The cerebellum, or 'little brain', handles movement, thought and social interaction. Unlike the rest of the brain, which primarily develops in the womb, most of its cells appear within the first year of our lives (or first few weeks in mice). This makes it vulnerable to injury around the time of birth.

We used to think that the brain could not replace damaged neurons, but when specific precursor cells in the cerebellum in the brains of newborn mice are removed, they are able to renew themselves. This is because specialized stem cells start to divide and produce the missing cells of the cerebellum.

Another type of cells in the cerebellum, called Purkinje neurons, are already produced in the embryo. They direct the development of several other cell types in the cerebellum after birth. They are also a crucial component of the circuits within the cerebellum, and losing them can cause loss of muscle coordination. Purkinje cells do not normally divide once an animal is born, but scientists want to know if they might be able to regrow after injury at birth.

Bayin et al. killed Purkinje cells in newborn mice with a toxin and used fluorescent markers to track the dying cells. Then, the remaining cells in the surrounding area were studied. This revealed that even when half of the Purkinje cells died a day after birth, the mice behaved normally. The cells regrew, and the cerebellum developed as it should. This happened because the loss of the Purkinje cells activated a population of immature Purkinje cells (iPCs). These cells would normally mature into adult Purkinje cells, but in their immature state they can still divide and make copies of themselves to replace lost neurons after injury.

As the mice grew older, the number of iPCs started to drop as the immature cells developed into adult Purkinje cells. When the iPCs ran out, any cells available to divide were gone and the mice could no longer replace any damaged Purkinje cells – the repair window had closed.

This work raises the possibility that other types of immature cells in the brain could be set aside to help repair damage during early development. A better understanding of these cells could reveal clues about conditions such as autism, which have been linked to damages or faults in the cerebellum. It may also help to gain new insights into how to regenerate the adult brain after injury.

Most neurons in the brain are generated at specific developmental time points, and once a neuron becomes postmitotic regeneration following injury is limited, except for in two forebrain regions that maintain neurogenesis (Chaker et al., 2016). In the context of injury, adult forebrain neurons undergo limited recovery that involves either reactive gliosis (Buffo et al., 2008; Robel et al., 2011; Sirko et al., 2013) or migration of neural stem cells from the neurogenic niches (Benner et al., 2013; Llorens-Bobadilla et al., 2015; López-Juárez et al., 2013; Martí-Fàbregas et al., 2010). The cerebellum (CB) of the hindbrain has a complex folded structure that houses the majority of neurons in the brain and is essential for balance and motor coordination, as well as higher order reasoning via circuits it forms throughout the forebrain (Fatemi et al., 2012; Steinlin, 2007; Tavano et al., 2007; Tsai et al., 2012; Wagner et al., 2017). For two weeks after birth, the postnatal mouse CB consists of both neurons generated in the embryo, and two neurogenic progenitor pools that produce late born neurons and glia. Interestingly, the proliferating granule cell progenitors can be replenished following injury by adaptive reprograming of the second Nestin-expressing progenitors (Wojcinski et al., 2017). However, once a neurogenic process has ended, the degree to which post mitotic neurons can undergo regeneration is poorly understood.

Purkinje cells (PC) are born by embryonic day (E) 13.5 in the mouse and during weeks 10–11 in humans (Rakic and Sidman, 1970; Wang and Zoghbi, 2001). After exiting the cell cycle in the ventricular zone, PCs express FOXP2 as they migrate to form a PC layer (PCL) under the cerebellar surface by E17.5, and turn on Calbindin1 (CALB1) in the late embryo and stop expressing FOXP2 by two weeks after birth. PCs play a central role in postnatal CB development by being the main source of sonic hedgehog (SHH), which is required for proliferation of granule cell progenitors and Nestin-expressing progenitors that produce interneurons and astrocytes (Corrales et al., 2006; Fleming et al., 2013; Lewis et al., 2004). PCs also are key for CB function by integrating the inputs that converge on the cerebellar cortex (Sillitoe and Joyner, 2007). Hence, PC loss is linked to cerebellar motor behavior syndromes and has also been implicated in autism (Fatemi et al., 2012; Tsai et al., 2012; Wang et al., 2014). In this study we determined the regenerative potential of PCs in neonatal mice.

To ablate and track PCs, the diphtheria toxin receptor (DTR) and a lineage tracer, TdTomato (TdT), were expressed in a subpopulation of PCs using a transgenic approach (Pcp2^Cre/+; R26^{LSL-DTR/LSL-TdT} or PC-DTR mice; LSL = lox stop-lox). We found that only 52.16 ± 21.84% of PCs (n = 5 mice), identified by expression of CALB1, expressed TdT and DTR at postnatal day (P) 1, and surprisingly the percentage and large variation remained similar at P5 and P30 (Figure 1—figure supplement 1). Strikingly, when DT was injected at P1 into PC-DTR pups (P1-PC-DTR), nearly all TdT+ PCs formed an ectopic layer below the PC layer (PCL) by 1 day post injection (dpi) (Figure 1A–M). The ectopic layer was absent by P8 (Figure 1K), and TdT+ cells in the ectopic layer were TUNEL positive starting at P3 with a peak at P5. These results show that almost all DTR-expressing TdT+ cells become misplaced, die and are cleared within 5–7 dpi of DT (Figure 1N,O).

