No description available.

The brain is our most complex organ, with an estimated 1011 neurons. With the spinal cord, it forms the central nervous system which controls our movements and our senses, holds our memories and creates our thoughts. Because of this, neurodegenerative disorders can be extremely distressing and a thorough understanding of how the nervous system develops is essential if progress is to be made in finding ways to treat them. Critically, this includes understanding how the nervous system forms, i.e., the nature of the signals that promote neural identity (neural induction) and determine correct positional information (patterning). The zebrafish (Danio rerio) has become established as a model for embryological studies due to ease of experimental manipulation. Taking advantage of this, the aims of this PhD were to contribute to unravelling the molecular mechanisms of neural induction and patterning, using a variety of embryological and molecular methods. In the first project, functional analyses of the eve1 gene identified a key factor for posterior neural development. Eve1 was found to be a critical posteriorising factor, with an additional role in posterior neural induction. An outstanding question in neural induction is the relative contribution to this process of two key developmentally important signalling pathways, Bmp and Fgf. In the second project, differential analyses of maternal versus zygotic Bmp and Fgf signalling revealed crucial maternal roles for these two pathways in neural development as neural and epidermal capacitators. The results further suggested that Fgf signalling may be the critical neural inducer. Finally, as a third project, a zebrafish ectodermal explant assay was developed using the organiser-deficient ichabod mutant. The aim was to develop a system to analyse how key molecules directly affect ectoderm and neural development, free of mesoderm and endoderm influences, as signalling from these layers can directly or indirectly influence neural development.

Vertebrates have evolved a unique way to sense their environment: placodallyderived sense organs. These sensory structures emerge from a crescent-shaped domain, the preplacodal domain, which surrounds the anterior neural plate and generates the paired sense organs as well as the cranial ganglia. For decades, embryologists have attempted to determine the tissue interactions required for induction of various placodal tissues. More recently, technological advances have allowed investigators to ask probing questions about the molecular nature of placodal development. In this dissertation I largely focus on development of the otic placode. I utilize loss-of-function techniques available in the zebrafish model system to demonstrate that two members of the fibroblast growth factors family of secreted ligands, Fgf3 and Fgf8, are redundantly required for otic placode induction. I go on to show that these factors are expressed in periotic tissues from the beginning of gastrulation. These findings are consistent with a model where Fgf3 and Fgf8 signal to preotic tissue to induce otic-specific gene expression. This model does not address other potential inducers in otic induction. A study using chick explant cultures suggests that a member of the Wnt family of secreted ligands also has a role in otic induction. I therefore test the relative roles of Wnt and Fgf in otic placode induction. The results demonstrate that Wnt functions primarily to correctly position the Fgf expression domain and that it is these Fgf factors which are directly received by future otic cells. Lastly, I examine the function of the muscle segment homeobox (msx) gene family expressed in the preplacodal domain. This study demonstrates that Msx proteins refine the boundary between the preplacodal domain and the neural plate. Further, msx genes function in the differentiation and survival of posterior placodal tissues (including the otic field), neural crest and dorsal neural cell types. Loss of Msx function results in precocious cell death and morphogenesis defects which may reflect perturbed BMP signaling.

