GCPs expand predominantly via symmetric cell division
The mode of neural precursor division has important implications for patterns of neurogenesis. In the cerebral cortex, for instance, neural precursors in the ventricular zone undergo characteristic changes of division pattern from early asymmetric division to later symmetric division (Langman et al., 1966
; Martin, 1967
; Chenn and McConnell, 1995
; Zhong et al., 1996
). What mode of division is used for GC neurogenesis has not been analyzed previously. Do GCPs undergo symmetric cell division and exponential expansion, asymmetric cell division and linear expansion, or a combination of the two? Does the cell division mode change over time? Analysis of MADM G2
-X events can be used to resolve these issues, because sister cells generated after G2
recombination followed by X segregation (G2
-X event) would be labeled by two different colors () (Zong et al., 2005
). A symmetric division model predicts that green and red cells that originate from the same precursor should be present in equal numbers, whereas an asymmetric division model predicts that clones are predominantly of one color (). In addition, the time of clone induction can be used to determine the birth date of GCPs giving rise to the clones.
Figure 1 GCPs divide predominantly symmetrically during perinatal and postnatal development. A, Schematic of the MADM method illustrating Cre-mediated interchromosomal recombination that results in reconstitution of two fluorescent markers, GFP and red fluorescent (more ...)
We used the β-Actin-CreER
transgene (Guo et al., 2002
) to induce G2
-X MADM clones at E17.5. In the absence of tamoxifen, CreER protein is sequestered in the cytoplasm and therefore cannot catalyze DNA recombination. When CreER binds tamoxifen, it translocates into the nucleus where Cre can catalyze site-specific recombination (Metzger and Chambon, 2001
). Previous experiments based on recombination efficiency and CreER subcellular localization indicated that Cre recombinase activity peaks within 24 h and subsides 36-48 h after tamoxifen administration (Hayashi and McMahon, 2002
). Therefore, interchromosomal recombination and consequent labeling of GCPs is limited to ~1 d after tamoxifen administration. Application of 8 mg of tamoxifen intraperitoneally allows us to maintain a low frequency of labeled cells (~1.6 cluster of GCs per half cerebellum). Statistical analyses suggest that each spatially restricted cluster of labeled GCs is derived from a single recombination event (Zong et al., 2005
We fixed and stained the cerebella from animals at different ages () and quantified the total number of green and red cells within each G2-X clone (). Green and red cells were intermingled or segregated independent of type of clone (EGL or IGL). We find that most G2-X clones have similar numbers of red and green cells (). This finding is inconsistent with an asymmetric model of cell division and supports the notion that at E17.5, or shortly afterward, GCPs predominantly undergo symmetric cell division, giving rise to two daughter cells with an equal potential of generating similar numbers of GC progeny. Although some clones are biased toward one color (e.g., C15), they are the exception to the trend and may result from the occasional GCP cell death.
To test whether the division mode changes over time, we extended this analysis to four different postnatal times by applying tamoxifen at P3, P6, P9, and P12, while always examining the clones at P21 (). At P21, GCP proliferation and GC differentiation are complete. At all injection time points, we find that most G2-X clones contain similar numbers of red and green cells within a clone (). We conclude that at these postnatal times, GCPs also undergo predominantly symmetric division leading to exponential expansion of progeny number.
The exponential expansion of GCPs makes it possible that each GCP at E17.5 gives rise to a median of 250 GCs during a period of, at most, 3 weeks of expansion (see Fig. 4A
below). A pure linear expan1
sion model requires the GCP cell cycle period to be ~2 h (21 d × 24 h/~250), much too fast for a mammalian neural precursor with a cell cycle period of at least 8 h (Caviness and Takahashi, 1995
; Takahashi et al., 1995
). The exponential model requires only eight cell divisions to expand from one labeled precursor to ~250 progeny (28
). If this were the case, the average length of the cell cycle would be 2.6 d (21 d per eight cell divisions), assuming that (1) there is minimal contribution by cell death and (2) all GCPs divide at the same rate all the time. Although assumption (1) is consistent with previous studies (Krueger et al., 1995
; Muzumdar et al., 2007
), assumption (2) does not apply (see below).
Most GCs stack their axons within the ML in a temporal sequence
We have previously conducted preliminary studies to examine the relationship between lineage and axon projection patterns of GCs (Zong et al., 2005
). We labeled GCPs by administering tamoxifen at E17.5 and analyzed the GC progeny at P21. Interestingly, these experiments revealed that single GCPs at E17.5 give rise to GC progeny that project axons to a restricted sublayer in the ML despite the fact that their cell bodies disperse throughout the IGL () (Zong et al., 2005
Figure 2 GCs stack their axons in the ML in a temporal sequence from deep to superficial sublayers.A, To induce MADMG2-X, G2-Z, or G1 labeling, tamoxifen was applied intraperitoneally at E17.5 in the pregnant mother. Progeny of the genotype GR/RG;β-Actin-CreER (more ...)