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Figure 1—source data 1

Summary of the antibodies used in the study.

https://doi.org/10.7554/eLife.39879.007

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Figure 1—source data 2

Summary of the statistics performed.

https://doi.org/10.7554/eLife.39879.008

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Unexpectedly, although the number of CALB1+ PCs in the PCL of P1-PC-DTR mice was significantly reduced at P2 compared to non-injected controls (No DT), it was not significantly reduced at P3 and later stages (Figure 1P). Furthermore, the total number of PCs (ectopic layer + PCL) was significantly greater in DT-injected cerebella than in No DT controls at P2 and P3, and the total number of PCs was down to normal levels at P5, overlapping with the time of clearance of the ectopic layer (Figure 1P). Although the number of TdT+ cells in the PCL increased between P8 and P30 in P1-PC-DTR brains, it remained significantly lower than in No DT controls at P30 (Figure 1Q). Given that there is no significant increase in the recombination induced by Pcp2^Cre after birth in the control postnatal CB (Figure 1Q, Figure 1—figure supplement 1F), the percentage of TdT+ cells in P1-PC-DTR brains at P30 (~25–30%) matched the predicted percentage if 50% of the PCs were killed by DT and then ~50% of the regenerated PCs underwent recombination. Interestingly, and consistent with the rapid recovery of PC numbers in the PCL, no significant decrease in the sectional area of the CB was observed between P1.5 and P30 (Figure 1R–T, Figure 1—figure supplement 2). Furthermore, the thickness of the outer (proliferating) and inner (differentiating) external granule cell layers remained normal (Figure 1—figure supplement 3). In summary, we uncovered that the CB can rapidly recover (within 24–48 hr) from the loss of ~50% of PCs at P1, by producing new PCs and resuming normal growth.

In order to document the rapid production of new PCs after ablation, we tested whether PCs that had recently undergone cell division could be detected at P3. P1-PC-DTR mice were divided into four groups; each group receiving three injections of BrdU (2 hr apart) during 4–26 hr after DT-injection (Figure 2A). As predicted, BrdU+ PCs (FoxP2+ and CALB1+) were observed in the PCL of all groups (Figure 2B, Figure 2—figure supplement 1), with the greatest incorporation being between 10–20 hr after DT (Figure 2C, Figure 2—figure supplement 1). Importantly, in No DT mice no incorporation of BrdU was observed in PCs (Figure 2—figure supplement 1). Curiously, FOXP2 and BrdU showed non-overlapping subnuclear localization in the nuclei of PCs. In addition, when we analyzed brains of P1-PC-DTR mice at P30 that had received BrdU 10–14 hr after DT injection, we observed BrdU+ mature PCs with similar cell bodies and dendritic trees to their neighbors, showing that the newly generated PCs differentiate and survive to adulthood (Figure 2—figure supplement 2). Furthermore, a lack of BrdU incorporation in the ectopic layer at P3 shows that the labeling of PCs is not due to DNA damage induced by DT-mediated cell death (Figure 2—figure supplement 1C).

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In order to further confirm that BrdU incorporation is not due to DNA damage following DT injection, we treated P1 pups either with 4Gy γ-irradiation or DT at P1 followed by three BrdU injections (2 hr apart) at 30 min or 10 hr after treatment, respectively. The brains were then analyzed 24 hr after the last BrdU injection. In the irradiated pups we observed extensive γ-H2AX foci, including in PCs, but BrdU incorporation was not detected in any PCs. In contrast, P1-PC-DTR mice injected with DT showed BrdU incorporation in PCs without any γ-H2AX foci (Figure 2—figure supplement 3). Thus, DNA damage does not account for the BrdU incorporation into PCs following ablation of ~50% of PCs at P1.

As a second means to specifically label dividing cells that give rise to new PCs, we intracranially injected GFP-expressing retrovirus into P1-PC-DTR pups and littermate controls 12 hr after DT injection, since retroviruses can only incorporate into the DNA of dividing cells and are widely used for clonal analysis of neural stem cells and progeny (Figure 2D)(Cepko, 1988; Yu et al., 2009). When the mice were analyzed at P21, we indeed observed rare GFP-labeled CALB1+ PCs in P1-PC-DTR animals near the site of injection, and not in No DT controls. As expected, GFP+ granule cells and Bergmann glia were observed in both the No DT and P1-PC-DTR mice (Figure 2E–I). These three sets of experiments thus reveal that a progenitor capable of proliferating produces the new PCs after ablation at P1.

Based on the rapid replenishment of PCs after ablation at P1, we hypothesized that a local progenitor in the PCL must be responsible for the response. The Nestin-expressing progenitors (NEPs) in the PCL were a candidate, as they display plasticity upon ablation of granule cell precursors in newborn mice (Wojcinski et al., 2017). Furthermore a putative rare Nestin+ cell in the adult CB was recently described as able to produce new neurons in response to exercise (Ahlfeld et al., 2017). However, when we tested the contribution of NEPs to PC regeneration using a Nes-CFP reporter allele that transiently maintains CFP protein after differentiation, no CFP+ cells were found to co-express FOXP2 or CALB1 at 12 hr and 2 days post DT injection in P1-PC-DTR mice and in No DT controls (Figure 2—figure supplement 4). Furthermore, inducible fate mapping of NEPs using Nestin-FlpoER/+; R26^FSF-TdT/+ (FSF = frt stop-frt) mice showed no TdT+ PCs at P30 in P1-PC-DTR and No DT control mice given tamoxifen at P0 (Figure 2—figure supplement 5). These results suggest that a progenitor other than NEPs mediates regeneration following PC depletion.

