Innovating genetic technology to map the origin of the precerebellar system.

摘要

The precerebellar afferent system, located in the ventral brainstem, plays an essential role in coordinating motor activity by providing the principal input to the cerebellum. Precerebellar afferents are comprised of two fiber subsystems: mossy fibers synapsing on cerebellar granule cells and climbing fibers contacting cerebellar Purkinje cells. Although indirect evidence has suggested that dorsal neuroepithelium contains precursors fated to become ventral precerebellar neurons, direct evidence for the long-distance origin of all precerebellar afferent neurons has been lacking. To test the hypothesis that precerebellar afferent neurons in mice indeed originate from dorsal neuroepithelium, we have developed and applied a novel site-specific recombinase (FLP)-based approach that switches on a reporter gene in targeted progenitor cells to irreversibly mark descendant cells in the adult nervous system. Using this approach, I have directly localized the origin of precerebellar afferent mossy fiber neurons to dorsal neural progenitors that transiently express the signaling molecule Wnt1. These results demonstrate, for the first time, that this novel FLP-based system can create a permanent map of Wnt1 descendant cells in the mature nervous system. To further enhance the capabilities of this system, we have characterized a new "enhanced" version of FLP recombinase called FLPe. Our in vivo studies have shown that FLPe has ten-fold improved activity as compared to previously available versions of FLP (FLP-L) and can achieve maximum target gene excision in both somatic and germ cells, demonstrating that FLPe is highly effective in mice. By comparing fate maps generated using FLP-L versus FLPe, we found that although morphologically similar, the cells of the precerebellar primordium can be distinguished molecularly: precursor populations that give rise to mossy and climbing fiber sub-systems appear to have intrinsic differences in the regulatory machinery that controls Wnt1 expression. This result suggests that these precursor populations may be specified much earlier than previously appreciated. Indeed, the potential differences in the regulatory machinery that controls Wnt1 expression in these two precursor populations suggests that this may be an instance where early, intrinsic cues play a role in determining cell fate.