Zebrafish Research at UCL

Because of its outstanding suitability for imaging and transgenesis approaches, the developing zebrafish becoming a leading vertebrate model for studies of brain circuitry, synaptic plasticity and behaviour. An essential prerequisite for studies of circuit connectivity and behaviour is a clear understanding of the neuroanatomy of the brain. However, there is a lack of detailed neuroanatomical information for this vertebrate model – this lack of knowledge presents a major bottleneck in the field. Although we have a reasonable understanding of the general principles of how neurons are generated and acquire identities, our knowledge of the circuitry subsequently established by the neurons is very fragmentary.

Furthermore, research to date has tended to focus on a small number of specific brain regions but even within relatively simple regions of the fish CNS, such as the spinal cord, there is still much to learn about the detailed neuroanatomy of the circuits that drive behaviour. If we do not know the normal connectivity of a class of neurons, we will not be able to address how the circuits they form are working.

Figure 1 - 4 day old zebrafish embryo labelled with SV2 and acetylated tubulin antibodies showing axon tracts(green) and neuropil(red)viewed from lateral(top) and dorsal(bottom) orientations.

Figure 2 - Triptych of a 5dpf zebrafish forebrain viewed from dorsal(anterior to the left), frontal and lateral(anterior to the right) aspects. The embryo has been labelled with SV2 and acetylated-tubulin antibodies showing axon tracts(blue) and neuropil(pink). In these images we can clearly see the different components of the zebrafish forebrain; the olfactory bulb is situated most anteriorly in front of the telencephalic lobes. We can also see the habenulae from dorsal and lateral view points in the dorsal diencephalon.

Despite the exceptional properties of the zebrafish embryo as a model system, neuroanatomical resources are scarce and most detailed neuroanatomical data has to be divined from histological atlases of larval and adult brains or from scanty information in primary research papers. Although paper atlases are undoubtedly an accurate and indispensable resource, they are not easy to use unless one is already expert in fish neuroanatomy. Traditional atlases standardly use annotated images from serially cut histological sections and so to fully understand the information presented, the user must already have a good understanding of the anatomical relationships and connectivity of the brain as this is difficult to interpret from 2D sections.

Figure 3 - A time-series of tubulin immunostaining through development from 36hpf to 5 day old larva. Note the initial simplicity of the network of tracts and commissures which rapidly increases in complexity through development. The network provides convenient and reproducible landmarks as points of reference for comparisons between specimens.

For the past several years, in collaboration with Jon Clarke's, lab in KCL, we have been developing an online, high-resolution atlas of the neuroanatomy of the developing zebrafish brain. We have named this resource zebrafishbrain.org

zebrafishbrain.org aims to be a user-friendly and highly flexible resource for presenting information about the neuroanatomy of the developing zebrafish brain.

To build the zebrafishbrain.org atlas, we have collated and curated the highest quality neuroanatomical data and information generated both in-house and by leading laboratories in the zebrafish neuroanatomy community. We employ these data to communicate the current state of knowledge about neuroanatomical structures in the developing zebrafish. Most of these data are from high-resolution confocal imaging of intact brains in which neuroanatomical structures are labelled by combinations of transgenes and antibodies. Our intention is to be intuitively accessible to all: not just those with expertise in neuroanatomy. This how we aim to complement the already available paper atlases: at zebrafishbrain.org we use a variety of media employed in creative and innovative ways to clearly explain the latest information on the neuroanatomy of the zebrafish brain to the end user.

Figure 4 - A-F Identification of early embryonic precursors of neuronal structures. A-C. High resolution confocal projections of Tg(tbr:YFP) transgenics at multiple stages. Continuous expression of GFP through development permits the tracing back of structures which can be morphologically delineated at later stages to place their early origins; eg. the olfactory bulb (OB) which originates in the dorsal early telencephalon (36hpf; A) but moves to the anterior pole of the brain by 4 days. (C) D-F. Tubulin immunohistochemistry (red) aids orientation of GFP expression in transgenics in this case Tg(tbr1:YFP) (Mione at al. 2008, Dev Neurosci;30:65-81)