Classical studies suggest an orderly differentiation of GCs, which we coin the “stacking model” (Altman and Bayer, 1997
). This hypothesis states that GCs that differentiate first leave their axons at the deepest sublayer in the ML; GCs that differentiate progressively later leave their axons in progressively more superficial sublayers in the ML. The supporting evidence for this hypothesis came primarily from developmental snapshots using Golgi staining: deepest projecting axons tend to belong to the most morphologically differentiated GCs () (Ramon y Cajal, 1911
). If this stacking model were correct, our observation that clonally related GCs project their axons to specific sublayers would imply that (1) all clonally related GCs “synchronously” differentiate within a narrow time window during postnatal development and (2) the time windows for differentiation occur earlier for deeply projecting clones than for the superficially projecting clones.
To test these predictions, we first performed a set of experiments to quantitatively test the stacking model in mice. Because there is no correlation between the depth of GC body position within the GC layer and the depth of their axon position in the ML () (Zong et al., 2005
), we need a method to correlate birth of individual GCs with their axonal positions.
In the first set of experiments, we injected tamoxifen at E17.5 to generate MADM clones and subsequently injected the thymidine analog CldU at P6 or P15 to label dividing cells in the S phase (Takahashi et al., 1992
; Vega and Peterson 2005
). We then analyzed P21 brains to determine the correlation of birth timing and axon projection of each MADM-labeled clone (). Strong CldU-positive cells should be born shortly after injection time because there is no dilution of the label resulting from the lack of further cell divisions. The stacking model would predict that superficially projecting clones are composed of cells born exclusively at a late developmental time point (e.g., P15), whereas deeply projecting clones are composed of cells born exclusively at an early developmental time point (e.g., P6). For example, a deep projecting clone composed of cells that were born at P15 would be incompatible with a strict stacking model.
When CldU was injected at either P6 or P15, we found examples in which CldU-positive cells in MADM-labeled GC clones project axons to both superficial and deep layers (). We quantified these events in two different ways. We took the position of the mean axonal projection of GC clones and bin into the deepest one-third, middle one-third, or most superficial one-third. First, we quantified the percentage of GC clones with at least one CldU-labeled cell for each type of clone (percentage of clonesCldU+) (). Second, we quantified the percentage of CldU-positive cells within these clones (percentage of GCsCldU+) (). Both analyses gave similar results: deep projecting GC clones injected with CldU at P6 had a higher frequency of clones (~50%) and cells per clone (~20%) labeled with CldU. In contrast, when CldU was injected at P15, labeled cells are found with higher frequency in superficial projecting GC clones (~50% of clones, ~15% of cells per clone). Multiplying the percentage of clones that have at least one CldU-labeled cell by the average percentage of CldU-labeled cells per clone gives us a labeling index for the likelihood of labeling a class of GC with CldU. We find that CldU injection at P6 will label ~0.1% (~10% × ~1%) of superficial projecting GCs and ~10% (~50% × ~20%), or 100-fold more, for deep projecting GCs. Conversely, injection of CldU at P15 will label ~7.5% (~50% × ~15%) of superficial projecting GCs and ~0.05% (~5% × ~1%), or 150-fold less, of deep projecting GCs. These results provide quantitative support for the stacking model. They also suggest that the stacking model is not absolute, because both P15 CldU-positive cells that project to deep layers and P6 CldU-positive cells that project to superficial layers exist, albeit at a very low frequency (see Discussion).
In a second set of experiments, we coinjected tamoxifen and CldU to induce MADM recombination and label dividing cells, respectively (). Tamoxifen injection during early postnatal development results in MADM-labeled postmitotic and mitotic cells; however, CldU labeling allowed us to identify which of these GCs were born shortly after the time of CldU injection. CldU is diluted with each round of division, so cells born later may also be labeled, but less intensely. We quantified only strong CldU-positive cells, which are likely to be born shortly after CldU injection. We found that coinjection with tamoxifen and CldU at P6 double labeled deep projecting GCs (n = 20 cells) () and coinjection with tamoxifen and CldU at P15 double labeled superficial projecting GCs (n = 22 cells) (). However, we found rare exceptions such as cells born at P6 projecting to superficial sublayers (n = 4 cells) () and cells born at P15 projecting to deep sublayers in the ML (n = 2 cells) (). Consistent with the first experiment, the stacking model applies to most GCs, with notable exceptions.