We next examined whether a progenitor exists after birth that expresses early (FOXP2) but not late (CALB1) PC markers. Indeed, at P1 we identified CALB1 negative/low and FOXP2-expressing cells that could be immature PCs (named iPC for immature Purkinje cells; Figure 3A–B, Figure 3—video 1). Possibly accounting for the regeneration of PCs, iPCs were not labeled by Pcp2^Cre as they were TdT and DTR negative in No DT controls, thus they escape DT-mediated cell death (Figure 3—figure supplement 1). Temporal analyses revealed a decrease in the number of iPCs from P1 (74.33 ± 5.69/midline sagittal section) to P5 (28.66 ± 7.51/midline sagittal section, Figure 3A, Figure 3—figure supplement 1), indicating the progenitors are a transient population. Interestingly, the few iPCs present at P5 were specifically enriched in the central and nodular zones of the CB, which are developmentally delayed at P5 (Legué et al., 2015; Sudarov and Joyner, 2007)(Figure 3A).

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In order to investigate the normal fate of iPCs, we tested whether there is an increase in the number of CALB1+ PCs from P1 to P30. In order to minimize variation across animals, we used the C57BL/6 inbred strain of mice and analyzed the entire half-vermis of each brain (every second section). As expected, a significant reduction in the number of iPCs was observed between P1 and P5, but in addition we detected a significant increase in the number of CALB1+ cells at P30 compared to P1 (Figure 3C). At P5 there was a trend towards an increase in the number of CALB1+ cells. There was also a trend towards a reduction in the total number of iPCs plus CALB1+ cells at P5 and P30 compared to P1 (Figure 3C), suggesting that some PCs may also undergo apoptosis during early postnatal development. These results provide evidence that iPCs are cells destined to become PCs, but normally undergo a delay in differentiation until the first week after birth.

We then asked whether the number of iPCs increases after DT treatment of P1-PC-DTR mice. Quantification of iPC numbers showed a significant increase 12 hr after DT injection in P1-PC-DTR mice (1.90 ± 0.05 fold, Figure 3D), correlating with the time window of highest BrdU incorporation after injury (Figure 2B). Interestingly, at P5 the number of iPCs was significantly lower in P1-PC-DTR animals than in No DT mice (Figure 3D), possibly reflecting an exhaustion of the progenitor population by production of new PCs. To further show that iPCs expand in number after their neighbors are killed, we used constitutive FLP-based fate mapping in FoxP2^Flpo/+; R26^FSF-TdT/+ mice to transiently mark and follow PCs and iPCs. We found that all CALB1+ PCs and iPCs expressed TdT at P1 (Figure 3—figure supplement 2), and as predicted, an increase in transiently fate mapped TdT+ iPCs was seen in P1-PC-DTR mice 12 hr after DT injection at P1 compared to No DT controls (1.86 ± 0.46–fold, n = 3, Figure 3—figure supplement 3). Thus, iPCs expand in number after damage to neighboring PCs.

To confirm that iPCs undergo proliferation upon PC depletion, we injected BrdU or EdU 10–14 hr after DT and collected cerebella 1 hr (~P1.5) later. Other than glial progenitors and microglia seen in No DT controls (Figure 3—figure supplement 4), all additional BrdU+ (or EdU+) cells in the PCL of P1-PC-DTR mice expressed FOXP2, and of these cells 45.5 ± 1.1% expressed CALB1 (Figure 3E–N, Figure 3—figure supplements 5,6). Furthermore, the total number of FOXP2+ cells in the PCL that were acutely labeled with BrdU, was similar to the number of BrdU+ cells that became PCs (CALB1+) at P3 (38.7 ± 9.1/section vs 40.3 ± 19.0/section, n = 3, Figure 3—figure supplement 5). In addition, FOXP2+ cells that were Ki67+ (Figure 3O–S, Figure 3—figure supplement 6C) or pH3+ (Figure 3T–X, Figure 3—figure supplement 6D) and EdU+ were detected at P1.5 in the PCL of P1-PC-DTR pups, confirming the presence of proliferative iPCs following PC ablation. In order to further study the cell-cycle state of iPCs in uninjured cerebella, we analyzed the expression levels of the cell cycle inhibitor P27^Kip1 (Watanabe et al., 1998) and KI67 in iPCs compared to CALB1 high FOXP2+ PCs at P1. Fluorescent intensity analyses revealed that PCs have higher P27 and lower KI67 expression levels compared to iPCs (Figure 3—figure supplement 7). Collectively, our data argues that the recovery of PCs in P1-PC-DTR mice is mediated by a previously unrecognized and age-dependent progenitor population (iPCs) that normally transitions to a CALB1+ PC, but in response to loss of PCs proliferates and differentiates to replace the lost cells.

Given that the population of iPCs is greatly reduced by P5 (Figure 3C), PCs should not be efficiently replaced if ablated at P5, when similar to at P1 Pcp2^Cre induces recombination (expression of DTR) in 40.5 ± 21.5% of CALB1+ PCs (Figure 1—figure supplement 1). As predicted, when DT was injected at P5 (P5-PC-DTR mice) (Figure 4—figure supplement 1A), the number of PCs was significantly reduced at P12 compared to No DT controls (Figure 4—figure supplement 1B–I,R). TdT+ PCs were TUNEL+ by P8 (Figure 4—figure supplement 1J–K) and the majority of TdT+ cells were cleared from the PCL by P12 (Figure 4—figure supplement 1G,P and S). Furthermore, in P5-PC-DTR mice at P8 and P12 the dendrites and cell bodies of the PCs were poorly organized compared to in controls (Figure 4—figure supplement 1B–G and L–P) and at P30 the cell bodies of some PCs were misplaced into the molecular layer (Figure 4—figure supplement 1N–R). Importantly, the reduction in PC numbers observed at P12 was maintained at P30 (Figure 4—figure supplement 1R), such that the number of PCs was reduced by 32.4 ± 6.5%. In summary, there is little replenishment of PCs when they are ablated at P5 (Figure 4A).

