Lineage-specific reporter expression segregates VZ/SVZ cells into distinct subpopulations
Using IUE to express fluorescent reporter molecules under the control of cell type or temporally specific DNA promoters is a powerful technique to map cell lineage and fate in the mammalian brain (Wang et al., 2007
; Chen and LoTurco, 2012
; Ohtaka-Maruyama et al., 2012
). Previously, we demonstrated that mitotic RGCs and SNPs can be labeled and distinguished via reporter expression driven by the Blbp/Glast
(pBlbp/pGlast) and Tubulin α 1 (
1) promoters, respectively (Gal et al., 2006
). We therefore developed an intersectional approach, which employs the combinatorial activity of two promoters to determine the lineal relationships between cells independently labeled by these reporters. Specifically, we paired lineage-specific Cre driver plasmids with a series of dual fluorescent reporter plasmids that we modified from the Cre Stoplight design (Yang and Hughes, 2001
). These reporter constructs, which we have named Blbp-Stoplight (pBlbp-SL), Glast-Stoplight (pGlast-SL), and Tα
1-SL), function as binary switches that drive the expression of ZsGreen in the absence of Cre activity or mCherry following Cre-dependent recombination ().
Figure 1 Schematic illustration of lineage-specific Cre driver and Stoplight reporter plasmids. A, In separate experiments, each Cre driver was coelectroporated in combination with each Stoplight reporter. Theoretical results of pGlast-Cre × pGlast-SL (more ...)
In theory, coelectroporation of the dorsal telencephalic VZ with different combinations of Stoplight and Cre driver plasmids labels cells green or red depending on the following: (1) whether or not both reporter and Cre driver promoters are active concurrently in the same cell or (2) their lineage of origin (whether or not they are derived from parent cells expressing Cre). For example, coelectroporation of cells with pGlast-Cre × pGlast-SL highlights RGCs red due to Cre-dependent recombination and expression of mCherry, while pGlast(−) cells, such as SNPs, are excluded from the fluorescent population due to pGlast promoter inactivity (). Alternatively, mixing Cre driver and Stoplight plasmids theoretically enables a hierarchical, time-dependent analysis of promoter expression in individual cell types. For instance, coelectroporation of cells with pGlast-Cre × pTα1-SL () should initially feature ZsGreen-expressing SNPs as well as unlabeled (pTα1 (−)) RGCs. However, pGlast-Cre expression in RGCs nevertheless results in recombination of the silent pTα11-SL plasmid, ensuring that any pTα1(+) cells subsequently generated via RGC cell division will express mCherry (). In this example, existing SNPs can be distinguished from newly generated SNPs based on fluorescent protein expression controlled by their parent cell of origin.
To test the validity of this approach (i.e., to measure recombination efficiency and to rule out differences in promoter strength), we electroporated dorsal VZ/SVZ cells with Cre and reporter plasmids containing matched promoter sequences at E14.5 in vivo
. We selected 24 h as our primary short-term end-point as it is sufficient time for electroporated cells to complete only one round of the cell cycle (Stancik et al., 2010
). Importantly in these pairings, reporter plasmids approached full recombination within 24h (). Specifically, pGlast-Cre × pGlast-SL or pBlbp-Cre × pBlbp-SL resulted in >96% mCherry(+)
cells 24 h following electroporation (). Similarly, >93% of cells electroporated with pTα
1-Cre × pTα
1-SL were mCherry(+)
in the same time frame. These results demonstrate that Cre expression and activity driven by each promoter is robust and that recombination of the target reporter plasmids occurs at the same rapid pace in all combinations.
Figure 2 Lineal fate mapping reveals heterogeneity of VZ/SVZ progenitors. A, Representative images of VZ/SVZ cells coelectroporated with pGlast-Cre or pTα1-Cre paired with pGlast-SL or pTα1-SL, 24 h after E14.5 IUE. B, Percentage of mCherry+ cells (more ...)
