Adult stem cells have fundamental importance in replenishment, regeneration and/or growth of animal bodies during post-embryonic stages. We can find such cells in our intestines, skin, bone marrow and in the male testes. Adult neural stem cells are scarce in humans (and mammals in general), but other vertebrates - birds, reptiles, amphibians and fish - contain several populations of continuously proliferating cells in their brain and eyes, and these can be used as models to study post-embryonic neurogenesis. Indeed, neurogenesis has been studied in the adults of several teleost fish species and previous research has described multiple neurogenic proliferating cell populations in the anterior central nervous system (CNS) and eyes.
Complementing our studies of eye induction, morphogenesis and differentiation (see eye development research pages), we are also studying cell behaviour in the retinal stem cell niche. In the zebrafish eye, all the stages of progression from stem cell to differentiated neuron are found near the margin of the eye in a region termed the ciliary marginal zone (CMZ). These stem cell niches are found in all non-mammalian vertebrates and contain perpetually self-renewing proliferative neuroepithelial cells that are spatially ordered with respect to cellular development and differentiation. The youngest and least determined cells are most peripheral, proliferative retinoblasts are located in the middle, and the quiescent, differentiating cells are most central (see Fig 1).
Progression of differentiation in the CMZ. Frontal transverse sections of 3 dpf zebrafish eyes either stained with b-catenin antiserum to highlight all cell membranes (A) or subjected to in situ hybridization with markers for undifferentiated stem retinal stem cells (B), proliferating progenitors (C), differentiating neuroblasts (D-E).
We are addressing questions of how cells in the CMZ exit the cell cycle, differentiate into specific types of retinal neurons, and integrate into the retino-tectal system to enable visual perception. To this end, we are examining the contribution of specific signalling pathways to the formation and maintenance of the CMZ by employing a variety of techniques including 4-D imaging of live zebrafish embryos, cell transplantation, and focal electroporation.
As the eyes grow through life, so does their central target, the midbrain optic tectum (OT), and so stem cells must also exist in this structure. In support of this, previous BrdU and [3H]thymidine incorporation experiments in zebrafish (Danio rerio), medaka (Oryzias latipes) and goldfish (Carassius auratus) have shown the presence of proliferating cells in the lateral, medial and caudal margins of the OT (Fig 2)
Proliferative cells are present in the lateral, medial and caudal margins of the OT, as marked by BrdU uptake (A), and the expression of the cell cycle marker ccnD1 (B). (A: dorsal view; B: cross section)
The OT is one of the most important processing centers of sensory (mainly visual) information in teleost fish. Its size and complexity depends on the behaviour and ecological niche of the. During embryonic development the OT develops from the simple neuroepithelium of the mesencephalic alar plate into a complex, multilayered structure (Fig 3), receiving and integrating inputs not only from the eyes, but also from the lateral line, telencephalon and the tori. In a typical adult teleost tectum, such as that in the goldfish, at least 11 to 15 morphologically different cell types can be distinguished. As the teleost OT grows during the whole lifetime of the fish, these different cell types have to be continuously generated, making he tectum an ideal model to study the biology of neural stem cells.
The OT develops from the neuroepithelium of the mesencephalic alar plate (green) (A) into a complex, multilayered organ (B) which contains several morphologically distinct neuronal populations. Using nuclear stains (blue) and antibodies against acetylated-tubulin (red) and GFP (green), the main layers of the adult tectum were visualized in a transgenic fish line (C). (SM = stratum marginale, SO = startum opticum, SGC = stratum grisea centrale, SPV = stratum periventriculare).
Despite decades of intensive research into the development of retinotectal axons and the formation of topographic retinotectal maps, our understanding of the molecular cues that drive the embryonic and post-embryonic development of the tectum is scant. Previous evidence suggests that ingrowing retinal fibers can have an effect on the proliferation of precursor cells in the tectum, but we have no information about the molecules that relay this effect. Similarly our knowledge is very limited about the signaling mechanisms that ensure the development of the 12+ cell types from a single pluripotent progenitor in the OT.
Image of a section through the tectum of a two day fish showing expression (green) of cells that have active Wnt signalling. Those on either side of the midline at the top of the picture are either in or adjacent to the stem cell niche while those lower down in the picture are subsets of neurons.
For all these reasons, we have started to study the development of the zebrafish optic tectum, trying to unswer some key questions: how can complicated, multilayered structures arise from a small set of pluripotent cells? What is the identity of the neural stem cells and what signalling pathways regulate their maintenance? We are using a combination of forward and reverse genetics, and sophisticated high-resolution imaging to answer these questions.