Zebrafish Properties

Transgenic animals are an excellent resource for resolving neuroanatomy. Traditional neuroanatomical tracing techniques, such as lipophilic dye labelling of axons, suffer from the problem that it is hard and time consuming to restrict labelling to unique subsets of neurons. Genetic approaches circumvent this problem by enabling the cell-type specific expression of transgenes encoding proteins that label the entire morphologies of the expressing cells. This approach can be used to label entire classes of neurons or indeed individual neurons. The embryonic zebrafish has outstanding optical properties that permit imaging of the entire intact brain at high resolution by confocal microscopy. Transgenic techniques in the zebrafish are constantly evolving and becoming more sophisticated. In recent years there has been a shift towards using transactivation systems e.g. the Gal4/UAS system to drive transgene expression. This system offers fantastic flexibility as transgenic lines where Gal4 is expressed under the control of a certain promoter can then be outcrossed to any UAS line, or injected with any UAS construct. This system allows the same set of neurons to be visualised and also manipulated in multiple ways: for example, by outcrossing to UAS-brainbow each neuron in the entire expression pattern could be resolved and its connectivity established.

Other UAS transgenics continually activate Gal4 expression in a feedback loop allowing cells to be followed into adulthood (‘Kaloop fish’). This form of fate-mapping is very useful from a neuroanatomical perspective allowing us to observe how neuronal structures develop and migrate to their positions within the adult brain. It is also important from a comparative neuroanatomical perspective.

In concert, these techniques allow us to visualise different neuronal subgroups within the CNS and characterize their connectivity.

Figure 5 - A-C Other staining methods which can be used for orientation. A SV2 immunohistochemistry (red), here combined with tubulin (green) shows areas of neuropil that can act as reference points. B & C. Nuclear dyes (grey (B); blue (C)) show areas of somal coalescence (brain nuclei) and also expose morphological boundaries between brain areas (B). Neuropil is not marked and is revealed as dark spaces (B). Thus nuclear staining is complementary to neuropil markers like SV2 or tract tracing methods (red and green; C).

Future Perspectives

Aside from maintaining continuity of funding to support the project, one of our biggest practical challenges is the annotation and web-presentation of 3D models. Almost all of the data thus far employed to build zebrafishbrain.org exist as multi-layer stacks that have the potential to be rendered in 3D as interactive models allowing the user to see structures in any and all orientations. Some of these are currently shown as small movies but there is enormous potential for interactive presentation of these data on the web. This is an area we wish to explore through collaborative work with software developers.

Recent innovations concerning the warping and registration of confocal data of zebrafish brains onto standardized frameworks may offer mechanisms by which we can offer standardized protocols and data pipelines to permit comparisons between datasets. We intend to incorporate standardised anatomical frameworks and datasets into zebrafishbrain.org, perhaps serving the vital function of ensuring the various models are interoperable.

Figure 6 - A & B Distinct populations of neurons are GFP positive (green) in Tg(brn3a-hsp70:GFP)(Okomoto et al. 2008, Dev Growth Differ. 2008 Jun;50) (A & B)and Tg(1.4dlx5a-dlx6a:GFP)transgenics(Mione et al. 2008, Dev Neurosci ;30:65-81 (C) counterstained with anti-tubulin immunostaining (red). D Calretinin immunohistochemistry (green) of neurons at the fore/midbrain boundary

Figure 7 - GFP expression in the ET11 enhancer-trap transgenic line (Choo et al. BMC Dev Biol. 2006 Feb 14;6-5). The box highlights a population of neurons in the dorsomedial tectum some of which are GABAergic.

Figure 8 - Single cell labelling. A. Focal electroporation of GFP in a single habenular neuron; the entire morphology is revealed from the cell body on the dorsal surface of the brain to the ventral terminals in the interpeduncular nucleus (IPN). B. Exquisite detail of the morphology of a pair of habenular neurons. C. The somal and dendritic morphology of a large reticulospinal neuron revealed by anti-calretinin antibody. Images in A and B adapted from Bianco et al. 2008, Neural Developent 3, 8.

Figure 9 - An overview of a typical region tutorial from the database

Funding

Funding from the EU FP6 ZF-MODELS integrated project (with additional Wellcome Trust support to Steve Wilson) allowed us to build a beta-version of the atlas and generate tools and develop protocols. We gathered pilot data to populate the atlas from various transgenic lines.

In 2010 we secured responsive-mode funding from the BBSRC for further development of the resource. These works have included a re-engineering of the database to permit web-editing and the collection and imaging of more transgenic lines.

The project also receives support from the EU via the FP7 projects ZF-Health and NeuroXsys.