The degree of cellular heterogeneity in the rodent neocortical ventricular zone (VZ) has been a source of contention among developmental neurobiologists for decades. The widely-held hypothesis that this germinal compartment contains a single, multipotent population of neural precursor cells contrasts with what is known about the human and primate VZ, in which multiple neuron- or glia-restricted precursor populations reside side-by-side. Radial glial cells (RGCs), the supposed sole occupants of the rodent VZ, have been shown to generate excitatory neurons, astrocytes and oligodendrocytes using in vivo and in vitro techniques. However, these studies do not show that RGCs alone comprise the rodent VZ. Indeed, morphological and molecular differences have recently been identified between RGCs and another neural precursor population—short neural precursors (SNPs)—within the mouse VZ, though acceptance of precursor diversity in rodents has not been quick to follow. In order to more fully elucidate the nature and degree of heterogeneity among neural precursor cell types within the rodent VZ, experiments were designed to examine the cell cycle kinetics, neuronal output and lineage potentials of RGCs and SNPs.
In utero electroporation (IUE) was used to label VZ precursors based on expression of specific promoters: the glutamate-aspartate transporter (GLAST) and brain lipid binding protein (Blbp) promoters are expressed in RGCs and the tubulin α-1 (Tα1) promoter is expressed by SNPs. The Nestin promoter, thought to label all neural stem cells (NSCs), was used as a control. For acute experiments, here defined as ≤ 48 hr from IUE to sacrifice, green fluorescent protein (GFP) reporter plasmids driven by cell type-specific promoters (Tα1 or GLAST) were used. For long-term fate mapping experiments (≥ 72 hr from IUE to sacrifice), co-electroporation of a Cre plasmid, driven by a cell type-specific promoter (Tα1, GLAST, Blbp or Nestin), and a flox-stopped GFP reporter plasmid was used to mediate Cre/loxP recombination in specific precursor populations.
We found that GLAST+ RGCs and Tα1+ SNPs have significantly different cell cycle kinetics, with the length of G1-phase and the total cell cycle duration being 4 hr shorter in RGCs than in SNPs. Based on a study in which cell cycle kinetics and mode of division of VZ precursor cells were compared, we hypothesized that SNPs would undergo more neurogenic divisions than RGCs. Indeed, immunostaining revealed that SNPs labeled on embryonic day 14.5 (E14.5) generate neurons (GFP/TUJ1 +) through divisions in the VZ, while RGCs more often produce intermediate progenitor cells (GFP/Tbr2+), a transit amplifying population residing in the subventricular zone, which then divide to generate neurons. This extra step in neuron generation for RGCs results in their neuronal progeny arriving in the neocortical wall later than, and thus laminating superficially to, progeny of SNPs labeled at the same time. Similar mechanisms of neuron generation and lamination patterns were observed when precursors were labeled at E12.5. Interestingly, Nestin+ NSCs labeled at E12.5 and E14.5 produced neurons whose laminar allocation was distinct from both RGC- and SNP-derived neurons. Moreover, there were significant differences in neuronal allocation from Blbp-expressing RGCs (bRGCs) and GLAST-expressing RGCs (gRGCs) labeled at E12.5 and E14.5. Lineage analyses of precursors labeled at E16.5 uncovered additional differences in these four populations: all four precursor types produced GFAP+ and S100β+ astrocytes and ependymal cells; SNPs and NSCs also generated DCX+ neuroblasts; but only NSCs produced Olig2+ oligodendrocyte precursors. In conclusion, the data presented here indicate that the mouse VZ is a heterogeneous germinal compartment, comprised by at least two distinct precursor populations which differ in their cell cycle kinetics, mechanism of neuron generation and the phenotypes of their neuronal and glial progeny.