In contrast to the results from matched promoter experiments, when VZ/SVZ cells were coelectroporated with mixed Glast and Blbp Cre and Stoplight plasmids (i.e., pGlast-Cre × Blbp-SL or pBlbp-Cre × Glast-SL) we detected a modest but significant decrease in the number of mCherry(+)
cells from E14.5 to E15.5. In particular, on average 85% of the cells in these pairings expressed mCherry while 15% expressed ZsGreen, suggesting that this latter group of cells expressed pGlast or pBlbp, but not both of these promoter sequences (). This finding highlights the heterogeneity among Glast- and Blbp-expressing cells, which has been previously described, and provides a new molecular labeling method with which to isolate individual cell types (Hartfuss et al., 2001
Next, we coelectroporated pGlast-Cre × pTα
1-SL. In sharp contrast to experiments with matched promoter sequences, we observed a significant decrease in the number of mCherry(+)
cells (56%), mirrored by an increase in the number of ZsGreen(+)
cells at E15.5 (44%) (). Similar results were obtained following pBlbp-Cre × pTα
1-SL coelectroporation (62% mCherry(+)
cells, 38% ZsGreen(+)
cells; ). To determine whether these subclasses of cells were spatially segregated, we counted labeled cells based on their position relative to the ventricle by dividing the VZ/SVZ into five 20 μ
m bins (). While we observed near complete recombination and an equal distribution of mCherry(+)
cells following co-IUE with matched plasmid pairs (pGlast-Cre × pGlast-SL or pBlbp × Blbp-SL), coelectroporation of pGlast-Cre × pTα
1-SL or pBlbp-Cre × pTα
1-SL resulted in a decrease in the number of mCherry(+)
cells that was apparent across all bins relative to matched promoter samples (). Interestingly, a larger proportion of pTa1 cells in the apical VZ (bins 1–2) expressed mCherry due to pBlbp-Cre activity compared with pGlast-Cre, again supporting the finding that a subgroup of VZ cells exhibits nonoverlapping expression of pGlast and pBlbp. Nevertheless, pTα
cells that do not express Cre driven by the pGlast or pBlbp promoters are present throughout the radial axis of the VZ/SVZ and are thus likely present in all phases of the cell cycle (Takahashi et al., 1993
). The pTα
cells resulting from pBlbp-Cre or pGlast-Cre recombination may reflect either overlapping expression of the pTα
1, pGlast, or pBlbp promoters in the same cell or, potentially, that some pTα
cells are derived from pGlast(+)
parent cells through cell division within 24 h.
Unexpectedly, when the reverse experiments were performed (pTα
1-Cre × pGlast-SLorpTα
1-Cre × pBlbp-SL), we observed near complete recombination 24 h post-IUE (). This strongly suggests that the pTα
1-Cre driver is sufficiently active to generate recombination in most pGlast(+)
RGC precursors, greatly increasing estimates of overlap in Tα
1, Glast, and Blbp expression noted in our previous study (Gal et al., 2006
). Nevertheless, when all of the cells labeled by these sensitive intersectional tools are considered together, the data indicate that the VZ and SVZ populations identified by the expression of pGlast or pBlbp are nested within a larger class of cells that express pTα
1, almost half of which express neither pGlast nor pBlbp (). In other words, while this dual reporter fate-mapping approach demonstrates that most of the VZ precursor cells exhibit pTα
1 activity, our multiplex methodology can be used to subdivide this large pTα
1-expressing population into pTα
populations that express mCherry, and pTα
populations that express ZsGreen. This fate-mapping approach therefore exploits the significant molecular expression differences between VZ and SVZ precursors in the mammalian telencephalon and the dual color reporter strategy enables a simultaneous cellular and molecular analysis of each defined cell type.
Blbp and Glast promoter-dependent Cre activity segregates pTα1(+) cells into morphologically distinct cohorts
To resolve whether the molecular differences we identified between precursor populations coincide with morphological distinctions, we scored fate-mapped VZ/SVZ cells as either “radial-like” or “multipolar” in 3D Z-stack reconstructions. In these counts, we confined our analysis to cells located within 100 μm of the ventricular surface and included unipolar and bipolar cells with apical processes in the radial-like group and cells with more than two processes into the multipolar group (, inset). As expected, mCherry(+) radial-like VZ cells were the primary group labeled by pGlast-SL and pBlbp-SL (1.8-fold for each vs pTa1-SL; ), and we noted a trend toward an increase in mCherry(+) radial-like cells at the expense of mCherry(+) multipolar cells identified by Blbp-SL compared with Glast-SL. In contrast, when combined with pGlast-Cre or pBlbp-Cre, we found that the pTα1-SL reporter labeled fivefold more ZsGreen(+) VZ cells with radial-like morphology and twofold more ZsGreen(+) multipolar cells than the Blbp-SL and Glast-SL reporters (). Interestingly, pGlast-Cre × pTα1-SL electroporated sections contained a 56% increase in ZsGreen radial-like cells relative to pBlbp-Cre × pTα1-SL. This finding is consistent with the observation of a greater number of mCherry(+) cells in the apical half of the VZ in pBlbp-Cre × pTα1-SL crosses (). Together, these morphological counts indicate that radial-like and multipolar precursor cells were labeled to varying degrees by each Cre/Stoplight combination. However, the pTα1-SL population included sizable cohorts of ZsGreen(+) radial-like and multipolar cells that do not express pGlast and pBlbp within the 24 h time period examined, suggesting that these cells are either separate from the RGC lineage entirely or that they are a population of molecularly distinct RGCs.
