Developmental timing and cell fate maintenance in Caenorhabditis elegans

The development of a multi-cellular organism begins with a single fertilized egg cell, and ends with a fully-grown adult with all kinds of specialized cell types. During this complex developmental process, cells undergo several important developmental events, such as cell fate differentiation, movement and growth. The correct timing of the occurrence and duration of those developmental events is one of the key challenges during development, in order to prevent malformation of tissues. After cells have reached their terminal cell fate, a new important challenge appears, that is the maintenance of their obtained terminally differentiated state, to prevent loss of their specialized functions. Robust maintenance of terminal cell fate is particularly important in cell types that are hardly renewed during the lifetime of an animal, such as neuronal cells. In this thesis, we focused on these two complementary key challenges, of correct developmental timing and long-term cell fate maintenance, that cells face during their lifetime. With the help of the model system Caenorhabditis elegans, we studied how cell growth is timed by gene expression oscillators during development, and what mechanism lies behind the long-term maintenance of terminal cell fate that is controlled by terminal selector genes

Human teneurin-1 is a direct target of the homeobox transcription factor EMX2 at a novel alternate promoter

<p>Abstract</p> <p>Background</p> <p>Teneurin-1 is a member of a family of type II transmembrane proteins conserved from <it>C.elegans </it>to vertebrates. Teneurin expression in vertebrates is best studied in mouse and chicken, where the four members teneurin-1 to -4 are predominantly expressed in the developing nervous system in area specific patterns. Based on their distinct, complementary expression a possible function in the establishment of proper connectivity in the brain was postulated. However, the transcription factors contributing to these distinctive expression patterns are largely unknown. Emx2 is a homeobox transcription factor, known to be important for area specification in the developing cortex. A study of Emx2 knock-out mice suggested a role of Emx2 in regulating patterned teneurin expression.</p> <p>Results</p> <p>5'RACE of human teneurin-1 revealed new alternative untranslated exons that are conserved in mouse and chicken. Closer analysis of the conserved region around the newly identified transcription start revealed promoter activity that was induced by EMX2. Mutation of a predicted homeobox binding site decreased the promoter activity in different reporter assays <it>in vitro </it>and <it>in vivo </it>in electroporated chick embryos. We show direct <it>in vivo </it>binding of EMX2 to the newly identified promoter element and finally confirm that the endogenous alternate transcript is specifically upregulated by EMX2.</p> <p>Conclusions</p> <p>We found that human teneurin-1 is directly regulated by EMX2 at a newly identified and conserved promoter region upstream of the published transcription start site, establishing teneurin-1 as the first human EMX2 target gene. We identify and characterize the EMX2 dependent promoter element of human teneurin-1.</p

Spatio-temporal control of the cytosolic redox environment in C. elegans

Compartmentalization of redox reactions is essential to all life forms. Protein activity can respond to changes in the local redox environment through the reversible oxidation of cysteine thiols. For the majority of cysteines in the proteome, this interaction takes place through equilibration with the glutathione pool; this raises the question whether this redox pool acts as a buffer, or instead as a sensitive media, transducing information from a local physiological state into protein function

Characterization of one of the the REF-1 Family Members, HLH-25, in C. elegans

The REF-1 family proteins are distinguished by the presence of two basic helix-loop helix domains. The REF-1 family members are considered functional homologs of the Hairy/Enhancer of Split in humans. HLH-25 is one of the six members of the REF-1 family. HLH-25 has not been studied extensively. In preliminary studies from our laboratory, genes identified by microarray analysis of hlh-25 mutants were essential for embryogenesis, larval development, and growth. Thus, the present study was designed to further characterize HLH-25 and to more precisely define its role during embryonic and larval development. The gene encoding HLH-25 is actively expressed in embryos, larvae and adults. In the absence of hlh-25, animals show a 54% embryonic lethality, a reduced brood size, an increased number of unfertilized eggs, a slower movement rate, a longer life span, and a longer dauer recovery. The human tumor suppressor PTEN homolog, daf-18 is one of the HLH-25 target genes. The regulation of daf-18 through HLH-25 is the responsible for changes to the life span and dauer recovery in hlh-25 mutant animals