Figure 3 Orderly differentiation of GC clones during postnatal development. A1, A schematic of the experiment: tamoxifen application at E17.5, and dissection time points from P3 to P21. A2-A4, Confocal images illustrating three main classes of clones found at (more ...)
Clonally related GCs differentiate within a narrow time window in an orderly sequence
Having verified that the stacking model is mostly applicable, we next examined the hypothesis that clonally related GCs exit the cell cycle within a specific time window and thus project axons to a similar sublayer in the ML. We followed GCP expansion during postnatal development by generating GC clones at E17.5 and analyzing their progeny at different time points during development ().
When examining MADM-labeled clones, we noted the position of cell bodies (EGL, ML, or IGL) using DAPI as a counterstain and the morphology of cells (polyhedral, nonpolyhedral, axonal, and dendritic processes). Examination of early postnatal time points (less than P4) revealed that all cells in all clones were located exclusively in the EGL (), indicating that this is a period consisting predominantly of GCP expansion. MADM clones analyzed at later postnatal time points (P6-P15) fell into three classes: class 1 (EGL), labeled cells are exclusively in the dividing EGL layer and characterized by a polyhedral shape (); class 2 (MIX), labeled cell bodies are found in EGL, migratory route to IGL, and in IGL (); class 3 (IGL), labeled cell bodies are predominantly in IGL ().
Given the orderly organization of cells at different stages of differentiation during postnatal cerebellar development (Hanaway, 1967
; Eccles, 1970
; Herrup and Kuemerle, 1997
), we interpret EGL clones to indicate that all labeled cells are still GCPs and none have undergone differentiation. IGL clones indicate that all labeled cells have already completed their division and migration through the ML to occupy their final destination in IGL; indeed, most labeled GCs in most IGL clones have elaborated their dendrites within the IGL. MIX clones are most likely caught in the process of differentiation: some cells have already completed their migration, some are in the process of migrating from EGL to IGL, and the rest are still dividing in the EGL. Interestingly, close examination of all MIX clones showed labeled axons extending at the ML/EGL border () as would be predicted by the stacking model. In contrast, all IGL clones examined from P6 to P12 project axons in sublayers below the ML/EGL border (). By P21, all clones have cell bodies in the IGL, consistent with the end of morphological development of the cerebellar cortex. Quantification of the frequencies for each of three clonal types at different developmental stages reveals an orderly transition from EGL to MIX to IGL types as development proceeds ().
The existence of EGL clones as late as P15 indicates that all progeny from these clones will differentiate after P15; the appearance of IGL clones as early as P6 indicates that all progeny from these clones have exited the cell cycle before P6. Together, these data rule out the possibility that all clonally related GCs differentiate throughout the entire period of postnatal neurogenesis and support the notion that clonally related GCs exit the cell cycle within specific time windows.
Because GC dendrites undergo a stereotyped maturation process from a single leading process (migrating) to multiple dendrites to few (three to seven) dendrites with claw-like terminal indicative of synapse maturation (Ramon y Cajal, 1911
), we also used dendrite morphology to assess the GC differentiation status for different clonal types (). We found that GCs that project axons deeper in the ML have more fully developed dendritic claws. In contrast, GCs from IGL clones that project axons near the ML/EGL border, and all MIX clones, have immature dendritic differentiation (). These observations indicate that GCs belonging to the MIX clones are the most recently born and leave their axons at the most superficial part of the ML during these developmental snapshots (see above), providing further proof that GCs stack their axons in chronological order.
Average expansion rate of GCPs gradually decreases during postnatal development
Our findings provide evidence that GC clones that project deep in the ML must undergo differentiation earlier than GC clones that project superficial in the ML. One prediction from these findings is that clonally related “deep projecting” GCPs will have less time to divide compared with clonally related “superficial projecting” GCPs and therefore one would expect deep projecting GC clones to be smaller, on average, than superficial projecting GC clones. We sorted GC clones induced at E17.5 and examined at P21 into one of three groups according to their average axon projection depth in the ML: the bottom one-third (deep), middle one-third (middle), top one-third (superficial) (). Next, we quantified the total number of GCs within each clone by counting the cell body number in consecutive sagittal sections that span the entire clone. Surprisingly, we found that the median of GCs per clone is similar regardless of whether their axon projection is to the deep, middle, or superficial sublayers in the ML (). In all three subgroups, we find heterogeneous clone size, ranging from <50 to >2000 cells, with a median of ~250. The heterogeneity in clone size may be caused by occasional cell death of early GCPs, or a further heterogeneity of GCP proliferation potential within each subgroup. However, these differences are independent of the axon projection type, suggesting that time of cell cycle exit is not a predominant variable.