Figure 3 Cre-Stoplight combinations uncover morphological distinctions between VZ and SVZ cells. A, Schematic illustrating the morphology of radial-like and multipolar cells (inset) and quantification of the percentage of each morphological class 24 h following (more ...)
To determine whether pTα1-ZsGreen and pTα1-mCherry(+) radial-like cells were RGCs (i.e., possessing long basal processes contacting the pial surface), we coelectroporated either pBlbp-Cre (+) pTα1-SL or pGlast Cre × pTα1-SL and performed a series of 3D reconstruction confocal imaging experiments. First, we imaged pial end feet as well as cell bodies in the VZ/SVZ of the same 60-μm-thick brain section to determine whether ZsGreen(+) and mCherry(+) cells extended pial-contacting basal processes. Interestingly, despite the fact that 38% of the cells in the VZ expressed ZsGreen but not mCherry (pBlbp-Cre × Tα1-SL; ), and 14% of the ZsGreen(+) cells possessed radial-like morphology (), we detected very few ZsGreen(+) basal fibers with end foot processes at the pia. Instead, the majority of pial projections were mCherry(+) ().
As a second approach, we applied DiI to the pia of electroporated brains to trace basal fibers of RGCs and label their somas (). For this experiment, we replaced the mCherry coding sequence in the pTα
1-SL plasmid with a STOP cassette to further highlight pTα
cells that express only ZsGreen but do not show pGlast or pBlbp-dependent Cre activity and expression. Consistent with the absence of ZsGreen(+)
pial end feet, we observed that many pTα
cells failed to incorporate DiI, including cells rounded up in M-phase at the surface of the ventricle (; pGlast-Cre × pTα
1-SL shown, similar results seen with pBlbp-Cre). These results support the previous observation that many VZ cells exhibit a “short” morphology defined by the absence of a basal process that spans the cortical plate (Gal et al., 2006
In addition to multipolar precursors in the SVZ, we noted a number of pTα
cells above the band of multipolar SVZ cells, which appeared to be bRG cells as they possessed long basal fibers but had no discernible apical processes. Interestingly, in time-lapse multiphoton imaging experiments, we were able to identify only pTα
cells dividing as bRG in the SVZ, indicating that the murine bRG population may be specifically generated from pGlast(+)
RGC precursors and that our fate-mapping technique can distinguish these lineage restricted events (Movies 1
Together, these results demonstrate that our dual fluorescent fate-mapping approach identifies all known classes of VZ and SVZ precursor cells and elucidates lineage transitions between subpopulations. For example, approximately half of the pTα1-ZsGreen(+) cells exhibit a short, radial morphology, and likely represent the SNP population, while half are multipolar. In addition, while some pTα1-mCherry(+) cells appear to be RGCs since they have basal processes contacting the pial surface, several additional non-RGC cell types express mCherry, including multipolar SVZ cells, bRG, and potentially some SNPs, which may indicate that they are the immediate progeny of Glast(+) RGCs.
