Characterisation of FIsh NEural STem cells in the midbrain – FINEST

We have identified the fish caudal midbrain as a very original cellular system to study NSCs in physiological conditions or during regeneration. A population of stem cells is indeed present there, forming the posterior midbrain layer (PML). It is juxtaposed to the proliferation zone of the edge of the optic tectum (OT). The OT indeed grows by a process of addition of columns of cells at its periphery. This growth is associated with a spatially oriented organization of the OT germinal zones, homologous to the telencephalic cortical hem.
FINEST should obviously lead us to move up at the technological level, thanks to the collaboration of Jean-Stéphane Joly (P1), whose primary knowledge is neuroanatomy, cis-regulation and regeneration, with Laure Bally-Cuif (P2) having skills in conditional expression of transgenes in fish and Nadine Peyrieras (P3), a leader in live imaging. We obtained very original results first because zebrafish offers many unique advantages and tools; first, it combines genetic approaches with imaging: the transparency of fish will indeed allow imaging of stem cells in embryos, at later stages and possibly in adults. Second, fish nervous system present the exceptional ability to regenerate. Using these models, cell types, mechanisms and pathways acting in brain repair can be studied. Third, the population of PML stem cells are spatially segregated and relatively big.

FINEST project brought the opportunity to unravel anatomical and molecular characteristics of neural stem cells biology.

FINEST should have a strong impact in biomedical research aiming at the efficient manipulation of neural stem cells in the human diseased or ageing brain.

Recher G, Jouralet J, Brombin A, Heuzé A, Mugniery E, Hermel JM, Desnoulez S, Savy T, Herbomel P, Bourrat F, Peyriéras N, Jamen F, Joly JS.
Zebrafish midbrain slow-amplifying progenitors exhibit high levels of transcripts for nucleotide and ribosome biogenesis.
Development. 2013 Dec;140(24):4860-9.

There are fundamental questions in the field of neural stem cells (NSCs) biology: how do NSCs keep their multipotent and semi-quiescent state? How NSCs are recruited in healthy or pathological conditions? To tackle these questions, we propose to use fish models, whose brains have the peculiarity to grow continuously during whole adult life. We have identified the fish caudal midbrain as a very original cellular system to study NSCs in physiological conditions or during regeneration. A population of stem cells is indeed present there, forming the posterior midbrain layer (PML). It is juxtaposed to the proliferation zone of the edge of the optic tectum (OT). The OT indeed grows by a process of addition of columns of cells at its periphery. This growth is associated with a spatially oriented organization of the OT germinal zones, homologous to the telencephalic cortical hem.

In FINEST, our first aim is to analyze this region at high resolution using TEM and correlative microscopy. We will also perform a transcriptomic approach using laser microdissection of the PML and by subsequent deep sequencing of the transcripts. Our second aim is to further document the functions of genes selected on the basis of their preferential expression in the PML. From a list of 32 such genes (unpublished data), we have selected for further study several genes essential for ribosome biogenesis and for the control of cell proliferation. Our third aim is to use the regulatory sequences which drive expression of the her5 gene in a subset of the PML cells
a) to express reporter genes in the PML for 3D+time imaging and b) to induce permanent expression of reporter genes in this structure, in order to study long-term NSC derivatives. We will also isolate new regulatory sequences specific of the whole PML. Finally, our fourth aim is to study the short- and long-term consequences of PML ablations and their potential effect at stimulating regeneration from adjacent regions. To this aim, the activity of the toxic nitroreductase protein will be conditionally triggered in the PML at different time points.

This project contains five WPs. WP1 (ultrastructure and transcriptomics), WP3 (isolation of active regulatory sequences) and WP4 (imaging) provide phenotyping tools and concepts for the functional analysis of genes (WP2), for lineage analysis (WP4) and for the study of degenerative and regenerative events (WP5).

FINEST should obviously lead us to move up at the technological level, thanks to the collaboration of Jean-Stéphane Joly ( P1), whose primary knowledge is neuroanatomy, cis-regulation and regeneration, with Laure Bally-Cuif (P2 ) having skills in conditional expression of transgenes in fish and Nadine Peyrieras (P3 ), a leader in live imaging and localtechnological platforms are also involved. Hence, although this project is ambitious, technical locks have been identified and preliminary experiments were performed in all cases. This ensures the success of the proposed experiments.

We expect very original results first because zebrafish offers many unique advantages and tools; first, it combines genetic approaches with imaging: the transparency of fish will indeed allow imaging of stem cells in embryos, at later stages and possibly in adults. Second, fish nervous system present the exceptional ability to regenerate. Using these models, cell types, mechanisms and pathways acting in brain repair can be studied. Third, the population of PML stem cells are spatially segregated and relatively big.

FINEST project should bring the opportunity to unravel anatomical and molecular characteristics of neural stem cells. Thus, FINEST should have a strong impact in biomedical research aiming at the efficient manipulation of NSCs in the human diseased or ageing brain.