Clonal Analysis Of Mammalian Neocortical Gliogenesis

Neurons and glia are the fundamental building blocks of the neocortex.Radial glial progenitors(RGPs)are responsible for producing the vast majority of neurons and glia in the neocortex.While RGP behavior and progressive generation of neocortical neurons have been delineated,the exact process of neocortical gliogenesis remains elusive.By performing systematic mosaic analysis with double markers(MADM),we delineated the precise progenitor behavior and cellular program of gliogenesis at single-cell resolution in the mouse neocortex.We found that RGPs undergo the transition from neurogenesis into gliogenesis progressively,with a peak at E16.Upon transition,individual RGPs proceed to generate astrocytes,oligodendrocytes,or both in the proportion of ?60%:15%:25%,respectively,leading to the existence of three types of gliogenic clones.Clonally related astrocytes or oligodendrocytes form discrete,unmixed local clusters,consistent with the existence of fate-restricted intermediate astrocyte or oligodendrocyte precursor cells originating from individual gliogenic RGPs.While the generation of fate-restricted intermediate precursor cells appears to be stochastic,individual precursor cells display clear patterns in number and subtype of glia that they generate.We found that the output of individual intermediate precursor cells is largely of the same subtype and forms a local subcluster.As a consequence,astrocyte and oligodendrocyte generated by individual RGPs exhibits layer and subtype-specific regulation.To gain insight into the molecular regulation of neocortical gliogenesis,we took advantage of the MADM design to perform single cell level loss-of-function analysis of Neurofibromatosis type 1(NF1),a tumor suppressor protein that has been linked to brain tumor development.While glial cells and neurons share the same progenitor cell origin of RGPs,removal of NF1 in RGPs leads to a significant increase in gliogenesis,but does not affect neurogenesis.Furthermore,while astrocyte and oligodendrocyte generation are both enhanced upon NF1 removal,the extent of change is clearly different.The average number of astrocytes generated by a single gliogenic RGP increases by ?2-fold in the absence of NF1.In comparison,the average number of oligodendrocyte increases by >15-fold.Interestingly,NF1 loss leads to distinct changes in astrogenesis and oligogenesis by individual RGPs.For astrogenesis,NF1 loss causes an increase in astrocyte intermediate precursor cell generation,but not the output of individual astrocyte intermediate precursor cells.On the other hand,for oligogenesis,NF1 loss results in an increase in oligodendrocyte intermediate precursor cells generation as well as the output of individual oligodendrocyte intermediate precursor cells.The increase of OPCs is especially drastic.We observed individual Nf1 mutant clones with hundreds to thousands of immature,proliferative OPCs.Given that Nf1 mutation is linked to brain tumorigenesis,such as pediatric glioma,these results suggest that oligodendrocyte lineage is particularly susceptible to tumor formation.Together,these results delineate the precise RGP behavior in vivo and the cellular program of gliogenesis in the mammalian neocortex,and suggest the cellular and lineage origin of primary brain tumor.