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.

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.

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.

Ontogeny of putative GABAergic neurons and their receptors in the nervous system of the crayfish Cherax destructor

Inhibitory neurons exert control the expression of many aspects of behaviour by regulating the effectiveness of excitatory neural function. By comparison with excitatory neural systems, relatively little is known about the development of inhibitory neurons and the influence which these neurons exert on the development of other neural systems. Two issues which relate to the development of inhibitory neurons are of particular interest. First, a paradox arises when inhibitory neurons are considered in terms of modern models of synaptic development which involve activity-dependent mechanisms of synaptic plasticity. Second, there is some evidence that inhibitory neurotransmitters may act in a special trophic manner during the early development of nervous systems. Investigations of these issues would be greatly facilitated in a neural system in which it was possible to experimentally control aspects of the development of individual pre- and postsynaptic cells. The aim of the results presented in this thesis was to characterise the normal development of one such system: the GABAergic inhibitory system of the Australian freshwater crayfish, Cherax destructor. The ontogeny of the inhibitory neurotransmitter GABA across the embryonic period of 30% to 100% development was investigated using immunohistochemical techniques. GABA-like immunoreactive cells and fibres were first detected in the embryonic brain region. The expression of GABA-like immunoreactivity progressed along a rostro-caudal gradient, with GABA-like immunoreactive cells being detected in the most anterior thoracic ganglia at 45% development and in all ganglia by 65% development. GABA-like immunoreactive fibres were evident in peripheral nerves as early as 55% development and ramified extensively throughout the neuropil of the nervous system by 65% development. By contrast, immunoreactivity to the primary excitatory neurotransmitter, glutamate, was not detected until 60-65% development. Glutamate-like immunoreactivity at 60-65% development was evident only in the form of punctate staining in the midline of the ventral nerve cord. Cell body staining was observed only at 90% development and was restricted to only a few cells on the periphery of the ventral nerve cord. Radio-labelled ligand binding methods and autoradiography were used to study the expression of putative GABA receptors in the Cherax embryos from 30% to 100% development. Specific binding was evident in the earliest embryos studies at 30% development. There was an initial increase in binding from 30% to 40% development, followed by a dramatic drop to almost zero binding at 50-55% development. This was followed by a gradual increase in binding levels with age, reaching a plateau at 85% development. Preliminary pharmacological evaluation of binding indicated that at least three GABA receptor types were expressed during embryonic development. Methods for culturing, dissociated neural tissues explanted form Cherax embryos at 85% development were established. The success of cultures was demonstrated by neurite extension, and neuronal networks in which neurons appeared to form connections with other neurons and with explanted muscle cells after two days in culture. Immunohistochemical studies demonstrated that some explanted neurons expressed GABA-like immunoreactivity within two days of explanting. These studies have provided a comprehensive description of the development of GABAergic neurons and their receptors in Cherax destructor embryos. The very early expression of GABA-like immunoreactivity, coupled with the early onset of specific GABA binding, strongly indicates that the GABAergic neurons are functional and able to exert an effect on other cells during much of the period of nervous system development in crayfish embryos. These results support the hypothesis that inhibitory neurons may play an important role as regulators of the overall process of assembly and maturation of the nervous system and provide a substantial basis for future experimental studies in which the specific action of inhibitory neurons on the development of discrete components of the crayfish nervous system may be investigated.

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.

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.

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.

Effects of foods and drugs on the development and function of the nervous system : methods for predicting toxicity /

Symposium organized by the Office of Health Affairs, Food and Drug Administration.

Animal education; an experimental study on the psychical development of the white rat, correlated with the growth of its nervous system,

Mode of access: Internet.