MRC Centre for Developmental Neurobiology

Identification of molecules and genes that regulate the survival and direction of axonal growth during development and regeneration.

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Pattern formation in the vertebrate central nervous system.

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Our group is interested in the mechanisms and molecules that direct the formation of the vertebrate head. This is a complex issue, as it involves multiple interactions between a number of disparate embryonic cell types, and our long term aim is to characterise these interactions and define how they are co-ordinated to produce the adult head - to ascertain how the nerves, muscles and skeletal components of the head are laid down and organised into a functioning apparatus. This is an important area of research, which has ramifications outside the immediate field of developmental biology. There are numerous inherited conditions that result in malformations of the head, and thus our work will help in understanding the basis of many birth defects. Our work also informs us of the how the vertebrates evolved. The real difference between the vertebrates and our nearest relatives lie in the organisation of the head, and these difference are thought to have their basis in alterations to the programme that underlies the development of the head. Consequently, our work also feeds into our understanding of the evolutionary origin of the vertebrates. The development of the vertebrate head differs in a number of significant ways from the trunk, and one of the most glaring examples that has often been offered lies in the deployment of the neural crest. In the head, it is the neural crest cells that are the source of bone and cartilage, and furthermore, the cranial neural crest are thought to pattern the head. Our recent results however suggest that these views should be tempered. Recently, we have shown that trunk crest cells possess skeleotgenic capabilities. Thus neural crest from all axial levels can generate the full repetoire of crest derivatives, but during normal development the skeletogenic ability of the trunk crest is suppressed. However, this potential is noteworthy as it was likely realised in early vertebrates, which had extensive post-cranial exoskeletal coverings. Furthermore, we have also shown the development of the pharyngeal apparatus is a lot more consensual than was previously believed, and besides the important role played by the neural crest, it now apparent that the pharyngeal endoderm also plays a prominent part. Again, this is of evolutionary significance, as it is known that segmented pharyngeal endoderm preceded the neural crest during evolution. Indeed, a clear example of the integrative nature of the development of this region of the body can be found in our studies of the epibranchial placodes, which provide the sensory neurons that inervate the mouth and throat. We have shown that these placodes are induced to form by the pharyngeal endoderm, and then that the neurons, and their axons, produced by these structure are guided inwards by the neural crest to establish the sensory ganglia and their innervation of the brain.

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My research focus on one of the fundamental questions in developmental biology namely how cells are specified during development. Using Drosophila as a model organism I am particularly interested in the molecular mechanisms underlying patterning of neurons in the brain. During late embryonic and larval development the Drosophila brain neuropile divides into compartments composed of axonal and dendritic arbors. It has been shown that these compartments prefigure discrete functional units in the adult brain. For example the mushroom body and antennal lobe can be traced as a neuropile compartment from late embryogenesis.
Whereas the genetic components involved in specifying broad regional identity of the early embryonic brain are well understood, little is known about the molecular mechansimsm underlying patterning of the smaller neuronal comartments. Consequently there is a large gap in our understanding about how most of the adult brain is patterned.

The aim of my work is to elucidate the molecular players involved in specifying sub-populations of neurons that give rise to individual compartments. I use a novel promoter trap to screen for genes expressed in restricted populations of developing neurons [3]. This unique approach allows me to label cells and address the function of the “trapped” gene within those cells using loss- and gain-of-function approaches. In addition, the promoter trap allows me to address the function of the neurons themselves within the intact brain. This is a valubale tool since we still know little about the function of large areas of the Drsosophila brain. Thus, my promoter trap allows me to address the function of genes in patterning neuronal sub-populations and to identify the function of novel neuronal circuits.

Using the promoter trap approach in a pilot screen we have identified an insertion into the transcription factor Odd-skipped (Odd). Odd is expressed in a small number of cells in the developing brain and their projections form a novel circuit which is part of the Mushroom body, the learning and memory centre of the Drosophila brain [5]. In addition, preliminary results suggest that Odd may also function non-autonomously in patterning the embryonic brain.

