Comparative analysis of midline guidance and axon pathfinding in the chelicerates Achaearanea tepidariorum and Cupiennius salei

Die Neurogenese und axonale Wegfindung sind in den vergangenen Jahrzehnten Thema einer Vielzahl wissenschaftlicher Untersuchungen in den verschiedensten Organismen gewesen. Die zusammengetragenen Daten in Insekten und Crustaceen geben eine gute Übersicht darüber, wie das Nervensystem in Arthropoden aufgebaut wird. Die entwicklungsbiologischen Prozesse, die daran beteiligt sind, sind in den beiden genannten Gruppen sehr gut verstanden. In den Gruppen der Cheliceraten und Myriapoden jedoch wurden ähnliche Analysen bisher kaum durchgeführt. Das Hauptanliegen dieser Arbeit war es daher, Mechanismen in den Spinnen Achaearanea tepidariorum und Cupiennius salei, zwei Vertretern der Cheliceraten, zu untersuchen, die eine Rolle im Leitsystem der ventralen Mittellinie und bei der axonalen Wegfindung spielen. Eine Vorraussetzung hierfür sind Kenntnisse über die Architektur des Zentralnervensystems. In einem ersten Schritt beschrieb ich daher grundlegend die Morphologie des Nervensystems im Verlauf der gesamten Embryoalentwicklung. Ich konnte zeigen, dass in Spinnen ein für Arthropoden typisches Strickleiternervensystem gebildet wird. Dieses wird von segmental angelegten Neuronen geformt, wobei sowohl Gruppen von Zellen als auch einzelne Neurone daran beteiligt sind, die primären axonalen Trakte zu etablieren. Im Besonderen konnte ich eine Zelle identifizieren, die in Position, Projektionsmuster und der Expression des Markergens even-skipped vergleichbar zum PR2 Neuron in Drosophila ist, welches die posteriore Wurzel des Segmentalnervs anlegt.rnrnIn einem zweiten Ansatz untersuchte ich die ventrale Mittellinie in Spinnen im Bezug auf ihre mögliche Funktion in der axonalen Wegfindung. Es konnte gezeigt werden, dass es sich beim Epithel der Mittellinie, das die Lücke zwischen beiden Keimstreifhälften während des gesamten Prozesses der Inversion überspannt, um eine transiente Struktur handelt, die keine neuralen Zellen hervorbringt. Es ist daher vergleichbar mit der so genannten Floor plate in Vertebraten, die ebenfalls nur vorübergehend existiert. Die Untersuchung von single minded (sim) zeigte, dass es, anders als in Drosophila, wo sim ein wichtiges regulatorisches Gen für die korrekte Spezifizierung von Mittellinienzellen ist, nicht in den Zellen der Mittellinie, sondern in diesen benachbarten Zellen, exprimiert wird. Das ist vergleichbar mit Vertebraten. Zusätzlich konnte ich Expression von sim an den Basen der Gliedmassen und im Kopf nachweisen. Wie in Vertebraten könnte sim an der Musterbildung dieser Gewebe beteiligt sein. Dennoch spielt die Mittellinie in Spinnen eine wichtige Rolle als Organisator für auswachsende, kommissurale Axone. Diese Funktion teilt sie mit anderen Invertebraten und Vertebraten.rnrnDie Signaltransduktionskaskade, die an der axonalen Wegfindung an der Mittellinie beteiligt ist, ist in den verschiedensten Organismen hoch konserviert. In der vorliegenden Arbeit konnte ich sowohl in Achaearanea als auch in Cupiennius ein netrin Homolog identifizieren und eine konservierte Funktion des Wegfindungsmoleküls während der Bildung der Kommissuren aufzeigen. RNAi Experimente belegen, dass, wird die Funktion von netrin herunterreguliert, das Strickleiternervensystem nicht korrekt gebildet wird, ins Besondere die kommissuralen Faszikel. Des Weiteren konnte ich eine neue Funktion von netrin, die bisher in anderen Organsimen noch nicht beschrieben wurde, identifizieren. Neben seiner Rolle in der axonalen Wegfindung, scheint netrin auch an der epithelialen Morphogenese im zentralen Nervensystem beteiligt zu sein. In dieser Funktion scheint netrin in Gliazellen, die die epithelialen Vesikel der Invaginationsgruppen umhüllen, wichtig zu sein, um neurale Vorläuferzellen in einem undifferenzierten Zustand zu halten. Der Abbau von netrin Transkript durch RNA Interferenz führt zu einer verfrühten Segregation neuraler Vorläuferzellen aus dem epithelialen Verband der Invaginationsgruppen und zu einer Zunahme an Zellen, die den frühen Differenzierungsmarker islet exprimieren.

