Periodic Reporting for period 1 - NEURORIGINS (Role of cell lineage in the generation of neuronal diversity in the mouse cerebral cortex)
Okres sprawozdawczy: 2020-06-01 do 2022-05-31
The nervous system is one of the more diverse in terms of cellular types. In the brain, this diversity includes glial and neuronal types spanning all the functional areas. For instance, hundreds of different neuronal types populate the different layers in the cerebral cortex, which is the brain region that controls the higher cognitive functions such as language or abstract thought. Understanding how different neuronal types originate in this area is probably one of the more interesting questions in neurodevelomental biology. Multiple diseases such as autism or intellectual disability arise as a consequence of errors in the generation of cell diversity in the cerebral cortex.
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 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.
NEURORIGINS started with the generation of a library of genetic enhancers that, based on previous evidence, are active in the cerebral cortex during embryonic stages. Previous work in transgenic lines encoding a reporter under the regulation of different enhancers suggested these enhancers might be expressed only in subsets of neural progenitors, which makes them very interesting candidates to mark different progenitor subpopulations. The library of genetic enhancers was tested by in utero electroporation (IUE) of the brain in E13 and E14 mouse embryos. In these experiments, the activity of the enhancers was much broader that expected, with most of enhancers being expressed in most neural progenitors in the cerebral cortex.
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.
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.
NEURORIGINS has proved that the IUE approach is not adequate for testing the activity of enhancers in the brain. This contradicts previous evidence that overlooked the enormous variability of this approach to examine enhancer activity. This project has created a new framework to screen for transcriptional enhancers that are integrated in a specific genomic locus. This allows the characterization of enhancer activity in a fixed context that does not vary across different experiments. For that, NEUROGENESIS has generated a new mouse transgenic line that, combined with a simple and innovative technique of transgenesis (iGONAD) allows for the simultaneous characterization of multiple enhancers in a single experiment. This approach is extremely simple and efficient, suggesting the community will rapidly adapt in once published. Although the unforeseeable pandemic has resulted in delays, the forthcoming publication of these results will allow other scientist to have access to this technology. Beyond the research community, the results obtained in NEUROGENESIS will allow to study a number of diseases in which the activity of specific enhancers is compromised (enhanceropathies).
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.
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.