur in cerebellar neurons of zebrafish embryos during migration. We show that depolarization increases and hyperpolarization reduces the speed of tegmental hindbrain neurons using optogenetic tools and advanced track analysis optimized for in vivo migration. Finally, we introduce a compound screening assay to identify acetylcholine (ACh), glutamate, and glycine as regulators of migration, which act regionally along the neurons' route. We summarize our findings in a model describing how different neurotransmitters spatially interact to control neuronal migration. The high evolutionary conservation of the cerebellum and hindbrain makes it likely that polarization state-driven motility constitutes an important principle in building a functional brain.

Twenty million years of evolution: The embryogenesis of four Caenorhabditis species are indistinguishable despite extensive genome divergence.

The four Caenorhabditis species C. elegans, C. briggsae, C. remanei and C. brenneri show more divergence at the genomic level than humans compared to mice (Stein et al., 2003; Cutter et al., 2006, 2008). However, the behavior and anatomy of these nematodes are very similar. We present a detailed analysis of the embryonic development of these species using 4D-microscopic analyses of embryos including lineage analysis, terminal differentiation patterns and bioinformatical quantifications of cell behavior. Further functional experiments support the notion that the early development of all four species depends on identical induction patterns. Based on our results, the embryonic development of all four Caenorhabditis species are nearly identical, suggesting that an apparently optimal program to construct the body plan of nematodes has been conserved for at least 20 million years. This contrasts the levels of divergence between the genomes and the protein orthologs of the Caenorhabditis species, which is comparable to the level of divergence between mouse and human. This indicates an intricate relationship between the structure of genomes and the morphology of animals.

Oriented Cell Division in the C. elegans Embryo Is Coordinated by G-Protein Signaling Dependent on the Adhesion GPCR LAT-1.

Orientation of spindles and cell division planes during development of many species ensures that correct cell-cell contacts are established, which is vital for proper tissue formation. This is a tightly regulated process involving a complex interplay of various signals. The molecular mechanisms underlying several of these pathways are still incompletely understood. Here, we identify the signaling cascade of the C. elegans latrophilin homolog LAT-1, an essential player in the coordination of anterior-posterior spindle orientation during the fourth round of embryonic cell division. We show that the receptor mediates a G protein-signaling pathway revealing that G-protein signaling in oriented cell division is not solely GPCR-independent. Genetic analyses showed that through the interaction with a Gs protein LAT-1 elevates intracellular cyclic AMP (cAMP) levels in the C. elegans embryo. Stimulation of this G-protein cascade in lat-1 null mutant nematodes is sufficient to orient spindles and cell division planes in the embryo in the correct direction. Finally, we demonstrate that LAT-1 is activated by an intramolecular agonist to trigger this cascade. Our data support a model in which a novel, GPCR-dependent G protein-signaling cascade mediated by LAT-1 controls alignment of cell division planes in an anterior-posterior direction via a metabotropic Gs-protein/adenylyl cyclase pathway by regulating intracellular cAMP levels.

Adhesion GPCRs Govern Polarity of Epithelia and Cell Migration.

In multicellular organisms cells spatially arrange in a highly coordinated manner to form tissues and organs, which is essential for the function of an organism. The component cells and resulting structures are often polarised in one or more axes, and how such polarity is established and maintained correctly has been one of the major biological questions for many decades. Research progress has shown that many adhesion GPCRs (aGPCRs) are involved in several types of polarity. Members of the two evolutionarily oldest groups, Flamingo/Celsr and Latrophilins, are key molecules in planar cell polarity of epithelia or the propagation of cellular polarity in the early embryo, respectively. Other adhesion GPCRs play essential roles in cell migration, indicating that this receptor class includes essential molecules for the control of various levels of cellular organisation.

The Wnt pathway controls cell death engulfment, spindle orientation, and migration through CED-10/Rac.

Wnt signalling pathways have extremely diverse functions in animals, including induction of cell fates or tumours, guidance of cell movements during gastrulation, and the induction of cell polarity. Wnt can induce polar changes in cellular morphology by a remodelling of the cytoskeleton. However, how activation of the Frizzled receptor induces cytoskeleton rearrangement is not well understood. We show, by an in depth 4-D microscopy analysis, that the Caenorhabditis elegans Wnt pathway signals to CED-10/Rac via two separate branches to regulate modulation of the cytoskeleton in different cellular situations. Apoptotic cell clearance and migration of the distal tip cell require the MOM-5/Fz receptor, GSK-3 kinase, and APC/APR-1, which activate the CED-2/5/12 branch of the engulfment machinery. MOM-5 (Frizzled) thus can function as an engulfment receptor in C. elegans. Our epistatic analyses also suggest that the two partially redundant signalling pathways defined earlier for engulfment may act in a single pathway in early embryos. By contrast, rearrangement of mitotic spindles requires the MOM-5/Fz receptor, GSK-3 kinase, and beta-catenins, but not the downstream factors LIT-1/NLK or POP-1/Tcf. Taken together, our results indicate that in multiple developmental processes, CED-10/Rac can link polar signals mediated by the Wnt pathway to rearrangements of the cytoskeleton.

ccz-1 mediates the digestion of apoptotic corpses in C. elegans.