Presently we are addressing the function of Odd in patterning the brain using gain and loss of function approaches. We are also trying to elucidate the function of the Odd expressing neuron in learning and memory. Finally we are also carrying out a large scale screen for genes expressed in subsets of neuronal cells in the larval and embryonic brain

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I am interested in the genetic network that regulates the development of the vertebrate forebrain. I am using gain- and loss-of-function approaches in chick and mouse embryos as well as in tissue culture to characterise the role of extracellular signals (such as Wnts and Sonic hedgehog) and their intracellular transducers in order to understand how they establish regional identity and how they regulate the balance between proliferation and neuronal differentiation. I am also interested in how boundaries are established between neighbouring cell populations once their regional identity has been set, such that cells from these different regions are prevented from intermingling.

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Our main focus is the development of the vertebrate forebrain, in particular, the  events leading to cell fate specification and organisation of the anterior neural plate into telencephalic and diencephalic territories and later neuronal circuits. We are exploring these events using genetics, molecular biology and cell biology. Although the zebrafish is our principal model organism, a set of experiments in other vertebrates will be undertaken to address evolutionary issues.

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Dendritic morphogenesis and the development of local networks in Drosophila.

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The vertebrate central nervous system (CNS) consists of thousands of distinct neuronal cells, each with unique molecular genetic profiles, organised into complex patterns of circuitry that prefigure appropriate functional output. A key goal of modern developmental neuroscience is to unravel the molecular logic that first establishes the diversity in neuronal cell type and subsequently dictates their development into integrated higher order networks. Recent years have seen considerable progress in establishing the initial framework that underpins the acquisition of neuronal identity. Several lines of evidence have shown that cells are specified in discrete steps within the neuroepithelium influenced by the anteroposterior (AP) and dorsoventral (DV) position of precursor cell. In general, in response to a range of diffusible biochemical cues a series of ‘high level’ patterning cues or ‘selector’ genes (e.g. homeodomain transcription factors) establish the first molecular co-ordinates that imprint progenitor cells with an outline fate. However, despite this being well-recognised and investigates paradigm, we still remain relatively ignorant of the repertoire of subsequent molecular ‘effectors’ then impart specific facets of neuronal behaviour and connectivity. The developing vertebrate hindbrain and the adjacent signalling centre provide the ideal accessible system in which to examine the molecular controls underpinning the emergence of neuronal diversity. Exploiting the relationship between early anatomical distinctions and key selector gene activities of these territories we have generated fully representative and validated genome-wide expression libraries for each division of the hindbrain and the caudal portion of the midbrain. We use this data to investigate how neuronal specificity is achieved by the recruitment of effector genes and address the following questions;

• What and how are the developmental genetic factors used in response the patterning influences of the mid-hindbrain signalling centre?
• How do Hox genes drive downstream networks of effector gene activity?
• Can we use this information to drive the differentiation of embryonic-derived stem cells towards specific fates?

A more complete understanding of the molecular logic of neuronal specification and how patterning instructions are relayed to a defined set of cellular cues is a prerequisite to expanding our knowledge of nervous system function in health and dysfunction. Furthermore, the genetic networks and effectors uncovered here will broadly impact on the more fundamental question of how a cell becomes ultimately specified during development and offers insights into the genetic basis of disease

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My interests are in the development of algorithms for microarray and high-throughput sequencing data analysis, and their software implementation. In particular, I am interested in developing Bayesian statistical methods to create consistent error models for microarray data, which will facilitate the integration of those data with other experimental techniques. I am also interested in the conservation of transcription regulation networks throughout evolution.

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The molecular basis of neural induction and anterior patterning during vertebrate embryonic development.