Down syndrome-critical region contains a gene homologous to Drosophila sim expressed during rat and human central nervous system development.

Many features of Down syndrome might result from the overdosage of only a few genes located in a critical region of chromosome 21. To search for these genes, cosmids mapping in this region were isolated and used for trapping exons. One of the trapped exons obtained has a sequence very similar to part of the Drosophila single-minded (sim) gene, a master regulator of the early development of the fly central nervous system midline. Mapping data indicated that this exonic sequence is only present in the Down syndrome-critical region in the human genome. Hybridization of this exonic sequence with human fetal kidney poly(A)+ RNA revealed two transcripts of 6 and 4.3 kb. In situ hybridization of a probe derived from this exon with human and rat fetuses showed that the corresponding gene is expressed during early fetal life in the central nervous system and in other tissues, including the facial, skull, palate, and vertebra primordia. The expression pattern of this gene suggests that it might be involved in the pathogenesis of some of the morphological features and brain anomalies observed in Down syndrome.

Fibroblast growth factor (FGF) homologous factors: new members of the FGF family implicated in nervous system development.

Four new members of the fibroblast growth factor (FGF) family, referred to as fibroblast growth factor homologous factors (FHFs), have been identified by a combination of random cDNA sequencing, data base searches, and degenerate PCR. Pairwise comparisons between the four FHFs show between 58% and 71% amino acid sequence identity, but each FHF shows less than 30% identity when compared with other FGFs. Like FGF-1 (acidic FGF) and FGF-2 (basic FGF), the FHFs lack a classical signal sequence and contain clusters of basic residues that can act as nuclear localization signals. In transiently transfected 293 cells FHF-1 accumulates in the nucleus and is not secreted. Each FHF is expressed in the developing and adult nervous systems, suggesting a role for this branch of the FGF family in nervous system development and function.

Immunolocalization of myosin Va in the developing nervous system of embryonic chicks

Myosins are molecular motors associated with the actin cytoskeleton that participate in the mechanisms of cellular motility. During the development of the nervous system, migration of nerve cells to specific sites, extension of growth cones, and axonal transport are dramatic manifestations of cellular motility. We demonstrate, via immunoblots, the expression of myosin Va during early stages of embryonic development in chicks, extending from the blastocyst period to the beginning of the fetal period. The expression of myosin Va in specific regions and cellular structures of the nervous system during these early stages was determined by immunocytochemistry using a polyclonal antibody. Whole mounts of chick embryos at 24-30-h stages showed intense immunoreactivity of the neural tube in formation along its full extent. Cross-sections at these stages of development showed strong labeling in neuroepithelial cells at the basal and apical regions of the neural tube wall. Embryos at more advanced periods of development (48h and 72 h) showed distinctive immunolabeling of neuroepithelial cells, neuroblasts and their cytoplasmic extensions in the mantle layer of the stratified neural tube wall, and neuroblasts and their cytoplasmic extensions in the internal wall of the optic cup, as well as a striking labeling of cells in the apparent nuclei of cranial nerves and budding fibers. These immunolocalization studies indicate temporal and site-specific expression of myosin Va during chick embryo development, suggesting that myosin Va expression is related to recruitment for specific cellular tasks.

Trinucleotide repeats and long homopeptides in genes and proteins associated with nervous system disease and development.

Several human neurological disorders are associated with proteins containing abnormally long runs of glutamine residues. Strikingly, most of these proteins contain two or more additional long runs of amino acids other than glutamine. We screened the current human, mouse, Drosophila, yeast, and Escherichia coli protein sequence data bases and identified all proteins containing multiple long homopeptides. This search found multiple long homopeptides in about 12% of Drosophila proteins but in only about 1.7% of human, mouse, and yeast proteins and none among E. coli proteins. Most of these sequences show other unusual sequence features, including multiple charge clusters and excessive counts of homopeptides of length > or = two amino acid residues. Intriguingly, a large majority of the identified Drosophila proteins are essential developmental proteins and, in particular, most play a role in central nervous system development. Almost half of the human and mouse proteins identified are homeotic homologs. The role of long homopeptides in fine-tuning protein conformation for multiple functional activities is discussed. The relative contributions of strand slippage and of dynamic mutation are also addressed. Several new experiments are proposed.

