Lifelong regeneration of cerebellar Purkinje cells after induced cell ablation in zebrafish
Curation statements for this article:-
Curated by eLife
eLife assessment
The present manuscript addresses the controversial issue of the regeneration potential of cerebellar Purkinje cells in zebrafish and their integration into functional circuits. The authors use interesting genetic models to induce Purkinje cell-specific ablation to demonstrate regeneration of Purkinje cells can occur until adulthood and is accomplished by ptf1a+ progenitors. They further show that regenerated neurons reestablish electrophysiological properties and support appropriate behavior. These are important results that may help understand why mammalian neurons do not have similar properties and fail to regenerate. The conclusions on the source of regenerated neurons will however need additional experimental support.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
Zebrafish have an impressive capacity to regenerate neurons in the central nervous system. However, regeneration of the principal neuron of the evolutionary conserved cerebellum, the Purkinje cell (PC), is believed to be limited to developmental stages based on invasive lesions. In contrast, non-invasive cell type-specific ablation by induced apoptosis closely represents a process of neurodegeneration. We demonstrate that the ablated larval PC population entirely recovers in number, quickly reestablishes electrophysiological properties, and properly integrates into circuits to regulate cerebellum-controlled behavior. PC progenitors are present in larvae and adults, and PC ablation in adult cerebelli results in an impressive PC regeneration of different PC subtypes able to restore behavioral impairments. Interestingly, caudal PCs are more resistant to ablation and regenerate more efficiently, suggesting a rostro-caudal pattern of de- and regeneration properties. These findings demonstrate that the zebrafish cerebellum is able to regenerate functional PCs during all stages of the animal’s life.
Article activity feed
-
-
Author Response
Reviewer #1 (Public Review):
This is a well-performed and carefully executed and quantified study. There is however a point that needs clarification:
We thank the reviewer for these motivating comments and appreciate the careful reflection of our work.
The authors state that acute regeneration occurs between 5-10dpt. However, the graphs in Fig 1D, F, and 2F indicate that most PC generation occurs from 20-30 days. What happens in this period? Does proliferation increase? Can the authors perform BrdU incorporation between 6 days and 1 month?
The reviewer is right that PC regeneration seems to be more intense from 20-30 days. Yet during this stage also wildtype larvae add a number of PCs to their PC population pool, thus we would consider only PCs being added in surplus to the number of regularly added PCs as a …
Author Response
Reviewer #1 (Public Review):
This is a well-performed and carefully executed and quantified study. There is however a point that needs clarification:
We thank the reviewer for these motivating comments and appreciate the careful reflection of our work.
The authors state that acute regeneration occurs between 5-10dpt. However, the graphs in Fig 1D, F, and 2F indicate that most PC generation occurs from 20-30 days. What happens in this period? Does proliferation increase? Can the authors perform BrdU incorporation between 6 days and 1 month?
The reviewer is right that PC regeneration seems to be more intense from 20-30 days. Yet during this stage also wildtype larvae add a number of PCs to their PC population pool, thus we would consider only PCs being added in surplus to the number of regularly added PCs as a contribution to regeneration, and here we see in quantified samples the largest increase of regenerating PCs during 8-10 days post-treatment with 20,9 and 23,2 additional (surplus) PCs on average respectively.
This question also relates to the first comment of reviewer 3 who asked for a combined BrdU and EdU labeling approach to address the cell cycle length of PC progenitors. We have therefore performed this experiment with the first pulse of BrdU-labeling at 18 days after PC-ablation to include the request stated here for a BrdU-labeling at later stages of regeneration. Again, no significant difference between BrdU-positive PC progenitors was found at this later stage of PC regeneration, but a small number of PC progenitors underwent additional rounds of proliferation compared to controls, which provide an explanation of how the entire PC population is replenished and why complete PC regeneration requires several months. Please see also our answer to question 1 of reviewer 3. These new findings are now presented in an additional Supplementary Figure (Figure 1-figure supplement 3) and have been added to the last paragraph of the section reporting the findings presented in Figure 1.
Related to this, as the authors indicate in lines 129-131, the regeneration of new PCs overlaps with normal development. Are other neuronal cell types generated in appropriate numbers?
This is an interesting question raised by the reviewer. But it is very general relating to all cerebellar neuronal cell types, which is out of our possibilities to address. We considered eurydendroid cells as the most likely cell population, which could be affected in their numbers by PC ablation and regeneration, because eurydendroid cells share the same ptf1a+-expressing progenitor cells with Purkinje cells. Eurydendroid cells – the zebrafish equivalents to deep nuclei neurons in mammals – can be identified by their expression of olig2. We have therefore quantified the number of eurydendroid cells in the cerebellum of double transgenic PC-ATTAC/olig2:GFP larvae 15 days after PC ablation. No significant difference in olig2:GFP positive cells could be observed between PC-regenerating and control zebrafish suggesting that eurydendroid cells are not affected in their quantity and are generated in appropriate numbers in PC regenerating larvae. These findings are presented in a new Supplementary Figure (Figure 3-figure supplement 3) and are described together with findings about eurydendroid cells presented in the main Figure 3.
-
eLife assessment
The present manuscript addresses the controversial issue of the regeneration potential of cerebellar Purkinje cells in zebrafish and their integration into functional circuits. The authors use interesting genetic models to induce Purkinje cell-specific ablation to demonstrate regeneration of Purkinje cells can occur until adulthood and is accomplished by ptf1a+ progenitors. They further show that regenerated neurons reestablish electrophysiological properties and support appropriate behavior. These are important results that may help understand why mammalian neurons do not have similar properties and fail to regenerate. The conclusions on the source of regenerated neurons will however need additional experimental support.