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My research is based on the need to understand the genetic mechanisms that control the establishment of the circuitry of the nervous system and for insights into the normal and pathological function of proteins that contribute to neurodegenerative disease. Through a better knowledge of how neural development normally occurs and how neurodegenerative pathologies develop we hope to provide information to aid neural regrowth and combat neural disease and injury. During development, growing axons are guided to their appropriate targets by extracellular cues, the response to which depends crucially on the repertoire of receptors expressed by each axon and the intracellular mechanisms that transduce and regulate the response to these cues. In the past ten-fifteen years this research area has been significantly advanced by the identification of several major families of guidance cues and their cognate receptors and the elucidation of many aspects of the molecular mechanisms of axon guidance. My group has played a significant role in these discoveries, notably we contributed to the original discovery of the highly conserved Roundabout family of receptors and have revealed important aspects of their regulation. However, globally we still have only a rudimentary understanding of how these axon guidance processes are coordinated to specify the precise wiring of billions of cells in thousands of tracts in the brain. To fully understand this process it will be necessary to identify additional genes involved in wiring, define their expression patterns over the course of development and elucidate their cellular functions and molecular interactions. My group has identified further molecules that may contribute to the development of the nervous system (Dolan et al., 2007; Araujo et al., 2005; Wakefield and Tear, 2006; Alsbury et al., unpub obs) and identified intracellular regulatory mechanisms (Gogel et al., 2007; van den Brink et al., unpub. obs) which are adding to the study of the molecular mechanisms that regulate the development of connectivity in the embryonic central nervous system (CNS) of Drosophila. In addition my research group has developed the use of Drosophila as a model system to identify the normal and pathological roles of proteins associated with neurodegenerative disease. We have built collaborations with groups at the Institute of Psychiatry to investigate elements of Alzheimer’s and Batten disease. The Alzheimer’s work has focused on the critical role played by Tau hyperphosphorylation in Alzheimer’s disease pathology where it has become clear that a small number of kinases are responsible for the majority of the phosphorylation sites on tau in AD brain. We have established Drosophila as a whole animal assay to investigate which of these are responsible for the generation of toxic forms of tau in vivo. This makes use of humanised Drosophila that express human tau and specific identified human kinases to create an AD-like pathology. Using this system we are beginning to discriminate the specific involvement of the different kinases alone or together in the creation of toxic forms of tau. Batten disease or the neuronal ceroid lipofuscinoses (NCLs) descibes a group of at least nine fatal monogenetic neurodegenerative disorders that primarily affect infants and children. The genes mutated in several forms of the disorder have been identified recently, but very little is known about the precise roles of these gene products in normal neuronal tissue and how their mutation contributes to the disease. We have begun to combat this by investigating the role of the transmembrane protein Cln3, which is affected in the most common form of NCL, using Drosophila. We have identified that the Drosophila Cln3 shares many properties with the vertebrate form, it is localised to the endosomal-lysosomal compartment in many cell type and found at the synapse. By manipulating Cln3 function in Drosophila we have identified that it interacts with Notch and JNK signalling (Tuxworth et al.,2009) and increases in its activity interferes with the normal function and development of the neuromuscular junction (Tuxworth et al unpub obs). We are also near completion of a large scale screen to identify genes that modify Cln3 activity and this has provided us with a significant amount of information on the possible molecular pathways that are affected by changes in Cln3 activity.

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Circuit development in the mammalian visual pathway.

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All land vertebrates, including humans, use lungs to breathe air. The inspiratory and expiratory movements of the lungs are driven by a complex neural circuitry that consists of a central network in the brainstem that generates breathing rhythms and an output layer of motor neurons (MNs) which connect to respiratory muscles. These respiratory circuits develop prenatally and have to become functional immediately after birth. While significant progress has been made in understanding the central pattern generator itself, very little is known about the formation of neural circuits that turn breathing rhythms into coordinated motor output. Understanding the genetic program of respiratory MN differentiation is desirable for two reasons: First, respiratory MNs evolved after the basic vertebrate body plan was established and became more diverse during the evolution from ancestral tetrapods to mammals. Hence, understanding the logic of their genetic program might shed light on how neuronal developmental programs diversify, as organisms grow more complex. Second, in humans, loss of respiratory motor function is a leading cause of death in MN diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis. Reconstitution of respiratory muscle innervation through cell replacement therapy is therefore of considerable medical interest and will require the generation of authentic respiratory MNs in vitro from embryonic stem cells or induced pluripotent cells. Elucidating how respiratory MN identity and connectivity is established during normal embryogenesis would greatly facilitate the development of such a therapeutic approach.

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Cell-cell signalling processes that direct embryonic brain development. We use a variety of approaches and experimental systems both in vivo and in vitro to understand how local signalling processes direct various crucial aspects of brain development. These include closure of the neural tube, giving regional identity to parts of the brain, controling growth and differentiation of neurons and directing cell movements and axon pathfinding.
We study these processes in a range of vertebrate model organisms: transgenic mice, chick embryos and zebrafish embryos, while also using explant or cell culture approaches where applicable.