pTα1 cells are heterogeneous for transcription factor expression
From this point forward, we explored the morphology, expression profiles, and division parameters of the VZ and SVZ precursors using only the pGlast-Cre × pTα
1-SL vector combination as this pairing produced the clearest separation between precursor subtypes. These populations are hereafter denoted by “pTα
,” respectively. As such, pTα
cells represent cells either coexpressing pGlast or derived from a pGlast(+)
parent cell; pTα
cells do not coexpress pGlast nor are they derived from a pGlast(+)
parent during the course of the experiment. Using this approach, we asked whether the pTα
populations expressed distinctive differences in transcription factor expression by staining E15.5 sections of coelectroporated brains for Pax6 and Tbr2 (). Overall, in counts conducted within 100 μ
m from the ventricular surface, we found that 59% of the total electroporated population expressed Pax6 while 54% expressed Tbr2 (). This result implies that ~13% of the electroporated cells form an overlapping group that expresses both Pax6 and Tbr2, as described previously () (Englund et al., 2005
Figure 4 Subpopulations of pTα1-ZsGreen + and pTα1-mCherry + cells express Pax6 or Tbr2. A, Schematic representation of the classes of cells observed 24 h post co-IUE with Glast-Cre × pTα1-SL(IUE at E14.5) immunostained for Pax6 (more ...)
Further subdividing the population by fluorescent protein expression, Pax6(+)/pTα1-ZsGreen(+) cells accounted for 21% of the total electroporated population. In comparison, the number of pT(+)1-mCherry(+) cells that also expressed Pax6 was significantly larger than the ZsGreen(+) cohort, representing 38% of the transfected population (). In contrast, we did not detect a difference between the percentage of the pTα1-ZsGreen(+) andpTα1-mCherry(+) cells that express Tbr2, which constituted 25 and 28% of the electroporated cells, respectively (). These results indicate that while pGlast-dependent recombination of the pTα1-SL plasmid separates VZ cells into RGC and non-RGC groups, the two resulting populations of pTα1-ZsGreen(+) and pTα1-mCherry(+) cells exhibit further molecular heterogeneity with respect to the expression of Pax6 and Tbr2. This finding is consistent with the observation that subsets of pTα1-ZsGreen(+) and pTa1-mCherry(+) cells exhibit either radial-like or multipolar morphologies ().
To determine whether these groups were further resolved by their distribution within the germinal zone, we quantified the percentage of labeled cells by position relative to the ventricle by binning the VZ/SVZ as before (). While the Pax6(+)/pTα1-mCherry(+) population was larger than the Pax6(+)/pTα1-ZsGreen(+) group, we noted that both groups exhibited a similar spatial profile (). However, Pax6(+)/pTα1-ZsGreen(+) cells were concentrated in a narrower portion of the apical VZ. By comparison, the arrangement of Tbr2/pTα1-mCherry(+) and Tbr2(+)/pTα1-ZsGreen(+) cells was nearly identical with the exception of a slight increase in the number of Tbr2(+)/pTα1-mCherry(+) in the basal VZ/SVZ ().
Together, these results reveal an increased level of molecular heterogeneity among VZ/SVZ cells than previously appreciated. First, our combinatorial fate-mapping strategy shows that pBlbp and pGlast-Cre activity is able to separate pTα
1 cells into defined lineage groups, which are further subdivided by morphology and transcription factor expression. In particular, we find that pTα
groups are each comprised by Pax6(+)
cells with either radial-like or multipolar morphologies. As expected, the data show that the RGCs present in the VZ, which are Pax6(+)
, are joined by their Tbr2(+)
IPCs progeny in the SVZ, which were likely generated from RGCs during the 24 h experimental period. This confirms the lineal origin of Tbr2(+)
IPCs as suggested by other groups (Englund et al., 2005
; Noctor et al., 2008
). In addition, there is a cohort of multipolar Tbr2(+)
cells that were either generated from RGCs before the electroporation or are the progeny of Pax6(+)
cells. However, in addition to these cell types, which have been characterized previously, we find that a primary component of the cycling population in the VZ is a group of bipolar pTα
1 cells that (1) does not exhibit active expression from the pGlast or pBlbp promoters, (2) lacks pia-contacting basal processes throughout the cell cycle, (3) expresses Pax6, and (4) appears to divide at surface of the ventricle in images of fixed tissue (, ). These characteristics strongly suggest that the Pax 6(+)
cohort of cells found in the apical half of the VZ is not equivalent to the basal Tbr2(+)
IPC population, nor are they RGCs.