Doctor of Philosophy

dissertationThe nervous system is comprised of an estimated 100 billion individual neurons, which are connected to one another to form a network that senses environmental stimuli and coordinates the organism's behavior. Because of the complexity of the nervous system, deciphering the developmental processes and adult wiring diagram has proved challenging. A number of axon guidance molecules have been identified; however, the means by which they guide billions of axons to their target cells in vivo remains poorly understood. Several axon guidance molecules have been found to be bifunctional, meaning they can elicit different growth cone responses depending on the presence or absence of other molecules, such as growth cone receptors, intracellular signal transduction molecules, or extracellular modulators. Axon sorting within axon tracts is perhaps a means by which axons are presorted to make a precise connection on their target cells. The zebrafish, Danio rerio, is an ideal model organism to study vertebrate axon guidance and axon sorting due to its external fertilization, optical transparency, amenability to forward genetics, and ease of making transgenic lines. In order to study axon guidance within the zebrafish retinotectal system, I developed a new method of misexpressing genes. Local misexpression can be induced by using a modified soldering iron in transgenic zebrafish in which a gene of interest is driven by a heat shock promoter. This method allowed me to examine the mechanisms by which Slit1a and Slit2 guide axons from the retina to the optic tectum. I determined the expression pattern of Slits in the zebrafish and used antisense morpholino technology to knock down Slit1a. The iv resultant axon guidance errors indicated that Slit1a acts to guide retinal axons through the optic tract. I then misexpressed Slit1a and Slit2 near the optic tract to observe their effect on axons. I found that both proteins appeared to attract retinal axons. Additionally, I saw that Slit2 seems to attract retinal axons earlier in the retinotectal pathway, at the optic chiasm. I also report on a new method, to whose development I contributed, for automated tracking of axons through electron microscopy datasets. Taken together, my results add new methods to the endeavor of mapping neural connectivity and development, and suggest a new role for Slits in axon guidance

On the evolution of cnidarian eyes

Cnidarians and their medusa stage are generally considered to be diploblasts and therefore ancestral to Bilaterians. They represent the most primitive phylum where striated muscle tissue, a complex system of nerve rings and different sense organs of high complexity, including eyes have evolved in the jellyfish stage. We demonstrated that jellyfish and the triploblast Bilateria use homologous gene cascades and developmental pathways to build these muscle systems. The expression of JellyD, a derived jellyfish homolog of the master regulator of muscle tissue MyoD, is correlated with that of bilaterian muscle determination factors. Furthermore, the eye determination genes of the Pax and Six families of cnidarians have bilaterian-like expression patterns. Although no bona fide Pax6 homolog could be found, it can be shown that among the four Pax genes characterized, cnidarians do have a Pax gene (PaxA-Cr) that is exclusively expressed in the maintenance and regeneration of eye tissue. Additionally the hypothesis of a loss of Pax genes within the cnidarians can be rebut as well as the claim that cubozoans would possess only one Pax gene. Cladonema jellyfish have three cognate members of the sine oculis/Six class family of which Six1/2-Cr and Six3/6-Cr are upregulated during eye regeneration. Analysis of gene expression patterns during eye regeneration shows that the cnidarian Pax gene is upregulated before the Six genes, indicating a possible upstream position in the gene regulatory network. The results are in agreement with monophyly of eye evolution and indicate that the common ancestor between Cnidaria and Bilateria had a more complex anatomy than commonly anticipated

Whole-body integration of gene expression and single-cell morphology

Animal bodies are composed of cell types with unique expression programs that implement their distinct locations, shapes, structures, and functions. Based on these properties, cell types assemble into specific tissues and organs. To systematically explore the link between cell-type-specific gene expression and morphology, we registered an expression atlas to a whole-body electron microscopy volume of the nereid Platynereis dumerilii. Automated segmentation of cells and nuclei identifies major cell classes and establishes a link between gene activation, chromatin topography, and nuclear size. Clustering of segmented cells according to gene expression reveals spatially coherent tissues. In the brain, genetically defined groups of neurons match ganglionic nuclei with coherent projections. Besides interneurons, we uncover sensory-neurosecretory cells in the nereid mushroom bodies, which thus qualify as sensory organs. They furthermore resemble the vertebrate telencephalon by molecular anatomy. We provide an integrated browser as a Fiji plugin for remote exploration of all available multimodal datasets

