During embryonic development, all the various cell types present in the nervous system are generated along the length of the body axis from either the neural tube or neural crest. In contrast, in adults neurogenesis is generally restricted to special microenvironments, termed adult stem cell niches. Adult stem cells have fundamental importance in replenishment, growth and/or regeneration of animal bodies during post-embryonic stages. However, how these niches are maintained or regulated is not well understood.
In adult mammals, neurogenesis is restricted to the dentate gyrus in the hippocampus and to the olfactory bulb, which makes these areas a main focus of stem cell research. However, as animals age, these cells progressively lose the ability to generate new neurons, something that is relevant to neurodegenerative diseases in humans. In contrast, in anamniotes, such as fish and amphibians, there are stem cell populations along the entire rostrocaudal axis of the adult brain, with a robust and continuous proliferative capacity, 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 and eyes.
Complementing our studies of eye formation and differentiation we are also studying cell behaviour in a retinal stem cell niche. In the post-embryonic zebrafish eye, all the stages of progression from stem cell to differentiated neuron can be found near the margin of the eye, in a region termed the ciliary marginal zone (CMZ). In this niche, perpetually self-renewing proliferative neuroepithelial cells are spatially ordered in such way that the youngest and least determined cells are the most peripheral, proliferative retinoblasts are located in the middle, and differentiating cells are most central (see figure 1).
(A) Transverse section of a 3 day old zebrafish eye stained with anti Β-catenin to highlight cell membranes. Brackets indicate the CMZ. (B-D) In situ hybridization showing that cells at different points along the transition from stem cell/proliferation to differentiation in the CMZ, express specific markers. Undifferentiated retinal stem cells are in the peripheral most compartment (mz98-expressing), highly proliferative progenitors in the middle (ccnD1-expressing), and committed precursors and differentiating cells (ath5 and cdkn1c-expressing, respectively) are sequentially organized centrally from the CMZ. A diagram showing this dynamic is at the bottom.
We are interested in exploring the molecular mechanisms that govern neurogenesis in the CMZ and how specific intrinsic and extrinsic signals are integrated to carry out this task and allow the growth and function of the eye. To this end we are using forward and reverse genetics approaches, molecular biology techniques, and taking advantage of the powerful 4-D imaging of live zebrafish embryos.
As the CMZ fuels the production of new neurons and contributes to the lifelong growth of the eyes, so does the visual processing centre in the midbrain, the optic tectum. The tectum is a multiprocessing centre organizing visual, sensory and somatosensory inputs into ‘maps’ of the external world, such that appropriate behavioural outputs are generated. The stem cell niche in this structure is located along the caudo-medial edge, as studies in zebrafish and other teleosts have shown the presence of proliferating cells in the margins of the tectum that contribute to the formation of new neurons. As with the CMZ, the caudo-medial stem cell niche in the tectum is spatially ordered, with the youngest, least determined cells found at the peripheral edge, adjacent to the ventricle, and differentiating neurons more central (see figure 2).
A cartoon dorsal view of the zebrafish visual system, depicting areas of proliferation in the retina and optic tectum. Stem cells (yelllow) give rise to more committed progenitors (blue), which in turn give rise to differentiating neurons (purple). New neurons integrate into the existing functional circuitry, including into the retinal ganglion cell layer, where RGCs (pink), the sole output of the retina, grow axons along the optic nerve and terminate in the optic tectum.
In teleosts, the main input into the tectum comes from retinal ganglion cells (RGC), which represent the output neurons of the retina. RGC axons cross the midline at the level of the optic chiasm and terminate in a topographically appropriate site in the optic tectum. As the retina grows, an increasing number of RGCs reach the optic tectum and those already present increase the size of their dendritic arbors. This results in older fish having a greater visual resolution than those that are younger. However, due to the disparate mode of growth between the retina and optic tectum (the CMZ adds new cells circumferentially, whilst the optic tectum adds new cells in crescents) the RGC synapses need to ‘shift’ in order to maintain the topographic map.
As well as investigating the neurogenesis in the CMZ, we are also interested in the process of co-ordinated growth between the retina and optic tectum, which is currently poorly explored. We know that if retinal input into the tectum is abrogated, growth in the structure is reduced, however many questions remain to be addressed. For instance, how is this growth co-ordinated? and what are the molecules and signalling pathways underlying this process? We are using a combination of surgical manipulations, RNA sequencing, transgenesis and high-resolution imaging to answer these questions.
Proliferation along the caudo-medial edge of the optic tectum, as marked by EdU uptake at 14dpf (A), and the expression of cell cycle markers ccnD1 (B) and cmyc (C) at 9dpf. All shown as dorsal views.