Currently our work focuses on signalling by FGFs, BMPs and through the planar cell polarity pathway. Our work on FGFs is set in context in the following review, which will give a flavour of our research interests:

Mason. I. (2007) Initiation to end-point: multiple roles of fibroblast growth factors in neural development. Nature Reviews Neuroscience 8, 583-596. PMID: 17637802

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Morphogenesis of the central nervous system and neurogenesis in the zebrafish embryo. Particularly the role of polarized cell divisions in organizing tissue architecture and assigning fates during symmetric and asymmetrically fated divisions.

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Synaptic plasticity and development.

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The laboratory uses Drosophila as a model to understand the molecular mechanisms of human inherited neurodegenerative diseases and the functions of the corresponding Drosophila ortologue genes.

The main research line in the lab is concerned with Atrophins, a family of transcriptional co-factors with several additional roles and implicated in many cellular and developmental processes. Atrophin-1 in humans is responsible for the polyglutammine (PolyQ) disease Dentatorubropallydoluisian atrophy (DRPLA). We have recently performed a genome-wide microarry study to investigate how polyglutamine Atrophins lead to neurodegeneration.
We are now investigating the functional relevance for neurodegeneration of the detected transcriptional alterations in the fly.

Our specific interests at the moment are:

1. The deregulation of the autophagic catabolic pathway.
2. The importance of cell cycle stimulation in post-mitotic neurones
3. The role of glial cells in mediating toxicity for the nervous system.

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Our lab combines molecular genetic techniques and time-lapse microscopy in vivo to examine the process of synapse formation in the zebrafish visual system. The role of neuronal activity and cell-cell adhesion in synapse formation and stability are two areas of particular interest.

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Molecular mechanisms of axon extension and growth cone guidance.

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Neuroendocrine regulation of ageing and physiology.

Our lab interested in how neuroendocrine circuits process environmental information to influence ageing and other physiololgical processes.

We exploit the unique combination of powerful genomic/genetic tools, stereotyped cellular anatomy and automated high-throughput imaging techniques in C. elegans to study the ageing process. The short natural lifespan of C. elegans (~2 weeks) allows us to complete experiments in days or weeks that may require months to decades in other animals.

Neuroendocrine circuits that influence ageing and physiology

In C. elegans and mice, ageing is influenced by communication between signalling centres in the nervous system and other tissues, but the precise neuroendocrine circuits are not fully detailed. To detail these circuits, we seek to identify molecular signals that modify C. elegans lifespan and other aspects of physiology, and pinpoint the cells from which they act.

Interpreting environmental and nutritional cues

Environmental and nutritional cues affect ageing and other processes, but how these cues are interpreted by neuroendocrine circuits is poorly understood. We use high-throughput quantitative fluorescence microscopy to investigate how environmental and nutritional information is relayed and integrated through these circuits. Because the conserved insulin/insulin-like growth factor (IGF) signalling pathway influences many conserved physiological processes (including ageing) across animal phyla, we have developed tools to measure and manipulate insulin/IGF signaling in live C. elegans. We are now refining these tools and developing new ones.

Through this research, we expect to understand how neuroendocrine circuits process and integrate information from environmental and nutritional cues to affect ageing and other physiological processes. Because ageing is modulated by conserved hormonal pathways, we anticipate that insights from our studies will reveal biological mechanisms relevant to human ageing and age-related diseases such as Alzhemier’s and sarcopenia.

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Induction and patterning of the rhombic lip and its role in cerebellar development.

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Establishing the appropriate functional connectivity is a fundamental process during brain development. Cells at the origin and in the target area have to be correctly specified to ensure an accurate wiring between them. We are interested in the molecular control of processes such as axon guidance, cell differentiation and synaptogenesis, and are using the mouse and chick as model systems. Visual perception is not only dependent on the correct assembly of the different cell types found in the retina, but also on the correct information transfer to the brain. The retinal ganglion cells send out axons to form connections between the eye and different brain centres. Along their path, these axons have to make several crucial decisions which way to take and with which cells in the target area they form synapses. We study molecules that are involved in these decisions and try to analyse their exact roles. Research in our laboratory has identified several important genes encoding transmembrane proteins, but more recently we have also isolated microRNAs, a novel class of small non-coding RNA factors, controlling axon guidance at the midline. Our current research focuses on the investigation of these molecules during brain development.