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We next tested whether the rare iPCs at P5 (Figure 3A) can still proliferate upon PC depletion. In contrast to P1-PC-DTR mice, very few iPCs/PCs were BrdU+ in P5-PC-DTR cerebella injected with BrdU at 10–14 hr post DT-injection at both 1 hr (5.55 ± 0.51/ midline sagittal section, n = 3) and 1.5 days (6.22 ± 1.07/ midline sagittal section, n = 3, Figure 4B) post BrdU-injection. The few BrdU+ iPCs/PCs present were concentrated in the central and the nodular zones that are enriched for iPCs at P5 (Figure 4—figure supplement 2). Interestingly, compared to P1-PC-DTR mice in which 52.29 ± 0.09% (n = 3) of iPCs incorporated BrdU, only 20.55 ± 0.07% (n = 3) incorporated BrdU in P5-PC-DTR animals. Overall, these results demonstrate that replenishment of PCs is not efficient at P5 because with age, iPCs both diminish in number and in their ability to proliferate in response to PC depletion.

We next examined whether the depletion of PCs in P5-PC-DTR mice had an effect on CB development. Indeed, the area of CB sections was significantly reduced at P12 but not P8 (Figure 4C, Figure 4—figure supplement 3 although the thickness of the external granule cell layer was significantly reduced in P5-PC-DTR mice at P8. By P12 the thickness of the external granule cell layer was similar in PC-ablated mice and controls (Figure 4—figure supplement 4A–E). Surprisingly, despite the lack of recovery of PC numbers we found that the area of the CB was normal at P16 and P30 (Figure 4C, Figure 4—figure supplement 3A–I). As a consequence, there was a reduction in PC density compared to No DT or to P1-PC-DTR mice (Figure 4—figure supplement 3J, Figure 4—figure supplement 2N,Q). The density of granule cells also was lower compared to No DT and P1-PC-DTR P30 mice (Figure 4—figure supplement 4F). Interestingly, PCs in P5-PC-DTR mice had a larger soma (Figure 4D) and longer primary and secondary dendrites (Figure 4E) compared to No DT or P1-PC-DTR mice, a cellular phenotype observed in some mouse mutants with PC loss (Castagna et al., 2016). Furthermore, compared to controls, the percentage of PCs present at P30 in P5-PC-DTR animals compared to No DT controls (~66%) did not match the percentage of granule cells that were produced (~81% of No DT controls). Thus, the ratio of PCs to granule cells is disrupted in P5-PC-DTR animals because granule cells are over-produced. These results reveal that independent of iPCs being stimulated to produce new PCs following their ablation, there are mechanisms of cell and organ size regulation that ensure recovery of CB size.

Finally, given that the circuitry (proportions of neurons) is disrupted in P5-PC-DTR mice and not P1-PC-DTR mice but CB size is normal in both, we tested whether either mutant has normal motor function at P30. Interestingly, P1-PC-DTR animals had no significant changes in their motor function compared to controls (Figure 4F–J), whereas P5-PC-DTR mice showed a significant reduction in their latency to fall from the rotarod and had a shorter stride compared to both No DT and P1-PC-DTR mice (Figure 4F–G and I–J) but no change in grip strength (Figure 4H). These results demonstrate that P5-PC-DTR mice, but not P1-PC-DTR mice, have motor behavior deficits. Thus, rapid production of new PCs by iPCs enables establishment of functional circuits following depletion of PCs at P1. Furthermore, achieving correct cell numbers and/or proportions appears to be more important than maintaining CB size for functional recovery after injury in P5-PC-DTR mice.

In summary, we discovered a new regenerative process in the developing CB involving a previously unidentified and normally dormant and immature PC progenitor (iPC) that is able to expand and produce additional PCs, likely to buffer against early postnatal loss of these postmitotic neurons due to injury. Proliferation of iPCs is stimulated by ablation of PCs at P1 and importantly the response is rapid (10–48 hr), ensuring other components of the developing CB that are dependent on PCs for their proliferation or differentiation are not compromised. However, iPCs decrease in number and their capacity to proliferate during the first postnatal week, and consequently PCs are poorly replenished when ablated at P5. The cerebella of P5-PC-DTR mice nevertheless try to adapt by attaining near normal dimensions through a mechanism that includes increasing cell size (Figure 4—figure supplement 5). The CB therefore has multiple mechanisms for regulating organ size following perinatal injury that depend on the precise stage of development. Furthermore, the motor deficits seen in P5-PC-DTR mice highlight the importance of maintaining the correct number of PCs and relative neuron proportions during development, not just organ size.

One possible reason for why iPCs differentiate into PCs after P1 and lose their ability to proliferate is that a critical component of the microenvironment that supports iPCs is diminished soon after birth, perhaps as a consequence of a developmental clock that the cells in the microenvironment follow. A second possibility is that the differentiation of iPCs is dictated by the timing of the establishment of the cerebellar circuitry. We speculate that efficient regeneration is possible at P1 because PCs still have an immature morphology and integration into the cerebellar circuitry, whereas at later stages the parallel fibers (axons of granule cells) synapse with PCs and climbing fibers (axons of the inferior olive neurons) refine their synapses and both cells promote PC maturation (Good et al., 2017; Hoxha et al., 2017). Thus, maturation and integration of a newly generated PC into the cerebellar circuitry might not be efficient or possible after P5. By extrapolation, the replenishment process has evolved such that developmental plasticity is tightly correlated with age-dependent maturation of the neural circuit.