Radial-like pTα1-ZsGreen(+) cells divide at the ventricular surface
To conclusively demonstrate that apical pTα
cells are a bona fide and unique progenitor class rather than merely a transitory stage of basal Tbr2 IPCs before delamination to the SVZ, as has been previously suggested (Pontious et al., 2008
; Kowalczyk et al., 2009
; Miyata et al., 2001
; Lui et al., 2011
), we sought to describe the location and kinetics of mitotic divisions following pGlast-Cre × pTα
1-SL coelectroporation. Using time-lapse multiphoton imaging in cultured slices (n
= 16), we excited dividing mCherry(+)
cells simultaneously at 995 nm and split each emission to separate detectors. As expected, we detected many pTα
RGCs dividing at the VZ surface ( see Movie 5
). In addition, we captured pTα
cells dividing into two separate daughter cells at the ventricular surface (; Movies 3
). These time-lapse slice experiments conclusively demonstrate that the apical pTα
population is bipolar and divides at the surface of the ventricle and is thus separate from the Tbr2(+)
IPC population based on morphology, division dynamics, and gene expression. Thus, the fate mapping, gene expression, morphology, and time-lapse data all characterize the apical pTα
cells as an independent SNP population.
Figure 5 Radial-like pTα1-ZsGreen + and pTα1-mCherry + cells divide at the surface of the ventricle. Multicolor multiphoton time-lapse imaging of organotypic brain slices (E15.5) coelectroporated with Glast-Cre × pTα1-SL(IUE at (more ...)
SNPs are lineally derived from RGCs
To determine the lineal origin of pTα1-ZsGreen(+) cells, and in particular whether they are derived from pGlast(+) RGCs, we performed longer term fate-mapping experiments to observe both their genesis and fate over time in vivo. Specifically, we coelectroporated pGlast-Cre × pTa1-SL into neural precursors of the dorsal telencephalic VZ at E13.5 and then imaged the distribution of mCherry(+) and ZsGreen(+) cells in the developing neocortex at E15.5. We selected E15.5 as our primary endpoint because prior experiments conducted between E14.5 and 15.5 revealed a sizable number of pTα1-ZsGreen(+) cells present in the VZ at this time point. We hypothesized two possible outcomes of this experiment: (1) if pTα1-ZsGreen(+) SNPs lineally diverge from pGlast(+) RGCs before E13.5 and self-renew as an independent population, we would detect an equal if not greater number of ZsGreen(+) SNPs in the VZ following 48 h of electroporation, relative to the number seen at 24 h post IUE (); (2) if, however, SNPs are lineally derived from pGlast(+) RGCs and they do not exhibit self-renewal properties, the VZ and SVZ would be filled with pTα1-mCherry(+) cells that replaced the cohort of ZsGreen(+) SNPs as they were exhausted by terminal divisions at the ventricular surface ().
Figure 6 SNPs are neuronal progenitors lineally derived from RGCs. A, Theoretical model of pGlast-Cre × pTα1-SL populations if SNPs are a self-renewing precursor group. ZsGreen + SNPs existing in the VZ at the time of (IUE) would be equal or increased (more ...)
We found that virtually all of the pTα
1 cells present in the VZ and SVZ expressed mCherry 48 h after pGlast-Cre × pTα
1-SL electroporation (). At this same time point, many pTα
neurons were found migrating through the intermediate zone and beginning to settle in the upper layers of the cortical plate (). Importantly, two types of mCherry(+)
cells were observed dividing at the ventricular surface 48 h after transfection: cells containing a basal process () and completely rounded mitotic cells lacking a basal process (; Movie 6
). This latter cell type is the previously determined morphology of dividing SNPs (Gal et al., 2006
), which is dramatically different from the morphology of dividing RGCs that retain their basal processes (Miyata et al., 2001
; Noctor et al., 2001
; Noctor et al., 2002
; Gal et al., 2006
While the 48 h fate map study () indicated that pTα
cells generate postmitotic migrating neurons, we sought to confirm that these neuronal daughter cells differentiate and integrate into postnatal neocortical circuits. We therefore performed a longer duration fate map by electroporating neocortical VZ at E14.5 and analyzing brain sections at postnatal day 15 (P15). However, since the Tα
1 promoter is downregulated during postnatal neuron differentiation (Gloster et al., 1994
), in this experiment we used a pCAG-SL reporter, which retains high expression in postnatal brain (Navarro-Quiroga et al., 2007
). Importantly, we confirmed that both the pTα
1 and pCAG reporters were interchangeable in terms of the numbers and types of cells labeled during embryonic development, which was expected since all VZ cells examined by this method express the Ta1 promoter (; data not shown). We found that both ZsGreen(+)
neurons specified from the pGlast-Cre × pCAG-SL cotransfections on E14.5 migrated to and differentiated within layer II/III at P15 (). Specifically, 22.5 ± 4.5% (±SEM, n
= 3) of the fate mapped neurons expressed ZsGreen at P15. These findings indicate that SNPs generate neurons that differentiate and survive in the postnatal neocortex and that the progeny of the initial cohort of SNPs remains separately labeled by the multiplex fate map procedure.
