The molecular basis underlying adult neurogenesis during regeneration and tissue renewal

The molecular basis underlying adult neurogenesis during regeneration and tissue renewal

Funding Type: 
New Faculty II
Grant Number: 
RN2-00940
Award Value: 
$1,712,620
Status: 
Active
Public Abstract: 
Regeneration of lost body parts has long fascinated humans, yet regeneration remains one of the great mysteries in biology. Forty years ago, studies on the mammalian brain provided evidence that new neurons are generated throughout life. It is now widely accepted that neurons are born (neurogenesis) in a wide range of animals, including humans, from neural stem cells maintained in the adult brain. Neural stem cells, however, do not readily compensate for lost neurons after injury or due to diseases of the nervous system, such as Parkinson’s or Alzheimer’s disease. The existence of neural stem cells has raised hopes that in the future we may be able to manipulate or promote stem cells in living organisms to divide, acquire the fate of specific cell types, migrate to the proper location and replenish lost neurons. Alternatively, another source of stem cells for tissue replacement could be stem cells derived from adult, embryonic, or cells re-programmed to acquire a stem cell-like state. All of these prospects will require that we fully understand how stem cells can be signaled to divide, acquire the desired cell fate and integrate into a functional nervous system. Our understanding of how repair of the nervous system can be achieved could benefit from studies of organisms that, in contrast to humans, are capable of regenerating the nervous system. For more than a century, scientists have been intrigued by freshwater planarians (flatworms); these animals, when cut into small pieces, have the remarkable ability to regenerate complete organisms from small body pieces. This ability to regenerate missing parts originates from a population of adult stem cells planarians maintain throughout life. Thus planarians are an excellent system in which to examine how stem cells are signaled to divide and to become all the different cell types during regeneration. It is now possible to apply advanced scientific methods to study planarians; we can visualize the stem cells, label the different organ types and inhibit the expression of specific genes. One of the truly amazing properties of planarians is their capability for rapid repair and regeneration of the central nervous system, a capacity that is limited in most animal models currently studied. In this study, we will use planarians to identify and analyze the function of genes implicated in neurogenesis during regeneration and normal cell turnover. Successful identification of novel genes would help to fill gaps in our knowledge of conserved biological mechanisms that stimulate proliferation and differentiation of stem cells in the central nervous system. This information has the potential to contribute to our ability to induce human embryonic or adult stem cells to divide and acquire neuronal fates, which would be valuable for transplantation therapies to treat nervous system injuries or neurodegenerative disorders.
Statement of Benefit to California: 
The inability to recover from loss of neuronal function afflicts a large number of people: in the U.S. alone, 4 million people have been diagnosed with Alzheimer’s disease, 1.5 million have Parkinson’s disease, and 0.25 million suffer from spinal cord injuries. The aim of this proposal is to establish a model of regeneration to study how stem cells can be directed to replace lost neurons after injury. Invertebrate organisms have provided powerful venues to investigate biological conserved mechanisms, and their study has led to discoveries of biomedical relevance. Our research on planarian stem cells and neural regeneration has the potential to make contributions to our knowledge of genetic pathways that control neuronal determination of stem cells or, if disrupted, could lead to neurological disorders. These studies provide a unique opportunity to examine how regeneration of the nervous system can be achieved at the molecular and cellular levels and have implications for the development of neural stem cells in regenerative medicine.
Progress Report: 

Year 1

The aim of our work is to understand how stem cells replace lost neurons after injury in a model organism of regeneration. Freshwater planarians (flatworms) are excellent models in which to investigate fundamental mechanisms regulating stem cells. These animals maintain a population of pluripotent stem cells in the adult, which serve to replace cells lost after wounding or during normal physiological turnover. One of the truly remarkable properties of planarians is their ability for repair and regeneration of the central nervous system (CNS), a capacity that is limited in most animal models currently studied. Thus, we are capitalizing on the molecular tools available to study planarian regeneration to identify and functionally characterize genes with critical roles in the repair of the CNS. To advance our knowledge of how regeneration and patterning is achieved in the CNS, we are analyzing when and where neurons are replaced during regeneration. During the reporting period we have begun to generate reagents that recognize planarian neural proteins. These protein markers will be used, in combination with staining of dividing cells, to analyze the transition of the stem cells to specific neuronal cell types. To develop protein markers we are isolating a cell fraction from the planarian head that is highly enriched with neurons, which will be used to immunize host animals to generate planarian-specific antibodies. Isolation of cell fractions is underway and we anticipate the generation of these reagents to be completed this year. To identify genes that are differentially expressed during regeneration of the CNS, we are performing high-throughput gene expression studies (microarray analyses) using samples obtained from tissue replaced in the early phases of regeneration of the planarian head after amputation. Initial experiments have revealed hundreds of genes that change their level of expression in the regenerating tissues compared to control tissues obtained from uninjured animals. We will examine the pattern of expression of genes with high levels of expression in the animal to determine which of these candidate genes are localized in the nervous system. Genes expressed in the CNS that share high similarity to human genes will be given high priority for studies aimed at characterizing their function during neural regeneration.