An additional cellular process to consider for the age-dependency of regeneration is the ability of neurons to enter back into the cell-cycle. Most differentiated neurons, including PCs, when forced to proliferate undergo apoptosis (Feddersen et al., 1992). However, previous reports have shown that following experimental manipulation or neurodegeneration, ectopic proliferation of adult retinal and pyramidal neurons can occur (Ajioka et al., 2007; Sage et al., 2005; Skapek et al., 2001; Yang et al., 2001). Our data indicate that iPCs, which lack the mature PC marker CALB1 and express the immature marker FOXP2, show low expression of P27 and weak but higher expression of KI67 compared to CALB1+ PCs, suggesting that their cell-cycle exit may be incomplete. CALB1+ PCs, likely ones that recently began making CALB1 protein, also appear to be able to re-enter the cell-cyle. However, the increase in the number of iPCs ~12 hr after DT administration suggests that the main regenerative response is achieved by the proliferation of iPCs.

The regenerative processes previously described in neuronal tissues involve adaptive reprograming of cells that are either actively proliferating or retain proliferative capacity and also have cell fate plasticity (Benner et al., 2013; Buffo et al., 2008; Jinnou et al., 2018; Lin et al., 2017; Llorens-Bobadilla et al., 2015; López-Juárez et al., 2013; Martí-Fàbregas et al., 2010; Robel et al., 2011; Samanta et al., 2015; Sirko et al., 2013; Wojcinski et al., 2017). Here we identified a distinct regenerative process that involves a local and normally dormant or immature progenitor. Unlike NEPs of the CB or astrocytes and neural stem cells in the forebrain that produce neurons upon injury, iPCs do not require reprograming and cell fate plasticity as our data indicates that they normally produce additional CALB1+ PCs after birth. Thus, iPCs maintain their lineage decision, but proliferate and then mature upon injury. An important question raised by our study is whether regeneration of postmitotic neurons by age-dependent progenitors is unique to the CB where protracted development might provide a conducive milieu, or whether all brain regions retain similar progenitors for a particular time window after each neuron subtype is generated. Furthermore, understanding the mechanisms of PC regeneration in newborn mice could provide insights into how regeneration in the adult brain can be enabled.

All data generated or analysed during this study are included in the manuscript and supporting files.

1. 1. Ahlfeld J
   2. Filser S
   3. Schmidt F
   4. Wefers AK
   5. Merk DJ
   6. Glaß R
   7. Herms J
   8. Schüller U

   (2017) Neurogenesis from Sox2 expressing cells in the adult cerebellar cortex

   Scientific Reports 7:6137.

   https://doi.org/10.1038/s41598-017-06150-x

   • PubMed
   • Google Scholar
2. 1. Ajioka I
   2. Martins RA
   3. Bayazitov IT
   4. Donovan S
   5. Johnson DA
   6. Frase S
   7. Cicero SA
   8. Boyd K
   9. Zakharenko SS
   10. Dyer MA

   (2007) Differentiated horizontal interneurons clonally expand to form metastatic retinoblastoma in mice

   Cell 131:378–390.

   https://doi.org/10.1016/j.cell.2007.09.036

   • PubMed
   • Google Scholar
3. 1. Benner EJ
   2. Luciano D
   3. Jo R
   4. Abdi K
   5. Paez-Gonzalez P
   6. Sheng H
   7. Warner DS
   8. Liu C
   9. Eroglu C
   10. Kuo CT

   (2013) Protective astrogenesis from the SVZ niche after injury is controlled by Notch modulator Thbs4

   Nature 497:369–373.

   https://doi.org/10.1038/nature12069

   • PubMed
   • Google Scholar
4. 1. Bikoff JB
   2. Gabitto MI
   3. Rivard AF
   4. Drobac E
   5. Machado TA
   6. Miri A
   7. Brenner-Morton S
   8. Famojure E
   9. Diaz C
   10. Alvarez FJ
   11. Mentis GZ
   12. Jessell TM

   (2016) Spinal inhibitory interneuron diversity delineates variant motor microcircuits

   Cell 165:207–219.

   https://doi.org/10.1016/j.cell.2016.01.027

   • PubMed
   • Google Scholar
5. 1. Buch T
   2. Heppner FL
   3. Tertilt C
   4. Heinen TJ
   5. Kremer M
   6. Wunderlich FT
   7. Jung S
   8. Waisman A

   (2005) A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration

   Nature Methods 2:419–426.

   https://doi.org/10.1038/nmeth762

   • PubMed
   • Google Scholar
6. 1. Buffo A
   2. Rite I
   3. Tripathi P
   4. Lepier A
   5. Colak D
   6. Horn AP
   7. Mori T
   8. Götz M

   (2008) Origin and progeny of reactive gliosis: A source of multipotent cells in the injured brain

   PNAS 105:3581–3586.

   https://doi.org/10.1073/pnas.0709002105

   • PubMed
   • Google Scholar
7. 1. Castagna C
   2. Merighi A
   3. Lossi L

   (2016) Cell death and neurodegeneration in the postnatal development of cerebellar vermis in normal and Reeler mice

   Annals of Anatomy - Anatomischer Anzeiger 207:76–90.

   https://doi.org/10.1016/j.aanat.2016.01.010

   • PubMed
   • Google Scholar
8. 1. Cepko C

   (1988) Retrovirus vectors and their applications in neurobiology

   Neuron 1:345–353.

