The brain is the most complex of organs and many of the mechanisms that control its development are poorly understood. We attempt to understand brain development by studying embryos of the relatively simple vertebrate the zebrafish. For us the advantages of the zebrafish embryo are that its transparency allows us to watch cell behaviour in the intact embryo and the relative ease with which we can test gene function.
One of our main experimental approaches is to use confocal microscopy to analyse cellular and subcellular details of the important events that help construct a brain. We are studying three fundamental processes in brain development: What mechanisms build and shape the brain and spinal cord? What mechanisms regulate neural progenitor cell divisions to balance brain growth and neural differentiation? What is the organization of neural circuits in the developing central nervous system?
Building and Shaping the Brain
Making a neural tube: Building the neural tube is an important early developmental event and has significant clinical relevance as defects in neural tube formation have a high frequency in human pregnancies. During formation of the zebrafish brain and spinal cord several important processes need to occur in a co-ordinated manner. Neural cells first form a solid rod primordium at the dorsal midline of the embryo. Cells within this rod then need to reorganize and build a lumen at the centre of the rod. To understand this process we need to resolve how cells are able to detect the centre of the rod and assemble the molecular machinery required for lumen formation at this location. We believe an unusual form of cell division plays an important role in cell reorganization and polarization (Tawk et al 2007) and we are now investigating the role that intracellular organelles and adjacent tissues may play in organizing this complex process.
Boundary zones: One of the distinctive features of the developing vertebrate brain is its division into specialized regions that are often separated from one another by morphologically distinct boundaries. This has been most thoroughly investigated in the hindbrain region where the process of compartition allows a highly conserved set of sub-regions to develop along independent paths of differentiation. Cells that lie at the boundaries between these units appear to be morphologically specialized and this morphological organization may be important for maintaining the strict separation of cells from adjacent units. We are investigating these boundary specializations and determining whether other regions of the brain share common principles.
The fish telencephalon: As well as investigating the generation of the neural tube in general, we are particularly interested in the morphogenesis of the forebrain and especially the telencephalon, which has a very unusual organisation in many fish compared to other vertebrates. The zebrafish telencephalon is said to be "everted" which means that the ventricular system and the proliferative cells that line the ventricular surface form a distinctive T-shape, rather than forming a pair of lateral ventricles within the two telencephalic lobes. Understanding the development of this organisation may be an important step towards understanding the functional specialisations of the zebrafish telencephalon and their relationship to functional units in other vertebrate brains
Growth and neurogenesis in the embryonic brain
Asymmetric cell division: The progenitor population of the developing central nervous system has to achieve two things: growth of the brain and spinal cord by expanding the number of cells and the generation of differentiated neurons and glia. One mechanism that can achieve both growth and differentiation during the same time period is asymmetric division. When a progenitor divides in this mode one daughter becomes a progenitor capable of further division while the other daughter becomes more committed and may differentiate into a neuron. How this asymmetry of daughter fates is achieved is not fully understood. In invertebrate brains, this process depends on the asymmetric inheritance of fate determinants during progenitor divisions.
A similar mechanism is widely believed to underlie asymmetrically fated divisions in vertebrates but compelling evidence for this is missing. We are using live imaging of individual progenitors in the intact zebrafish embryo CNS to test this hypothesis. We use a fluorescently tagged version of the protein Par3 to outline the apical (ventricular) surface of progenitors. By observing this domain through progenitor divisions we provide evidence that asymmetric inheritance of a subcellular domain is strongly correlated with asymmetric daughter fates. Furthermore we see an unexpected feature of this process.
Previous hypotheses had suggested that the daughter that inherits the apical domain is most likely to retain progenitor characteristics while the more basal daughter becomes the differentiated cell. However our time-lapse analyses shows that the cell destined to become a neuron is derived from the more apical of the two daughters, while the more basal daughter replenishes the apical progenitor pool (Alexandre et al 2010). These observations suggest a revision of the conventional view of asymmetric divisions in the vertebrate neural tube is required. We are continuing to use live imaging to understand how this process is regulated.
Neuroanatomy of the developing brain
Online Atlas project: We are collaborating with Steve Wilson's group at UCL to build an online, open-access, high-resolution atlas of the neuroanatomy of the developing zebrafish brain. The zebrafish embryo is small enough and sufficiently transparent that the entire intact brain can be imaged at high resolution by confocal microscopy. Coupled with the availability of many transgenic lines in which subsets of neurons are labelled by fluorescent reporters, this makes the zebrafish an excellent model for resolving the developmental neuroanatomy of the vertebrate CNS. Because the zebrafish is a small and relatively simple vertebrate, we hope to be able to identify most of the major neuronal subgroups in the developing zebrafish brain, their patterns of dendritic and axonal projections and their likely connections.
We aim to make this information available via an online, open-access atlas of the neuroanatomy of the developing zebrafish brain. The atlas will be populated with high resolution images of subsets of neurons revealed by analysis of transgenic lines. The atlas will be searchable by reference to neuroanatomical structure or region, neuronal sub-type, gene expression, neurotransmitter expression and in some cases gene/transgene expression.