Year 2

The aim of our work is to understand how stem cells replace lost neurons after injury utilizing freshwater planarians (non-parasitic flatworms) as model organisms. Adult planarians maintain a population of pluripotent stem cells, which serve to replace cells lost after wounding or during normal physiological turnover. One of the truly remarkable properties of planarians is their ability for repair and regeneration of the central nervous system (CNS), a capacity that is limited in most animal models currently studied. Thus, planarians are excellent models in which to investigate fundamental mechanisms regulating stem cells. We are capitalizing on the molecular tools available to study planarian regeneration to identify and functionally characterize genes with critical roles in the CNS repair. To advance our knowledge of how regeneration and patterning is achieved in the CNS, we are characterizing when and where neurons are born during regeneration. During the reporting period, we have successfully generated reagents that recognize planarian proteins. We are currently characterizing these markers and will use them, in combination with staining of proliferating cells, to analyze the transition of stem cells to specific neuronal cell types. To identify genes that are differentially expressed during regeneration of the CNS, we have carried out high-throughput gene expression studies (microarray analyses) using samples obtained from newly replaced tissues during early regeneration of the planarian head. Our experiments have revealed >1000 genes changing their level of expression in the regenerating tissues compared to control tissues obtained from uninjured animals. We have begun to examine the pattern of expression of these genes in the animal; the on-going expression screen has revealed more than one hundred genes expressed in the CNS, regenerating tissue or the stem cell population. Finally, based on their pattern of expression, we have targeted genes for studies aimed at characterizing their function during regeneration. Successful identification of genes with no known or emerging roles in stem cell regulation would help to fill gaps in our knowledge of conserved biological mechanisms orchestrating proliferation and differentiation of stem cells in the CNS. This information has the potential to contribute to our ability to induce human embryonic or adult stem cells to divide and acquire neuronal fates, which would be valuable for therapeutic applications.

Year 3

The aim of our work is to use freshwater planarians (non-parasitic flatworms) as model organisms to understand how stem cells replace lost neurons after injury. Adult planarians possess a population of pluripotent stem cells, which serve to replace cells lost after wounding or during normal physiological turnover. One of the truly remarkable properties of planarians is their ability for repair and regeneration of the central nervous system (CNS), a capacity that is limited in most animal models currently studied. Thus, planarians are excellent models in which to investigate fundamental mechanisms regulating stem cells. We are capitalizing on the molecular tools available to study planarian regeneration to identify and functionally characterize genes with critical roles in CNS repair. To advance our knowledge of how regeneration and patterning is achieved in the CNS, we are analyzing when and where neurons are replaced during regeneration. During the reporting period we have generated antibodies that recognize planarian neurons and their projections. These antibodies are being used in combination with staining of dividing cells to analyze the transition of the stem cells to specific neuronal cell types. To identify genes that are differentially expressed during regeneration of the CNS, we have performed high-throughput gene expression studies (microarray analyses) using samples obtained from tissue replaced in the early phases of regeneration of the planarian head after amputation. Our experiments have revealed approximately 700 genes changing their level of expression in the regenerating tissues compared to control tissues obtained from uninjured animals. We have begun to examine the pattern of expression of these genes in the animal; thus far, screening of 289 genes has revealed 50 genes expressed in the CNS, 71 expressed in the stem cells (another 68 genes are potentially expressed in the stem cells and will be confirmed), and 225 are expressed in the early regenerating tissue. Based on the patterns of expression and sequence annotation from comparisons to genes of other organisms (including humans), we will analyze close to 225 genes in studies aimed at characterizing their function during regeneration. Results from these on-going experiments have already revealed interesting genes with potential roles in stem cell regulation that share high similarity with human genes. Successful identification of genes with no known or emerging roles in stem cell regulation would help to improve our knowledge of biological mechanisms regulating proliferation and differentiation of stem cells in the CNS. This information has the potential to contribute to our ability to manipulate pluripotent stem cells to divide and acquire neuronal fates in vivo, which would be valuable for therapeutic applications.