   https://doi.org/10.1016/0896-6273(88)90184-5

   • PubMed
   • Google Scholar
9. 1. Chaker Z
   2. Codega P
   3. Doetsch F

   (2016) A mosaic world: puzzles revealed by adult neural stem cell heterogeneity

   Wiley Interdisciplinary Reviews: Developmental Biology 5:640–658.

   https://doi.org/10.1002/wdev.248

   • PubMed
   • Google Scholar
10. 1. Corrales JD
    2. Blaess S
    3. Mahoney EM
    4. Joyner AL

    (2006) The level of sonic hedgehog signaling regulates the complexity of cerebellar foliation

    Development 133:1811–1821.

    https://doi.org/10.1242/dev.02351

    • PubMed
    • Google Scholar
11. 1. Fatemi SH
    2. Aldinger KA
    3. Ashwood P
    4. Bauman ML
    5. Blaha CD
    6. Blatt GJ
    7. Chauhan A
    8. Chauhan V
    9. Dager SR
    10. Dickson PE
    11. Estes AM
    12. Goldowitz D
    13. Heck DH
    14. Kemper TL
    15. King BH
    16. Martin LA
    17. Millen KJ
    18. Mittleman G
    19. Mosconi MW
    20. Persico AM
    21. Sweeney JA
    22. Webb SJ
    23. Welsh JP

    (2012) Consensus paper: pathological role of the cerebellum in autism

    The Cerebellum 11:777–807.

    https://doi.org/10.1007/s12311-012-0355-9

    • PubMed
    • Google Scholar
12. 1. Feddersen RM
    2. Ehlenfeldt R
    3. Yunis WS
    4. Clark HB
    5. Orr HT

    (1992) Disrupted cerebellar cortical development and progressive degeneration of Purkinje cells in SV40 T antigen transgenic mice

    Neuron 9:955–966.

    https://doi.org/10.1016/0896-6273(92)90247-B

    • PubMed
    • Google Scholar
13. 1. Fleming JT
    2. He W
    3. Hao C
    4. Ketova T
    5. Pan FC
    6. Wright CC
    7. Litingtung Y
    8. Chiang C

    (2013) The Purkinje neuron acts as a central regulator of spatially and functionally distinct cerebellar precursors

    Developmental Cell 27:278–292.

    https://doi.org/10.1016/j.devcel.2013.10.008

    • PubMed
    • Google Scholar
14. 1. Good JM
    2. Mahoney M
    3. Miyazaki T
    4. Tanaka KF
    5. Sakimura K
    6. Watanabe M
    7. Kitamura K
    8. Kano M

    (2017) Maturation of cerebellar purkinje cell population activity during postnatal refinement of climbing fiber network

    Cell Reports 21:2066–2073.

    https://doi.org/10.1016/j.celrep.2017.10.101

    • PubMed
    • Google Scholar
15. 1. Gu Y
    2. Janoschka S
    3. Ge S

    (2011) Studying the integration of adult-born neurons

    Journal of Visualized Experiments.

    https://doi.org/10.3791/2548

    • PubMed
    • Google Scholar
16. 1. Hoxha E
    2. Lippiello P
    3. Scelfo B
    4. Tempia F
    5. Ghirardi M
    6. Miniaci MC

    (2017) Maturation, refinement, and serotonergic modulation of cerebellar cortical circuits in normal development and in murine models of autism

    Neural Plasticity 2017:6595740.

    https://doi.org/10.1155/2017/6595740

    • PubMed
    • Google Scholar
17. 1. Jinnou H
    2. Sawada M
    3. Kawase K
    4. Kaneko N
    5. Herranz-Pérez V
    6. Miyamoto T
    7. Kawaue T
    8. Miyata T
    9. Tabata Y
    10. Akaike T
    11. García-Verdugo JM
    12. Ajioka I
    13. Saitoh S
    14. Sawamoto K

    (2018) Radial glial fibers promote neuronal migration and functional recovery after neonatal brain injury

    Cell Stem Cell 22:128–137.

    https://doi.org/10.1016/j.stem.2017.11.005

    • Google Scholar
18. 1. Legué E
    2. Riedel E
    3. Joyner AL

    (2015) Clonal analysis reveals granule cell behaviors and compartmentalization that determine the folded morphology of the cerebellum

    Development 142:1661–1671.

    https://doi.org/10.1242/dev.120287

    • PubMed
    • Google Scholar
19. 1. Lewis PM
    2. Gritli-Linde A
    3. Smeyne R
    4. Kottmann A
    5. McMahon AP

    (2004) Sonic hedgehog signaling is required for expansion of granule neuron precursors and patterning of the mouse cerebellum

    Developmental Biology 270:393–410.

    https://doi.org/10.1016/j.ydbio.2004.03.007

    • PubMed
    • Google Scholar
20. 1. Lin B
    2. Coleman JH
    3. Peterson JN
    4. Zunitch MJ
    5. Jang W
    6. Herrick DB
    7. Schwob JE

    (2017) Injury induces endogenous reprogramming and dedifferentiation of neuronal progenitors to multipotency

    Cell Stem Cell 21:761–774.

    https://doi.org/10.1016/j.stem.2017.09.008

    • Google Scholar
21. 1. Llorens-Bobadilla E
    2. Zhao S
    3. Baser A
    4. Saiz-Castro G
    5. Zwadlo K
    6. Martin-Villalba A

    (2015) Single-Cell transcriptomics reveals a population of dormant neural stem cells that become activated upon brain injury

    Cell Stem Cell 17:329–340.

    https://doi.org/10.1016/j.stem.2015.07.002

    • PubMed
    • Google Scholar
22. 1. López-Juárez A
    2. Howard J
    3. Ullom K
    4. Howard L
    5. Grande A
    6. Pardo A
    7. Waclaw R
    8. Sun YY
    9. Yang D
    10. Kuan CY
    11. Campbell K
    12. Nakafuku M