-
Reviewer #1 (Public Review):
According to current knowledge, zebrafish neurons maintain the capacity of regenerating with the exception of adult cerebellar Purkinje cells (PC), which are thought to have lost this property. Regeneration instead occurs at larval stages but whether newly generated PC form fully functional circuits is still unclear. This elegant and well-performed study takes advantage of a transgenic zebrafish line that enables inducing apoptosis under a tamoxifen-inducible system and at the same time visualizes PCs morphology through a membrane tagged RFP. Using this line (and other lines that tag radial glial and ventricular progenitors) in combination with morphological and functional analysis, the authors show that ventricular progenitors retain the lifelong ability to regenerate PCs. At larval stages, the newly …
Reviewer #1 (Public Review):
According to current knowledge, zebrafish neurons maintain the capacity of regenerating with the exception of adult cerebellar Purkinje cells (PC), which are thought to have lost this property. Regeneration instead occurs at larval stages but whether newly generated PC form fully functional circuits is still unclear. This elegant and well-performed study takes advantage of a transgenic zebrafish line that enables inducing apoptosis under a tamoxifen-inducible system and at the same time visualizes PCs morphology through a membrane tagged RFP. Using this line (and other lines that tag radial glial and ventricular progenitors) in combination with morphological and functional analysis, the authors show that ventricular progenitors retain the lifelong ability to regenerate PCs. At larval stages, the newly regenerated PCs form fully functional circuits that lead to normal behavior. In adults, PC regeneration is less efficient (and PCs are also less prone to undergo apoptosis) but sufficient to support exploratory behavior. This study resolves the controversial issue of whether adult PC regeneration is possible and demonstrates that newly formed PCs at larval and adult stages can form functional circuits that support normal behavior.
This is a well-performed and carefully executed and quantified study. There is however a point that needs clarification:
The authors state that acute regeneration occurs between 5-10dpt. However, the graphs in Fig 1D, F, and 2F indicate that most PC generation occurs from 20-30 days. What happens in this period? Does proliferation increase? Can the authors perform BrdU incorporation between 6 days and 1 month? Related to this, as the authors indicate in lines 129-131, the regeneration of new PCs overlaps with normal development. Are other neuronal cell types generated in appropriate numbers?
-
Reviewer #2 (Public Review):
In this paper, Pose-Méndez and colleagues have investigated the lifelong ability of zebrafish for functional Purkinje cell regeneration after selective ablation. Previous studies have determined that the adult zebrafish cerebellum lacks the capacity to regenerate Purkinje cells after traumatic injury. The authors use an elegant approach to determine whether selective ablation of Purkinje cells, a scenario closer to neurodegenerative disease, would allow for regeneration. The overall message is, that Purkinje cell regeneration is accomplished at every age after targeted ablation. The authors find in a series of well-executed functional and behavioral experiments that selective loss of Purkinje cells leads to a change in neuronal circuit activity and behaviors. During the regeneration process and interestingly …
Reviewer #2 (Public Review):
In this paper, Pose-Méndez and colleagues have investigated the lifelong ability of zebrafish for functional Purkinje cell regeneration after selective ablation. Previous studies have determined that the adult zebrafish cerebellum lacks the capacity to regenerate Purkinje cells after traumatic injury. The authors use an elegant approach to determine whether selective ablation of Purkinje cells, a scenario closer to neurodegenerative disease, would allow for regeneration. The overall message is, that Purkinje cell regeneration is accomplished at every age after targeted ablation. The authors find in a series of well-executed functional and behavioral experiments that selective loss of Purkinje cells leads to a change in neuronal circuit activity and behaviors. During the regeneration process and interestingly before the full recovery of Purkinje cell numbers compared to controls neuronal activity as well as behaviors are recovered.
-
Reviewer #3 (Public Review):
In "Lifelong regeneration of cerebellar Purkinje neurons after induced cell ablation in zebrafish" by Pose-Mendez and colleagues, the authors followed the regenerative properties that Purkinje cells have in larvae and adult Zebrafish. These properties common in teleostean and other animals are rare in mammals and, therefore, their study is of great interest to the neurodevelopmental community.
In this work, the authors use an already established animal model (PC-ATTACTM) to selectively ablate Purkinje cells in the larvae and adult Zebrafish, in a temporal control manner, that is by administering 4-OHT at defined stages. In doing so, the authors show that a full recovery of an ablated Purkinje cell population can be achieved when the ablation is induced in the larval stage, but this recovery is more modest …
Reviewer #3 (Public Review):
In "Lifelong regeneration of cerebellar Purkinje neurons after induced cell ablation in zebrafish" by Pose-Mendez and colleagues, the authors followed the regenerative properties that Purkinje cells have in larvae and adult Zebrafish. These properties common in teleostean and other animals are rare in mammals and, therefore, their study is of great interest to the neurodevelopmental community.
In this work, the authors use an already established animal model (PC-ATTACTM) to selectively ablate Purkinje cells in the larvae and adult Zebrafish, in a temporal control manner, that is by administering 4-OHT at defined stages. In doing so, the authors show that a full recovery of an ablated Purkinje cell population can be achieved when the ablation is induced in the larval stage, but this recovery is more modest when the ablation is induced in the adult stage, albeit very significant. The authors also show that regenerated Purkinje cells quickly elaborate their native electrical properties and integrate into functional circuits, which allow for the recuperation of motor behaviors produced by the loss of ablated Purkinje cells.
Overall, the work by Pose-Mendez and colleagues contributes to our understanding of neuronal regeneration in non-mammals. Technically, this study is well conducted and the provided data support most of the conclusions made by the authors.
-