The overexpression of homeotic complex gene Ultrabithorax in the post-embryonic neuronal lineages of the ventral nervous system in Drosophila melanogaster

This study uses a molecular technique called MARCM (Mosaic Analysis with a Repressible Cell Marker) to label neuronal lineages that overexpress the Hox gene Ultrabithorax (Ubx) in an unlabeled, wild type background. The results indicate that the overexpression of Ubx is sufficient to transform more anterior neuronal lineages to themorphology of their more posterior counterparts. The data presented here begin to elucidate the role that the Hox genes have in shaping segment-specific neural connections in the post-embryonic ventral nervous system.

Cooperative functions of the reaper and head involution defective genes in the programmed cell death of Drosophila central nervous system midline cells

In Drosophila, the chromosomal region 75C1–2 contains at least three genes, reaper (rpr), head involution defective (hid), and grim, that have important functions in the activation of programmed cell death. To better understand how cells are killed by these genes, we have utilized a well defined set of embryonic central nervous system midline cells that normally exhibit a specific pattern of glial cell death. In this study we show that both rpr and hid are expressed in dying midline cells and that the normal pattern of midline cell death requires the function of multiple genes in the 75C1–2 interval. We also utilized the P[UAS]/P[Gal4] system to target expression of rpr and hid to midline cells. Targeted expression of rpr or hid alone was not sufficient to induce ectopic midline cell death. However, expression of both rpr and hid together rapidly induced ectopic midline cell death that resulted in axon scaffold defects characteristic of mutants with abnormal midline cell development. Midline-targeted expression of the baculovirus p35 protein, a caspase inhibitor, blocked both normal and ectopic rpr- and hid-induced cell death. Taken together, our results suggest that rpr and hid are expressed together and cooperate to induce programmed cell death during development of the central nervous system midline.

Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation.

The folate pathway plays a crucial role in the regeneration and repair of the adult CNS after injury. Here, we have shown in rodents that such repair occurs at least in part through DNA methylation. In animals with combined spinal cord and sciatic nerve injury, folate-mediated CNS axon regeneration was found to depend on injury-related induction of the high-affinity folate receptor 1 (Folr1). The activity of folate was dependent on its activation by the enzyme dihydrofolate reductase (Dhfr) and a functional methylation cycle. The effect of folate on the regeneration of afferent spinal neurons was biphasic and dose dependent and correlated closely over its dose range with global and gene-specific DNA methylation and with expression of both the folate receptor Folr1 and the de novo DNA methyltransferases. These data implicate an epigenetic mechanism in CNS repair. Folic acid and possibly other nontoxic dietary methyl donors may therefore be useful in clinical interventions to promote brain and spinal cord healing. If indeed the benefit of folate is mediated by epigenetic mechanisms that promote endogenous axonal regeneration, this provides possible avenues for new pharmacologic approaches to treating CNS injuries.

Development of the nervous system in Dugesia tigrina during regeneration after fission and decapitation

In the present study of Dugesia tigrina the development of the nervous system is followed and compared during regeneration after fission and after decapitation. Immunocytochemistry was used, with antisera raised against the biogenic amine, 5-hydroxytryptamine (5-HT) and the two neuropeptides, neuropeptide F (NPF), and FMRF amide. The results indicate that two processes are involved in the formation of the new cerebral ganglion. First, new processes sprouting from the original main longitudinal nerve cords bend transversely, indicating the position of the developing horseshoe-shaped anterior cerebral commissure. Then new nerve cells in front of the commissure differentiate from neoblasts and their growth cones fasciculate with the fibres from the old main longitudinal nerve cords. In the cerebral ganglion, 5-HT-IR cells appear before NPF-IR cells, in contrast to the pharynx where NPF-IR cells differentiate before the 5-HT-IR cells. In the peripheral nervous system, NPF-IR fibres and cells appear at a very early stage and dominate the whole regeneration process. A role for the PNS in early pattern formation is suggested.

Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.

Investigating the role of retinoic acid in the vertebrate and invertebrate nervous system

Please consult the paper edition of this thesis to read. It is available on the 5th Floor of the Library at Call Number: Z 9999.5 B56 D64 2007