During development, the processes of cell division, differentiation and apoptosis must be precisely coordinated in order to maintain tissue homeostasis. The nematode C. elegans is a powerful model system in which to study cell death and its control. C. elegans apoptotic cells condense and form refractile corpses under differential interference contrast (DIC) microscopy. Activation of the GTPase CED-10 (Rac) in a neighbouring cell mediates the recognition and engulfment of the cell corpse. After inclusion of the engulfed corpse in a phagosome, different proteins are sequentially recruited onto this organelle to promote its acidification and fusion with lysosomes, leading to the enzymatic degradation of the cell corpse. We show that CCZ-1, a protein conserved from yeasts to humans, mediates the digestion of these apoptotic corpses. CCZ-1 seems to act in lysosome biogenesis and phagosome maturation by recruiting the GTPase RAB-7 over the phagosome.

Mass spectrometric comparison of N-glycan profiles from Caenorhabditis elegans mutant embryos.

The free-living nematode Caenorhabditis elegans is a well-characterized eukaryotic model organism. Recent glycomic analyses of the glycosylation potential of this worm revealed an extremely high structural variability of its N-glycans. Moreover, the glycan patterns of each developmental stage appeared to be unique. In this study we have determined the N-glycan profiles of wild-type embryos in comparison to mutant embryos arresting embryogenesis early before differentiation and causing extensive transformations of cell identities, which allows to follow the diversification of N-glycans during development using mass spectrometry. As a striking feature, wild-type embryos obtained from liquid culture expressed a less heterogeneous oligosaccharide pattern than embryos recovered from agar plates. N-glycan profiles of mutant embryos displayed, in part, distinct differences in comparison to wild-type embryos suggesting alterations in oligosaccharide trimming and processing, which may be linked to specific cell fate alterations in the embryos.

Functional dissection of Caenorhabditis elegans CLK-2/TEL2 cell cycle defects during embryogenesis and germline development.

CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2-null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development.

A conserved function for a Caenorhabditis elegans Com1/Sae2/CtIP protein homolog in meiotic recombination.

Genome stability relies on faithful DNA repair both in mitosis and in meiosis. Here, we report on a Caenorhabditis elegans protein that we found to be homologous to the mammalian repair-related protein CtIP and to the budding yeast Com1/Sae2 recombination protein. A com-1 mutant displays normal meiotic chromosome pairing but forms irregular chromatin aggregates instead of diakinesis bivalents. While meiotic DNA double-strand breaks (DSBs) are formed, they appear to persist or undergo improper repair. Despite the presence of DSBs, the recombination protein RAD-51, which is known to associate with single-stranded DNA (ssDNA) flanking DSBs, does not localize to meiotic chromosomes in the com-1 mutant. Exposure of the mutant to gamma-radiation, however, induces RAD-51 foci, which suggests that the failure of RAD-51 to load is specific to meiotic (SPO-11-generated) DSBs. These results suggest that C. elegans COM-1 plays a role in the generation of ssDNA tails that can load RAD-51, invade homologous DNA tracts and thereby initiate recombination. Extrapolating from the worm homolog, we expect similar phenotypes for mutations in the mammalian tumor suppressor CtIP.

Centriolar SAS-5 is required for centrosome duplication in C. elegans.

Centrosomes, the major microtubule-organizing centres (MTOCs) of animal cells, are comprised of a pair of centrioles surrounded by pericentriolar material (PCM). Early in the cell cycle, there is a single centrosome, which duplicates during S-phase to direct bipolar spindle assembly during mitosis. Although crucial for proper cell division, the mechanisms that govern centrosome duplication are not fully understood. Here, we identify the Caenorhabditis elegans gene sas-5 as essential for daughter-centriole formation. SAS-5 is a coiled-coil protein that localizes primarily to centrioles. Fluorescence recovery after photobleaching (FRAP) experiments with green fluorescent protein (GFP) fused to SAS-5 (GFP-SAS-5) demonstrated that the protein shuttles between centrioles and the cytoplasm throughout the cell cycle. Analysis of mutant alleles revealed that the presence of SAS-5 at centrioles is crucial for daughter-centriole formation and that ZYG-1, a kinase that is also essential for this process, controls the distribution of SAS-5 to centrioles. Furthermore, partial RNA-interference (RNAi)-mediated inactivation experiments suggest that both sas-5 and zyg-1 are dose-dependent regulators of centrosome duplication.

Two pathways converge at CED-10 to mediate actin rearrangement and corpse removal in C. elegans.