La crête neurale (CN) est une structure transitoire apparaissant en bordure de la plaque neurale chez les embryons de vertébrés. Au cours du développement embryonnaire, les cellules de la CN prolifèrent, subissent une transition épithélio-mésenchymateuse, migrent et se différencient en de nombreux types cellulaires tels que des neurones et cellules gliales du système nerveux périphérique, des mélanocytes, des cellules musculaires lisses ou des élements du squelette cranio-facial. Afin de mieux comprendre les mécanismes moléculaires contrôlant la prolifération et la spécification des cellules de la CN, nous avons étudié le rôle de deux facteurs de transcription, Hairy2 et Stat3, via des expériences de perte et gain de fonction chez l’embryon de xénope. Le gène Hairy2 code pour un facteur de transcription bHLH-O répresseur. Il est exprimé précocement au niveau de la bordure de la plaque neurale incluant la CN présomptive. Nous avons montré que Hairy2 est requis pour la prolifération des cellules de la CN en aval de signaux FGFs et qu’il maintient les cellules dans un état indifférencié en réprimant l’expression précoce des gènes spécifiques de la CN. Hairy2 réprime aussi la transcription du gène Id3 codant pour un facteur HLH essentiel à la prolifération des cellules de la CN. Id3 affecte également Hairy2. Nous avons observé que la protéine Id3 interagit physiquement avec Hairy2 et bloque son activité, démontrant que les interactions entre Hairy2 et Id3 jouent un rôle important dans la prolifération et la spécification des cellules de la CN. Afin de comprendre le mode d’action de Hairy2 dans la CN, nous avons comparé les propriétés de la protéine Hairy2 sauvage à celle d’une version mutée de la protéine incapable de lier l’ADN. Nos résultats ont montré que Hairy2 fonctionne selon deux mécanismes distincts. La capacité de Hairy2 à promouvoir la survie et la maintenance des cellules progénitrices de la CN dans un état non spécifié et indifférencié est dépendante de sa liaison à l’ADN. A l’inverse, sa capacité à stimuler la prolifération cellulaire et l’expression des gènes spécifiques de la CN est indépendante de sa liaison à l’ADN mais nécessite l’activation du ligand du récepteur Notch, Delta1. De plus, nous avons également montré que la capacité de Hairy2 d’induire Delta1 dans la CN requiert Stat3. Le gène Stat3 code pour un facteur de transcription latent dans le cytoplasme pouvant être activé par de nombreux signaux extracellulaires. Nos résultats ont montré que Stat3 joue un rôle crucial dans la prolifération cellulaire et dans l’expression des gènes de la bordure de la plaque neurale et de la CN. Stat3 est phosphorylé directement par la voie de signalisation FGF via FGFR4 et est requis in vivo en aval de FGFR4. Nous avons aussi montré que Hairy2 et Id3 sont des régulateurs positifs et négatifs de l’activité de Stat3 qui facilite et inhibe la formation du complexe Stat3-FGFR4, respectivement. De plus, Stat3 contrôle la transcription des gènes Hairy2 et Id3 de manière dose dépendante. Nous avons observé que Hairy2 est activé à faible dose et Id3 à forte dose de Stat3, suggérant que Stat3 s’auto-régule de manière indirecte via l’activation d’une boucle de rétro-contrôle positive (Hairy2) et une négative (Id3). Stat3 régule également de manière dose dépendante la prolifération et la différenciation des cellules de la CN. Une faible activité de Stat3 stimule la prolifération cellulaire et l’expression des gènes spécifiques de la CN tandis qu’une forte activité de Stat3 ralentit le cycle cellulaire, inhibe l’expression des gènes de la CN et maintient les cellules de l’ectoderme dans un état non spécifié et indifférencié. En conclusion, nous montrons pour la première fois que Stat3, en aval des FGFs et sous le rétro-contrôle de Hairy2 et Id3, joue un rôle essentiel dans la coordination de la progression du cycle cellulaire et de la spécification de la CN au cours du développement embryonnaire du xénope.

No description available.

The inner ear is a complex sensory organ of interconnected chambers, each with a sensory epithelium comprised of hair cells and support cells for detection of sound and motion. This dissertation focuses on the development and regulation of sensory epithelia in zebrafish and utilizes loss of function, gain of function and laser ablation techniques. Hair cells and support cells develop from an equivalence group specified by proneural genes encoding bHLH transcription factors. The vertebrate Atoh1 bHLH transciption factor is a potential candidate for this role. However, data in mouse has led some researchers to conclude it does not have a proneural activity, but, rather, is involved in later stages of hair cell differentiation. In addition, the factors regulating Atoh1 are mostly unknown. We address these issues in zebrafish and show that the zebrafish homologs atoh1a and atoh1b are required during two developmental phases, first in the preotic placode and later in the otic vesicle. They interact with the Notch pathway and are necessary and sufficient for specification of sensory epithelia. Our data confirm atoh1 genes have proneural function. We also go on to show Atoh1 works in a complex network of factors, Pax2/5/8, Sox2, Fgf and Notch. Misexpression of atoh1 alters axial patterning and leads to expanded sensory epithelia, which is enhanced by misexpression of either fgf8 or sox2. Lastly, we examine the role of sox2 in sensory epithelia development and regeneration. Sox2 has been implicated in maintainence of pluripotent stem cells as well as cell differentiation. In the inner ear, Sox2 is initially expressed in the prosensory domain and is required for its formation. Eventually, Sox2 is downregulated in hair cells and maintained in support cells; however, its later role has not been determined. We show that in the zebrafish inner ear, sox2 is expressed after sensory epithelium development has begun and, like in mouse, expression is down regulated in hair cells and maintained in support cells. Our data demonstrate a role for sox2 in maintenance of hair cells and in transdifferentation of support cells into hair cells after laser ablation. Additionally, sox2 is regulated by Aoth1a/1b, Fgf, and Notch.