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Molecular mechanisms involved in differentiation and axon guidance of cranial motor neurons.

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My main research focus is to understand the mechanisms and molecules involved in axon guidance and the formation of topographic maps in the nervous system. I have chosen as model systems the visual and olfactory systems, both of which exhibit topographic mapping of axon projections.

My first independent work was as a group leader at the Max-Planck-Institute for Developmental Biology, Tübingen (Germany). Here my first major achievement was to show a fundamental role for the EphA/ephrin-A family of axon guidance molecules in topographic mapping of the retinotectal projection. I purified and cloned the ephrin-A5 molecule (then termed RAGS), and characterised for the first time its gradient on the tectum and role in the topographic targeting of retinal axons (Cell 82, 359-370 (1995)). This study essentially founded a field, since it assigned for this first time a function to the EphA/ephrin-A family - the guidance of retinal ganglion cell (RGC) axons by a repellent mechanism

I then dissected in vitro the role of ephrin-A5 and the related molecule ephrin-A2 in organising the pattern of retinal axon projections (EMBO J.16,1258-1267 (1997)), and by showing that the EphA family is the predominant axon guidance family controlling this projection (Eur.J.Neurosci. 10, 1574-1588 (1998)). Since multiple Ephs and ephrins are expressed by both retinal axons and the target, it turned out to be challenging to unravel the mechanisms underlying connectivity. We went on to pinpoint the EphA receptors which mediated responses to the ephrin-As, especially for the guidance of nasal RGC axons (Mol.Cell. Neurosci.16, 365-375 (2000)). In addition, we discovered an unexpected function of ephrin-A molecules expressed on RGC axons, in desensitising nasal retinal axons to exogenous ephrin ligands in vitro (Neuron 22,731-742 (1999)) and in vivo (Dev. Biol. 216, 297-311(1999)). In a study which was initiated while in Tübingen, we were also able to show that ephrin-A5 induces the turning of retinal axons in vitro (Development 130,1635-1643(2003)), which uncovered the mechanism by which ephrin-A5 guides RGC axons.

During this time I also contributed to the finding that the transcription factor Engrailed is involved in controlling expression of ephrin-A genes via its patterning effects on the optic tectum (Curr. Biology 6, 1006 – 1014 (1996)), and that the EphA family plays an important role in the retinotectal projection in zebrafish (Development 124,655-664 (1997)). We have furthermore shown the expression of ephrin-A/EphA molecules after optic nerve lesion in adult mice, which is of potential relevance for regeneration-associated processes, as it indicates that in case of a (not yet possible) regeneration after optic nerve lesion, retinal axons would find appropriate guidance information in the target area to re-establish a functional map (Mech. Dev. 106,119-127 (2001)).

Since becoming a group leader at the MRC Centre in 2000, I have started to elucidate signalling pathways of Ephs/ephrins, showing their localisation to lipid rafts (J. Biol. Chem. 276, 6689-6694 (2001)) and demonstrating an important role of Src family kinases as downstream signalling molecules in EphA-mediated repulsion (J. Neurosci. 24, 6248-6257 (2004)). For some time it has been suspected that a gradient of another molecule(s) running contrary to that of the ephrin-As might control topographic branching of retinal axons on the tectum. Crucially, we have recently succeeded in demonstrating that this gradient consists of EphAs, acting as ligands and signalling via ephrin-As on retinal axons ('reverse signalling') to control branching in correct locations (Neuron 47, 57-69 (2005)). In vivo data derived from mice lacking EphA7 showed the presence of ectopic termination zones of retinal axons, consistent with a normal role of this molecule in inhibiting branch formation.

Recently our group has identified the TrkB receptor as a candidate ephrinA5 co-receptor. Thus there is a ligand (BDNF) -dependent interaction between TrkB and ephrinA5. Activation of ephrinA5 on retinal axons via application of EphA7-Fc (as a ligand) suppressed the BDNF-induced branching concentration-dependently, and locally in the stripe assay. An increase in ephrinA expression on retinal axons led to an increase in axon branching, an effect which could be diminished by RNAi-mediated knockdown of trkB. TrkB interacts with ephrinAs via its second, cysteine rich domain (CC2), which is necessary and sufficient for binding to ephrinA5 (J. Neuroscience 28, 12700 (2008)).

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