    (2013) Gsx2 controls region-specific activation of neural stem cells and injury-induced neurogenesis in the adult subventricular zone

    Genes & Development 27:1272–1287.

    https://doi.org/10.1101/gad.217539.113

    • PubMed
    • Google Scholar
23. 1. Madisen L
    2. Garner AR
    3. Shimaoka D
    4. Chuong AS
    5. Klapoetke NC
    6. Li L
    7. van der Bourg A
    8. Niino Y
    9. Egolf L
    10. Monetti C
    11. Gu H
    12. Mills M
    13. Cheng A
    14. Tasic B
    15. Nguyen TN
    16. Sunkin SM
    17. Benucci A
    18. Nagy A
    19. Miyawaki A
    20. Helmchen F
    21. Empson RM
    22. Knöpfel T
    23. Boyden ES
    24. Reid RC
    25. Carandini M
    26. Zeng H

    (2015) Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance

    Neuron 85:942–958.

    https://doi.org/10.1016/j.neuron.2015.02.022

    • PubMed
    • Google Scholar
24. 1. Madisen L
    2. Zwingman TA
    3. Sunkin SM
    4. Oh SW
    5. Zariwala HA
    6. Gu H
    7. Ng LL
    8. Palmiter RD
    9. Hawrylycz MJ
    10. Jones AR
    11. Lein ES
    12. Zeng H

    (2010) A robust and high-throughput Cre reporting and characterization system for the whole mouse brain

    Nature Neuroscience 13:133–140.

    https://doi.org/10.1038/nn.2467

    • PubMed
    • Google Scholar
25. 1. Martí-Fàbregas J
    2. Romaguera-Ros M
    3. Gómez-Pinedo U
    4. Martínez-Ramírez S
    5. Jiménez-Xarrié R Marín E
    6. J-L Martí-Vilalta R
    7. Garcia-Verdugo JM

    (2010) Proliferation in the human ipsilateral subventricular zone after ischemic stroke: neurology

    Annals of Neurosciences 17:134–135.

    https://doi.org/10.5214/ans.0972-7531.1017308

    • PubMed
    • Google Scholar
26. 1. Mignone JL
    2. Kukekov V
    3. Chiang AS
    4. Steindler D
    5. Enikolopov G

    (2004) Neural stem and progenitor cells in nestin-GFP transgenic mice

    The Journal of Comparative Neurology 469:311–324.

    https://doi.org/10.1002/cne.10964

    • PubMed
    • Google Scholar
27. 1. Rakic P
    2. Sidman RL

    (1970) Histogenesis of cortical layers in human cerebellum, particularly the lamina dissecans

    The Journal of Comparative Neurology 139:473–500.

    https://doi.org/10.1002/cne.901390407

    • PubMed
    • Google Scholar
28. 1. Robel S
    2. Berninger B
    3. Götz M

    (2011) The stem cell potential of glia: lessons from reactive gliosis

    Nature Reviews Neuroscience 12:88–104.

    https://doi.org/10.1038/nrn2978

    • PubMed
    • Google Scholar
29. 1. Sage C
    2. Huang M
    3. Karimi K
    4. Gutierrez G
    5. Vollrath MA
    6. Zhang DS
    7. García-Añoveros J
    8. Hinds PW
    9. Corwin JT
    10. Corey DP
    11. Chen ZY

    (2005) Proliferation of functional hair cells in vivo in the absence of the retinoblastoma protein

    Science 307:1114–1118.

    https://doi.org/10.1126/science.1106642

    • PubMed
    • Google Scholar
30. 1. Samanta J
    2. Grund EM
    3. Silva HM
    4. Lafaille JJ
    5. Fishell G
    6. Salzer JL

    (2015) Inhibition of Gli1 mobilizes endogenous neural stem cells for remyelination

    Nature 526:448–452.

    https://doi.org/10.1038/nature14957

    • PubMed
    • Google Scholar
31. 1. Sillitoe RV
    2. Joyner AL

    (2007) Morphology, molecular codes, and circuitry produce the three-dimensional complexity of the cerebellum

    Annual Review of Cell and Developmental Biology 23:549–577.

    https://doi.org/10.1146/annurev.cellbio.23.090506.123237

    • PubMed
    • Google Scholar
32. 1. Sirko S
    2. Behrendt G
    3. Johansson PA
    4. Tripathi P
    5. Costa M
    6. Bek S
    7. Heinrich C
    8. Tiedt S
    9. Colak D
    10. Dichgans M
    11. Fischer IR
    12. Plesnila N
    13. Staufenbiel M
    14. Haass C
    15. Snapyan M
    16. Saghatelyan A
    17. Tsai LH
    18. Fischer A
    19. Grobe K
    20. Dimou L
    21. Götz M

    (2013) Reactive glia in the injured brain acquire stem cell properties in response to sonic hedgehog. [corrected]

    Cell Stem Cell 12:426–439.

    https://doi.org/10.1016/j.stem.2013.01.019

    • PubMed
    • Google Scholar
33. 1. Skapek SX
    2. Lin SC
    3. Jablonski MM
    4. McKeller RN
    5. Tan M
    6. Hu N
    7. Lee EY

    (2001) Persistent expression of cyclin D1 disrupts normal photoreceptor differentiation and retina development

    Oncogene 20:6742–6751.

    https://doi.org/10.1038/sj.onc.1204876

    • PubMed
    • Google Scholar
34. 1. Steinlin M

    (2007) The cerebellum in cognitive processes: supporting studies in children

    The Cerebellum 6:237–241.