The vertebrate inner ear is a complex structure responsible for hearing and balance. The inner ear houses sensory epithelia composed of mechanosensory hair cells and non-sensory support cells. Hair cells synapse with neurons of the VIIIth cranial ganglion, the statoacoustic ganglion (SAG), and transmit sensory information to the hindbrain. This dissertation focuses on the development and regulation of both sensory and neuronal cell populations. The sensory epithelium is established by the basic helixloop- helix transcription factor Atoh1. Misexpression of atoh1a in zebrafish results in induction of ectopic sensory epithelia albeit in limited regions of the inner ear. We show that sensory competence of the inner ear can be enhanced by co-activation of fgf8/3 or sox2, genes that normally act in concert with atoh1a. The developing sensory epithelia express several factors that regulate differentiation and maintenance of hair cells. We show that pax5 is differentially expressed in the anterior utricular macula (sensory epithelium). Knockdown of pax5 function results in utricular hair cell death and subsequent loss of vestibular (balance) but not auditory (hearing) defects. SAG neurons are formed normally in these embryos but show disorganized dendrites in the utricle following loss of hair cells. Lastly, we examine the development of SAG. SAG precursors (neuroblasts) are formed in the floor of the ear by another basic helix-loophelix transcription factor neurogenin1 (neurog1). We show that Fgf emanating from the utricular macula specifies neuroblasts, that later delaminate from the otic floor and undergo a phase of proliferation. Neuroblasts then differentiate into bipolar neurons that extend processes to hair cells and targets in the hindbrain. We show evidence that differentiating neurons express fgf5 and regulate further development of the SAG. As more differentiated neurons accumulate, increasing level of Fgf terminates the phase of neuroblast specification. Later on, elevated Fgf stabilizes the transit-amplifying phase and inhibits terminal differentiation. Thus, Fgf signaling regulates SAG development at various stages to ensure that proper number of neurons is generated.

Vertebrates have evolved a unique way to sense their environment: placodallyderived sense organs. These sensory structures emerge from a crescent-shaped domain, the preplacodal domain, which surrounds the anterior neural plate and generates the paired sense organs as well as the cranial ganglia. For decades, embryologists have attempted to determine the tissue interactions required for induction of various placodal tissues. More recently, technological advances have allowed investigators to ask probing questions about the molecular nature of placodal development. In this dissertation I largely focus on development of the otic placode. I utilize loss-of-function techniques available in the zebrafish model system to demonstrate that two members of the fibroblast growth factors family of secreted ligands, Fgf3 and Fgf8, are redundantly required for otic placode induction. I go on to show that these factors are expressed in periotic tissues from the beginning of gastrulation. These findings are consistent with a model where Fgf3 and Fgf8 signal to preotic tissue to induce otic-specific gene expression. This model does not address other potential inducers in otic induction. A study using chick explant cultures suggests that a member of the Wnt family of secreted ligands also has a role in otic induction. I therefore test the relative roles of Wnt and Fgf in otic placode induction. The results demonstrate that Wnt functions primarily to correctly position the Fgf expression domain and that it is these Fgf factors which are directly received by future otic cells. Lastly, I examine the function of the muscle segment homeobox (msx) gene family expressed in the preplacodal domain. This study demonstrates that Msx proteins refine the boundary between the preplacodal domain and the neural plate. Further, msx genes function in the differentiation and survival of posterior placodal tissues (including the otic field), neural crest and dorsal neural cell types. Loss of Msx function results in precocious cell death and morphogenesis defects which may reflect perturbed BMP signaling.

