Final Activity Report Summary - NEUROGENESIS CONTROL (Post-embryonic neurogenesis in the ciliary marginal zone of medaka.)
The eyes of fishes and amphibians grow during their entire life, mainly because new neurons are permanently generated in a peripheral region of the retina, known as ciliary marginal zone (CMZ), which hosts the retinal progenitor cells (RPCs). The work performed during the period covered by the Marie Curie fellowship was embodied in the general field of retinal progenitor cells (RPCs) and focussed on how the equilibrium between proliferation and differentiation was maintained in the CMZ.
The proposal was based on the characterisation of the bean-shaped eye mutant (bns), which displayed eyes that looked normal during the early development but failed to grow afterwards due to absence of proliferation in the CMZ. Using fast-degradable proteins, we observed that neurogenesis was retarded in mutants since embryonic day four, when RPCs took more time to exit the CMZ. Later on the CMZ stopped proliferating, as revealed by the lack of incorporation of BrdU and the absence of phosorilated-histone3, which were both markers of proliferating cells. Bns was found to be a member of the integrin family of proteins, molecules known to be involved in both attachment of cells to the extracellular matrix and regulating classical signalling pathways. By performing transplantation experiments we observed that, in the presence of both wild type and bns cells, wild-type cells could proliferate much faster that the mutant cell in the retina but not in other tissues. In fact, wild type cells could populate virtually the entire mutant retina and rescue the CMZ functionality. On the contrary, they could not rescue the size of the eye, revealing some non-cell specific role for integrins which we were investigating by the time of the project completion.
In addition, we created a medaka transgenic line expressing green fluorescent protein (GFP) ubiquitously and permanently. This line was of particular interest in lineage studies. By transplanting these cells in wild type eyes, we could now follow the lineage of cells in the CMZ during the entire life, and initial experiments favoured a common progenitor for multiple cell types in the adult retina. We complemented these observations with new ways to label CMZ cells by using inducible tools that allowed not only for cells labelling but also for the control of gene expression.
The proposal was based on the characterisation of the bean-shaped eye mutant (bns), which displayed eyes that looked normal during the early development but failed to grow afterwards due to absence of proliferation in the CMZ. Using fast-degradable proteins, we observed that neurogenesis was retarded in mutants since embryonic day four, when RPCs took more time to exit the CMZ. Later on the CMZ stopped proliferating, as revealed by the lack of incorporation of BrdU and the absence of phosorilated-histone3, which were both markers of proliferating cells. Bns was found to be a member of the integrin family of proteins, molecules known to be involved in both attachment of cells to the extracellular matrix and regulating classical signalling pathways. By performing transplantation experiments we observed that, in the presence of both wild type and bns cells, wild-type cells could proliferate much faster that the mutant cell in the retina but not in other tissues. In fact, wild type cells could populate virtually the entire mutant retina and rescue the CMZ functionality. On the contrary, they could not rescue the size of the eye, revealing some non-cell specific role for integrins which we were investigating by the time of the project completion.
In addition, we created a medaka transgenic line expressing green fluorescent protein (GFP) ubiquitously and permanently. This line was of particular interest in lineage studies. By transplanting these cells in wild type eyes, we could now follow the lineage of cells in the CMZ during the entire life, and initial experiments favoured a common progenitor for multiple cell types in the adult retina. We complemented these observations with new ways to label CMZ cells by using inducible tools that allowed not only for cells labelling but also for the control of gene expression.