The actin-binding protein Drebrin and its implications for Alzheimer's Disease using the model organism C. elegans

Patients of Alzheimer’s Disease (AD) showed reduced levels of the actin-binding protein Drebrin in their neurons. The here presented work was set out to analyse the interaction between Drebrin and the disease-associated peptide Amyloid-β. To analyse the interaction in vivo two novel models were designed, employing the nematode C. elegans. The Amyloid-β pathology was modelled by overexpressing the disease causing peptide pan-neuronally and employing a genetic sub-stoichiometric labelling method to be able to follow the aggregation in vivo and in situ in a non-invasive manner. A second model, expressing human Drebrin pan-neuronally was generated to analyse Drebrin stability, localization and phosphorylation as well as analysing the effect of Drebrin overexpression on the nematodes’ vitality and fitness. The third project combined both generated models to obtain a genetic cross expressing Aβ(1-42) and Drebrin simultaneously. This model was sought to study the interaction between Aβ(1-42) and Drebrin. I could show, that Aβ(1-42) aggregates with the progression of ageing and exhibits multiple disease phenotypes that can be correlated to observations obtained in murine neurons as well as observations of AD patients’ brain tissues. Furthermore, I observed, that a distinct subset of head neurons of the anterior ganglion, the IL2 neurons, exhibits the first aggregates and that a cell-type specific suppression of Aβ(1-42) in IL2 neurons could delay the disease onset. Drebrin was observed to be regulated by phosphorylation at Serine-647 by Ataxia telangiectasia mutated kinase and render nematodes more resistant towards chronic oxidative stress. The genetic cross of Aβ(1-42) and Drebrin unravelled that overexpression of Drebrin can ameliorate Aβ(1-42) aggregation and toxicity and that this beneficial effect is dependent on phosphorylation of Drebrin-S647

Investigating the role of the fusogen eff-1 and natural genetic variation in Caenorhabditis elegans seam cell development

Robustness is the ability of biological systems to produce invariant phenotypes despite perturbations. Development is especially robust to internal perturbations, like stochastic gene expression or mutations, and external perturbations, such as changes in environmental factors including temperature and nutrition. The highly invariant developmental patterning in Caenorhabditis elegans offers an ideal system to study the genetic and molecular mechanisms underlying developmental robustness. This work describes an experimental paradigm to discover the mechanistic basis and consequences of developmental robustness using the C. elegans seam cells as a model. Seam cells are lateral epidermal cells that are stem cell-like in their ability to produce differentiated cells and maintain proliferative potential. Through a forward genetic screen, I describe a novel role for the fusogen gene eff-1, which was previously known to drive cell fusion events, in the robustness of seam cell patterning. Furthermore, I show that eff-1 is not required for differentiation of seam cells, therefore I demonstrate that fusion is uncoupled from the differentiation programme. In another set of experiments, I show for the first time that the terminal number of seam cells in C. elegans is robust to standing genetic variation. A consequence of developmental robustness is the acquisition of cryptic genetic variation that does not modify the phenotype under normal conditions but manifests phenotypically upon perturbation. I demonstrate that the genetic background affects seam cell number at a higher developmental temperature of 25 C or upon mutations in the GATA transcription factor and target of the Wnt pathway, egl-18. CB4856 (Hawaii) suppressed the effect of temperature on the seam cell number compared to the lab reference N2 (United Kingdom), as well as lowered the expressivity of egl-18 mutations. Multiple regions of the genome were found to interact epistatically to modify egl-18 mutation expressivity, suggesting that a complex genetic architecture underlies seam cell development. Taken together, this work increases our knowledge on the robustness of seam cell patterning to various sources of variation.Open Acces