    https://doi.org/10.1080/14734220701344507

    • PubMed
    • Google Scholar
35. 1. Sudarov A
    2. Joyner AL

    (2007) Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers

    Neural Development 2:26.

    https://doi.org/10.1186/1749-8104-2-26

    • PubMed
    • Google Scholar
36. 1. Tavano A
    2. Grasso R
    3. Gagliardi C
    4. Triulzi F
    5. Bresolin N
    6. Fabbro F
    7. Borgatti R

    (2007) Disorders of cognitive and affective development in cerebellar malformations

    Brain 130:2646–2660.

    https://doi.org/10.1093/brain/awm201

    • PubMed
    • Google Scholar
37. 1. Tsai PT
    2. Hull C
    3. Chu Y
    4. Greene-Colozzi E
    5. Sadowski AR
    6. Leech JM
    7. Steinberg J
    8. Crawley JN
    9. Regehr WG
    10. Sahin M

    (2012) Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice

    Nature 488:647–651.

    https://doi.org/10.1038/nature11310

    • PubMed
    • Google Scholar
38. 1. Wagner MJ
    2. Kim TH
    3. Savall J
    4. Schnitzer MJ
    5. Luo L

    (2017) Cerebellar granule cells encode the expectation of reward

    Nature 544:96–100.

    https://doi.org/10.1038/nature21726

    • PubMed
    • Google Scholar
39. 1. Wang SS
    2. Kloth AD
    3. Badura A

    (2014) The cerebellum, sensitive periods, and autism

    Neuron 83:518–532.

    https://doi.org/10.1016/j.neuron.2014.07.016

    • PubMed
    • Google Scholar
40. 1. Wang VY
    2. Zoghbi HY

    (2001) Genetic regulation of cerebellar development

    Nature Reviews Neuroscience 2:484–491.

    https://doi.org/10.1038/35081558

    • PubMed
    • Google Scholar
41. 1. Watanabe G
    2. Pena P
    3. Shambaugh GE
    4. Haines GK
    5. Pestell RG

    (1998) Regulation of cyclin dependent kinase inhibitor proteins during neonatal cerebella development

    Developmental Brain Research 108:77–87.

    https://doi.org/10.1016/S0165-3806(98)00032-7

    • PubMed
    • Google Scholar
42. 1. Wojcinski A
    2. Lawton AK
    3. Bayin NS
    4. Lao Z
    5. Stephen DN
    6. Joyner AL

    (2017) Cerebellar granule cell replenishment postinjury by adaptive reprogramming of Nestin⁺ progenitors

    Nature Neuroscience 20:1361–1370.

    https://doi.org/10.1038/nn.4621

    • PubMed
    • Google Scholar
43. 1. Yang Y
    2. Geldmacher DS
    3. Herrup K

    (2001) DNA replication precedes neuronal cell death in Alzheimer's disease

    The Journal of Neuroscience 21:2661–2668.

    https://doi.org/10.1523/JNEUROSCI.21-08-02661.2001

    • PubMed
    • Google Scholar
44. 1. Yu YC
    2. Bultje RS
    3. Wang X
    4. Shi SH

    (2009) Specific synapses develop preferentially among sister excitatory neurons in the neocortex

    Nature 458:501–504.

    https://doi.org/10.1038/nature07722

    • PubMed
    • Google Scholar
45. 1. Zhang XM
    2. Ng AH
    3. Tanner JA
    4. Wu WT
    5. Copeland NG
    6. Jenkins NA
    7. Huang JD

    (2004) Highly restricted expression of Cre recombinase in cerebellar Purkinje cells

    Genesis 40:45–51.

    https://doi.org/10.1002/gene.20062

    • PubMed
    • Google Scholar
46. 1. Zhao C
    2. Teng EM
    3. Summers RG
    4. Ming GL
    5. Gage FH

    (2006) Distinct morphological stages of dentate granule neuron maturation in the adult mouse hippocampus

    Journal of Neuroscience 26:3–11.

    https://doi.org/10.1523/JNEUROSCI.3648-05.2006

    • PubMed
    • Google Scholar

Author details

1. N Sumru Bayin

   Developmental Biology Program, Sloan Kettering Institute, New York, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4371-855X

2. Alexandre Wojcinski

   Developmental Biology Program, Sloan Kettering Institute, New York, United States

   Present address

   John Wayne Cancer Institute, Santa Monica, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

3. Aurelien Mourton

   Developmental Biology Program, Sloan Kettering Institute, New York, United States

   Present address

   Center for Interdisciplinary Research in Biology, College de France, Paris, France

   Contribution

   Conceptualization, Data curation

   Competing interests

   No competing interests declared

4. Hiromitsu Saito

   Department of Animal Functional Genomics of Advanced Science Research Promotion Center, Organization for the Promotion of Regional Innovation, Mie University, Tsu, JAPAN

   Contribution

   Resources

   Competing interests

   No competing interests declared

5. Noboru Suzuki

   Department of Animal Functional Genomics of Advanced Science Research Promotion Center, Organization for the Promotion of Regional Innovation, Mie University, Tsu, JAPAN

   Contribution

   Resources

   Competing interests

   No competing interests declared

6. Alexandra L Joyner

   1. Developmental Biology Program, Sloan Kettering Institute, New York, United States
   2. Biochemistry, Cell and Molecular Biology Program, Weill Cornell Graduate School of Medical Sciences, New York, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Writing—review and editing

   For correspondence

   joynera@mskcc.org

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7090-9605

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