Figure 4 Cell number matching among GC clones with distinct axonal projection patterns. A, Box plot representative of IGL clones grouped by the corresponding axonal projection pattern. The box demarcates the lower quartile, median (number), mean (asterisk), and (more ...)
We set out to resolve this discrepancy: GC clones projecting axons to deep sublayers have less time to divide because they differentiate earlier in development; however, they are similar in number compared with GC clones projecting axons to superficial sublayers. We examined GCP expansion rates during postnatal development, because differences in expansion rates during development may partially explain this finding. We induced MADM clones at E17.5 and quantified the number of labeled cells per clone at different postnatal time points as shown in . We first examined EGL clones, which represent the stage of GCP division. We found that the expansion rate decreases as development proceeds (). The average rate of GCP doubling is about once per day between E18 and P3, declines to once per 2 d from P3 to P6, and further declines afterward (). The progressively slow GCP expansion rate later in development therefore can contribute to, but does not by itself explain, the clone size discrepancy.
Departure from the trend: GCPs speed up division shortly before the onset of GC differentiation
The analysis in EGL clones represents an overall trend of GCP expansion but does not take into consideration that at any given time there could be different subpopulations of GCPs exhibiting different behavior. Indeed, when we include MIX clones into the cell expansion analysis, we found, strikingly, that MIX clones are much larger than EGL clones examined at the same developmental stage. Examples for two different stages of P9 and P12 are quantified in . Because all clones that contain differentiated cells derive from EGL clones at an earlier stage (e.g., EGL clones at P6 should give rise to EGL, MIX, and/or perhaps IGL clones at P9), these data suggest that shortly before onset of GC differentiation, GCPs expand faster to account for the differences between the cell numbers in EGL clones and those that contain differentiated cells. Interestingly, even the number of GCPs (counting only cells in EGL) in a MIX clone is larger than the number of GCPs in an EGL clone for the same developmental time point ().
This increase in cell number expansion could be caused by a reduction in GCP death or an increase in cell proliferation. Because the contribution of cell death is not a major contributor to the decrease in expansion during postnatal development (see Discussion), it is more likely caused by increased cell proliferation or speedup of the GCP cell cycle. To experimentally test this hypothesis, we generated MADM clones by tamoxifen injection at E17.5 and determined the proportion of GFP cells labeled with a pulse of CldU (S phase marker) () or pH3 (M phase marker) (). In general, the duration of the S and M phases has little variation (Cameron and Greulich, 1963
; Prescott, 1968
; Smith and Martin, 1973
). Therefore, the percentage of cells in the S and M phases should reflect the cell cycle length: the higher the percentage, the faster the cell cycle. , show two representative confocal images of MADM-labeled clones examined 2 h after CldU was injected at P9. P9 was chosen to maximize the chance of having both EGL clones () and MIX clones containing labeled cells in EGL/ML/IGL (). As expected, CldU or pH3 does not label cells residing in the ML and IGL, because they have already exited the cell cycle.
Figure 5 Clonally related GCPs divide faster shortly before differentiation. A1, A schematic of the experiment: tamoxifen application at E17.5, followed by a pulse injection of CldU at P9 to label the S phase. Mice were killed 2 h after CldU injection, and clonally (more ...)
Within the proliferative region of the outer EGL, we counted the number of CldU-positive or pH3-positive cells in each MADM clone. We divided these numbers by the total number of cells within that MADM clone to derive a percentage of cells within each clone that have been in the S phase in the past 2 h or in the M phase at dissection time. Strikingly, MIX clones (containing cells in the EGL, ML, and IGL) display more than twice as many CldU-positive or four times as many pH3-positive cells in the EGL compared with EGL clones (). As controls, we also quantified the percentage of CldU-positive or pH3-positive non-MADM-labeled cells within the EGL near each MADM clone, using the DNA dye DAPI as a nuclear marker. We find that 40% are CldU positive and 10% are pH3 positive for MADM-negative cells in the EGL region demarcated by the border of the MADM clone, regardless of whether the cell bodies are near EGL or MIX MADM clones ().
The above data provide direct evidence that EGL clones have a longer cell cycle compared with MIX clones, supporting a two-phase model of GCP expansion: a slow phase while expanding exclusively within EGL and a fast phase close to the onset of differentiation. Furthermore, given that non-MADM-labeled EGL cells have the same percentage of CldU-positive (40%) and pH3-positive (10%) cells as MADM mix clones, the population of dividing cells in EGL at P9 are mostly consisting of those that will differentiate shortly. Thus, the total cell number derived from a single GCP at E17.5 may primarily be contributed by the rapid expansion phase regardless of the timing of cell cycle exit. This, in combination with the slow GCP cell cycle during most of the proliferation period within EGL (last section), can now explain the similar clone sizes regardless of the timing of cell cycle exit.