Year 4

The past decade has witnessed major breakthroughs in our ability to generate and manipulate embryonic stem (ES) cells or ES-like cells in vitro, and these cells are being used to model animal development, drug screening, and cell transplantation studies. Despite these advances, major gaps exist in our understanding of how adult stem cells are regulated in most organs and to what extent these cells could be directed to replace lost cells in vivo after injury or during disease. In the human central nervous system (CNS), neural stem cells, which reside in specific areas of the adult brain, are not capable of compensating for cells lost after injury or disease. Studies of organisms that can regenerate the CNS can improve our understanding how adult stem cells can be harnessed for therapeutic purposes could benefit from. The aim of our work is to gain insights into how pluripotent stem cells are regulated in vivo using freshwater planarians (non-parasitic flatworms) as model organisms. Adult planarians possess a large population of somatic stem cells, which serve to replace cells lost after wounding or during normal physiological turnover. One of the truly remarkable properties of planarians is their ability for repair and regeneration the CNS. Thus, planarians are excellent models in which to investigate fundamental mechanisms regulating stem cells. We are capitalizing on the molecular tools available to study planarian regeneration to identify and functionally characterize genes with critical roles in CNS repair. To advance our knowledge of how regeneration and patterning is achieved in the CNS, we are analyzing when and where neurons are replaced during regeneration. During the reporting period, we have identified neural-specific transcription factor genes that are co-expressed with stem cell or progeny markers and are gamma irradiation sensitive, which strongly suggests these transcription factors label cells with characteristics of neural progenitors. We also performed experiments to label cells replicating their DNA (cycling cells) followed by in situ hybridization and found we can readily detect cycling cells expressing neural-specific genes. This raises the possibility that, similar to vertebrates, planarians may maintain a population of neural progenitor cells that act as a source for new neurons during normal physiological cell turnover. Our new results represent a step forward because we can now examine how external stimulation or genetic perturbations might influence neurogenesis in planarians and gain insights into the gene networks involved in adult neurogenesis, both of which could have important implications for understanding basic mechanisms regulating human neural stem cells in vivo. To identify genes that are differentially expressed during regeneration of the CNS, we completed high-throughput gene expression studies (microarray and RNA-seq) using samples obtained from tissue replaced in the early phases of regeneration of the planarian head after amputation. To date, we have performed gene inhibition studies for 168 genes chosen from the microarray list based on expression pattern or homology. Knockdown of 25 of these genes produced phenotypes, including loss of the stem cells, reduced or delayed regeneration, and patterning defects. We are currently performing extended knockdown experiments for 42 of the genes. Results from these on-going experiments have already revealed interesting conserved genes with potential roles in stem cell regulation. This information has the potential to contribute to our ability to manipulate pluripotent stem cells to divide and acquire neuronal fates in vivo.

Year 5

The human central nervous system (CNS) has very limited capacity for renewing cells and is not capable of compensating for cells lost after injury or disease. Studies of organisms that can regenerate the CNS could improve our understanding of how adult stem cells can be harnessed for therapeutic purposes. The goal of this work is to establish planarians as models to gain insights into mechanisms regulating stem cell differentiation in vivo. We are specifically interested in understanding how the remarkable ability of these animals to regenerate the central nervous system after injury or amputation. We have capitalized on the molecular tools available to study planarian regeneration to identify and functionally characterize genes with critical roles in nervous system repair, patterning and function. To advance our knowledge of how regeneration and patterning is achieved in the CNS, we are analyzing when and where neurons are replaced during regeneration. During the reporting period, we identified neural-specific transcription factor belonging to the basic Helix-Loop-Helix (bHLH) gene family. We found bHLH genes that were expressed in stem cells and neurons and were also required for normal CNS regeneration. Our laboratory has identified some of the first neural progenitor populations in planarians and has contributed to the hypothesis that planarians possess lineage-committed neural progenitor cells that might serve to generate new neurons in uninjured and regenerating animals. To identify genes that are differentially expressed during regeneration of the CNS, we completed high-throughput gene expression studies using samples obtained from tissue replaced in the early phases of regeneration of the planarian head after amputation. We have performed gene inhibition studies for 168 genes chosen from the microarray list based on expression pattern or homology and found that knockdown of 25 of these genes produced phenotypes such as loss of the stem cells, reduced or delayed regeneration, and patterning defects. Results from these experiments have revealed interesting conserved genes with potential roles in stem cell regulation. In addition, we have examined in detail the function of the transcription factor COE. COE proteins have been recently implicated in CNS diseases in adult organisms. Using RNA interference, we found that COE is essential for regeneration and to maintain nervous system architecture in planarians. Using genomic techniques, we examined gene expression changes following inhibition of COE expression and uncovered conserved genes required for CNS regeneration. We also found that COE is important for stem cell homeostasis, suggesting that studies using planarians could provide insights into how COE dysfunction leads to CNS diseases such as cancer.

© 2013 California Institute for Regenerative Medicine