The removal of apoptotic cells is essential for the physiological well being of the organism. In Caenorhabditis elegans, two conserved, partially redundant genetic pathways regulate this process. In the first pathway, the proteins CED-2, CED-5 and CED-12 (mammalian homologues CrkII, Dock180 and ELMO, respectively) function to activate CED-10 (Rac1). In the second group, the candidate receptor CED-1 (CD91/LRP/SREC) probably recognizes an unknown ligand on the apoptotic cell and signals via its cytoplasmic tail to the adaptor protein CED-6 (hCED-6/GULP), whereas CED-7 (ABCA1) is thought to play a role in membrane dynamics. Molecular understanding of how the second pathway promotes engulfment of the apoptotic cell is lacking. Here, we show that CED-1, CED-6 and CED-7 are required for actin reorganization around the apoptotic cell corpse, and that CED-1 and CED-6 colocalize with each other and with actin around the dead cell. Furthermore, we find that the CED-10(Rac) GTPase acts genetically downstream of these proteins to mediate corpse removal, functionally linking the two engulfment pathways and identifying the CED-1, -6 and -7 signalling module as upstream regulators of Rac activation.

The HLH-6 transcription factor regulates C. elegans pharyngeal gland development and function.

The Caenorhabditis elegans pharynx (or foregut) functions as a pump that draws in food (bacteria) from the environment. While the "organ identity factor" PHA-4 is critical for formation of the C. elegans pharynx as a whole, little is known about the specification of distinct cell types within the pharynx. Here, we use a combination of bioinformatics, molecular biology, and genetics to identify a helix-loop-helix transcription factor (HLH-6) as a critical regulator of pharyngeal gland development. HLH-6 is required for expression of a number of gland-specific genes, acting through a discrete cis-regulatory element named PGM1 (Pharyngeal Gland Motif 1). hlh-6 mutants exhibit a frequent loss of a subset of glands, while the remaining glands have impaired activity, indicating a role for hlh-6 in both gland development and function. Interestingly, hlh-6 mutants are also feeding defective, ascribing a biological function for the glands. Pharyngeal pumping in hlh-6 mutants is normal, but hlh-6 mutants lack expression of a class of mucin-related proteins that are normally secreted by pharyngeal glands and line the pharyngeal cuticle. An interesting possibility is that one function of pharyngeal glands is to secrete a pharyngeal lining that ensures efficient transport of food along the pharyngeal lumen.

Embryology of a planktonic tunicate reveals traces of sessility.

A key problem in understanding deuterostome evolution has been the origin of the chordate body plan. A biphasic life cycle with a sessile adult and a free-swimming larva is traditionally considered ancestral in chordates with subsequent neotenic loss of the sessile adult stage. Molecular phylogenies challenged this view, suggesting that the primitive life cycle in chordates was entirely free-living as in modern day larvaceans. Here, we report the precise cell lineage and fate map in the normal embryo of the larvacean Oikopleura dioica, using 4D microscopy technique and transmission electron microscopy. We document the extraordinary rapidity of cleavage and morphogenetic events until hatching and demonstrate that--compared with ascidians--fate restriction occurs considerably earlier in O. dioica and that clonal organization of the cell lineage is more tightly coupled to tissue fate. We show that epidermal cells in the trunk migrate through 90 degrees, reminiscent of events in ascidian metamorphosis and that the axis of bilateral symmetry in the tail rotates in relation to the trunk. We argue that part of the tail muscle cells are ectomesodermal, because they are more closely associated with prospective epidermis than with other tissues in the cell lineage. Cladistic comparison with other deuterostomes suggests that these traits are derived within tunicates strengthening the hypothesis that the last common ancestor of tunicates had a sessile adult and thus support traditional morphology-derived scenarios. Our results allow hypothesizing that molecular developmental mechanisms known from ascidian models are restricted to fewer, yet identifiable, cells in O. dioica.

Identification of essential genes in Caenorhabditis elegans through whole-genome sequencing of legacy mutant collections.

It has been estimated that 15%-30% of the ∼20,000 genes in C. elegans are essential, yet many of these genes remain to be identified or characterized. With the goal of identifying unknown essential genes, we performed whole-genome sequencing on complementation pairs from legacy collections of maternal-effect lethal and sterile mutants. This approach uncovered maternal genes required for embryonic development and genes with apparent sperm-specific functions. In total, 58 putative essential genes were identified on chromosomes III-V, of which 52 genes are represented by novel alleles in this collection. Of these 52 genes, 19 (40 alleles) were selected for further functional characterization. The terminal phenotypes of embryos were examined, revealing defects in cell division, morphogenesis, and osmotic integrity of the eggshell. Mating assays with wild-type males revealed previously unknown male-expressed genes required for fertilization and embryonic development. The result of this study is a catalog of mutant alleles in essential genes that will serve as a resource to guide further study toward a more complete understanding of this important model organism. As many genes and developmental pathways in C. elegans are conserved and essential genes are often linked to human disease, uncovering the function of these genes may also provide insight to further our understanding of human biology.