Perlecan is the major heparan sulfate proteoglycan (HSPG) in growth plate cartilage and is critical for growth plate chondrocyte proliferation and proper skeletal development. Its core protein and attached chondroitin sulfate (CS) and heparan sulfate (HS) chains mediate interactions with many diverse proteins. Fibroblast growth factor (FGF)-2 and FGF-18 are other regulators of chondrocyte proliferation in the growth plate. Additionally, FGF-18 controls the hypertrophy and cartilage vascularization necessary for endochondral ossification. The research presented in this dissertation aimed to identify known and novel perlecan-binding proteins that are endogenous to the growth plate and to characterize their interactions with perlecan. FGF-2 (known to bind HSPGs) bound to perlecan in both a cationic filtration (CAF) assay and an immunoprecipitation (IP) assay primarily via the HS chains on perlecan. When digested with chondroitinase ABC to remove its CS chains, perlecan augmented binding of FGF-2 to the FGFR-1 and FGFR-3 receptors and increased FGF-2 -stimulated proliferation in BaF3 cells expressing these FGF receptors. Thus, growth plate perlecan binds to FGF-2 by its HS chains but can only deliver FGF-2 to FGF receptors when its CS chains are removed. FGF-18 (known to bind to heparin and to heparan sulfate from some sources) bound to growth plate perlecan. This binding was unchanged by chondroitinase or heparitinase digestion of perlecan, indicating that perlecan GAGs are not involved in FGF-18 binding. FGF-18 bound equally to recombinant domains I-III of perlecan (Alt1) and to full-length perlecan purified from the growth plate. Additionally, FGF-18 bound equally to recombinant domain III of perlecan, to Alt1 and to Alt2 (a domain I-III variant with no heparan sulfate). Therefore, binding sites for FGF-18 are present in domain III of perlecan. Affinity chromatography isolated histone H3 as a perlecan-binding protein from the chondrocyte matrix. CAF assays confirmed the interaction as specific, dependent primarily on HS chains of perlecan, although CS chains and the perlecan core were also involved. Immunohistochemistry detected perlecan and histone H3 colocalized in growth plate cartilage. These results can help us better understand the growth factor-independent control that perlecan exerts on endochondral ossification and, therefore, long bone growth.

<p>Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the embyronal tumor neuroblastoma, where differentiation is a validated treatment strategy, remain unclear. The neuroblastoma tumor stroma is thought to suppress neuroblast growth via release of soluble differentiating factors. Here we identify critical components of the differentiating stroma secretome and describe preclinical testing of a novel therapeutic strategy based on their mechanism of action.</p><p>Expression of heparan sulfate proteoglycans (HSPGs), including T&#946;RIII, GPC1, GPC3, SDC3, and SDC4, is decreased in neuroblastoma, high in the stroma, and suppresses tumor growth. High expression of T&#946;RIII, GPC1, and SDC3 is associated with improved patient prognosis. HSPGs signal via heparan sulfate binding to FGFR1 and FGF2, which leads to phosphorylation of FGFR1 and Erk MAPK, and upregulation of the transcription factor inhibitor of DNA binding 1 (Id1). Surface expression and treatment with soluble HSPGs promotes neuroblast differentiation via this signaling complex. Expression of individual HSPGs positively correlates with Id1 expression in neuroblastoma patient samples and multivariate regression demonstrates that expression of HSPGs as a group positively correlates with Id1 expression, underscoring the clinical relevance of this pathway. HSPGs also enhance differentiation from FGF2 released by the stroma and FGF2 is identified as a potential serum prognostic biomarker in neuroblastoma patients. </p><p>The anticoagulant heparin has similar differentiating effects to HSPGs, decreasing neuroblast proliferation and reducing tumor growth while extending survival in an orthotopic xenograft model of neuroblastoma. Dissection of individual sulfation sites identifies 2-O-, 3-O-de-sulfated heparin (ODSH) as a differentiating agent that suppresses orthotopic xenograft growth and metastasis in two models while avoiding anticoagulation. These studies form the preclinical rationale for a multicenter clinical trial currently being proposed.</p><p>In conclusion, these studies translate mechanistic insights in neuroblast HSPG function to identify heparins as differentiating agents for clinical development in neuroblastoma, while demonstrating that tumor stroma biology can inform design of targeted molecular therapeutics.</p> / Dissertation