Altogether, these data suggest that all pTα
cells, including SNPs, are lineally derived from pGlast(+)
RGCs. Furthermore, SNPs do not undergo an appreciable number of self-renewing cell divisions, as they rapidly divide to generate neurons that exit the VZ (; Stancik et al., 2010
). Importantly, the above results also indicate that neurons generated from SNPs survive in the postnatal neocortex and are thus likely to play a significant role in brain maturation and function.
SNP production is reduced in embryonic Ts65Dn neocortex
The above results, that SNPs are a transient VZ dividing population continuously derived from RGCs, demonstrate that SNPs join Tbr2(+) IPCs and bRG as an independent class of neuronal progenitors during neocortical development. However, a question remained as to whether these three indirect streams of neuronal production are individually necessary or are perhaps redundant during neocortical development. If each precursor class is necessary for proper cortical growth, we reasoned that the relative quantities of particular neural precursors maybe altered in neurodevelopmental disorders, especially those exhibiting microcephaly or dysplasia. To test this, we measured the specification of each intermediate progenitor group in a mutant animal with well characterized defects in neocortical expansion.
Previously, we determined that neocortical growth in the Ts65Dn mouse model of DS is significantly delayed from E12.5 to E18.5 due to slower cell cycle kinetics and reduced neurogenesis from VZ precursors (Chakrabarti et al., 2007
). While the overall thickness of the VZ/SVZ is not remarkably different from Euploid controls over much of the neurogenic interval, differences in the cycling properties and/or proportions of VZ/SVZ cells are thought to underlie the stunted neocortical growth observed in Ts65Dn embryos. Interestingly, a compensatory expansion of Tbr2(+)
abventricular divisions has been noted during late prenatal Ts65Dn development (Chakrabarti et al., 2007
), but the cellular changes that underpin the differences in neurogenic output before E16.5 remain unresolved.
To determine whether specification and development of the multiple groups of VZ/SVZ neural precursors is altered during abnormal neocortical development, we measured their number and distribution in the Ts65Dn embryonic neocortex 24 h after electroporation of pGlast-Cre × pTα1-SLplasmidsatE14.5. Our dual fluorescent reporter fate-mapping assay revealed a 20% increase in pTα1-mCherry(+) cells and a corresponding reduction in pT(+)1-ZsGreen cells in Ts65Dn versus Euploid littermate embryos ().
Figure 7 SNPs are specifically reduced during Ts65Dn neurogenesis. A, Euploid and Ts65Dn littermate embryos were cotransfected with pGlast-Cre × pTα1-SL on E14.5. Twenty-four hours later, the VZ/SVZ contained ZsGreen + and mCherry + cells. B, The (more ...)
Interestingly, immunohistochemistry did not reveal a difference in the total numbers of electroporated cells that expressed Pax6 or Tbr2 in the Ts65Dn neocortical wall (). However, when the pTa1(+) cells were further subdivided based on fluorescent protein expression regulated by pGlast-Cre-dependent recombination, there was a 28% increase in the relative number of RGCs (Pax6(+)/pTα1-mCherry(+) cells) and a 57% decrease in SNPs (Pax6(+)/pTα1-ZsGreen(+) cells) in the Ts65Dn VZ (). While we did not observe significant differences in the percentage of Tbr2(+)/pTα1-mCherry or Tbr2(+)/ZsGreen(+) cells between Ts65Dn and Euploid samples, the groups exhibited a shift similar to that seen in the Pax6(+) populations, with more Tbr2(+)/pTα1-mCherry cells at the expense of Tbr2(+)/ZsGreen(+) cells (). Altogether, the data from electroporations of prenatal Ts65Dn neocortex demonstrate that subpopulations of neural precursors are specifically altered during development. In particular, Pax6(+) SNPs, and to a lesser degree Tbr2(+)/ZsGreen cells, are substantially reduced. These data strongly suggest that individual streams of indirect neurogenesis are affected in the trisomic brain and that their loss contributes to improper brain development.