Periodic Reporting for period 1 - NEURORIGINS (Role of cell lineage in the generation of neuronal diversity in the mouse cerebral cortex)
Período documentado: 2020-06-01 hasta 2022-05-31
The determinants of neuronal diversity are multiple but mostly unknown. Based on other models in which the generation of neural diversity has been studied in depth, a very heterogeneous pool of neural progenitor cells seems to exist and have a pivotal role on the generation of different neuronal types. However, this remains a matter of debate in the cerebral cortex, where we have not been able to prove this hypothesis. NEURORIGINS aims to identify and characterize different neural progenitors that give rise to diverse neurons, also characterizing how each of these progenitor types produces different neurons at different times.
The lack of specific patterns of activity for at least some of these enhancers suggested that IUE was a poor system to screen for enhancers with specific activity in subsets of neural progenitors. The fact that with the IUE, multiple plasmids enter the cell and remain as episomes, increases the number of copies of the same enhancer in each cell. This is probably the reason behind the broader patterns observed by IUE: while in transgenic lines there is only one copy inserted into the genomic DNA, the multiple copies of the same enhancer probably lowers the threshold for a cell to exhibit enhacer activity. In this way, those cells not exhibiting enhancer activity with one copy, may exhibit activity when containing multiple copies. For this reason, I decided to use the LiON system, in which the expression of a reporter only occurs when integrated into the genomic DNA via a Piggybac transposon. This eliminates the expression coming from episomes. To test the library of enhancers with this new system, I activated a contingency plan to generate again the same library of enhancers encoded in LiON plasmids.
After the generation of the library in LiON plasmids, I repeated the screening for activity of the enhancers in specific neural progenitors. This time I found that for all the enhancers tested, their activity was variable across different experiments. Because the insertion site of the Piggybac transposon is random, the same enhancer may exhibit a different activity depending on the locus of insertion. This led me to activate a second contingency plan, seeking to generate transgenic lines to test the specific expression of each enhancer in the locus R26. For that I implemented iGonad, a novel approach for transgenesis based on the electroporation of the oviduct of a pregnant female mouse. I used this method to generate embryos in which a first enhancer was controlling the expression of the reporter gene tdTomato, with the construct inserted in a specific location in R26 via CRISPR.
In addition, NEUROGENESIS has had a great impact in the career of the fellow. It has allowed him to secure a tenure track position in Spain as Cajal Fellow, the most prestigious position of its kind. This MSCA also helped him to be awarded with several start-up grants that allowed him to open his lab at the Cajal Institute - CSIC.