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The molecular basis underlying adult neurogenesis during regeneration and tissue renewal

Funding Type: 
New Faculty I
Grant Number: 
Funds requested: 
$1 555 370
Funding Recommendations: 
Not recommended
Grant approved: 
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.
Review Summary: 
SYNOPSIS: This is a proposal to identify signaling factors that induce neuronal differentiation of stem cells, using the planarian Schmidtea mediterranea as a model animal. Planarian flatworms are tractable models to examine stem cell biology and tissue replacement. These animals can restore lost body parts, including the CNS, from a population of adult pluripotent stem cells (neoblasts). Neoblasts are the only proliferating cells in planarians, and serve to replace cells lost after injury or during normal cell turnover. The goal of the present study is to use the plenarian system to identify genes that play critical roles in neuronal proliferation and differentiation of CNS stem cells. In Specific Aim 1, the applicant will generate monoclonal antibodies that recognize planarian neural proteins. Fluorescence-activated cell sorting will be used to isolate a cell fraction that is highly enriched with neurons; these cells will be used to immunize host animals to create hybridomas. Antibodies determined to specifically label neurons will be used, in combination with bromodeoxyuridine (BrdU) staining, for in vivo lineage tracing to analyze the transition of neoblasts into diverse neuronal subtypes during regeneration and tissue maintenance. In Specific Aim 2, the applicant will use microarrays to study genes expressed during regeneration and locate these by in situ hybridization. The genomic resources available for S. mediterranea have allowed the design and manufacturing of high-density oligonucleotide microarrays, which will be used to examine global gene expression profiles and to identify differentially expressed genes at different stages of regeneration of the CNS. Tissue-specific expression of genes that are significantly upregulated will be screened by high-throughput whole-mount in situ hybridization. Finally in Aim 3, the applicant proposes to look at the functional role of highly conserved genes identified in Aim 2, by using RNAi to knock down these genes and assess the effect of gene knock-down on neurogenesis in intact and regenerating planaria. STRENGTHS AND WEAKNESSES OF THE PROPOSAL: Employing stem cells to replace lost tissue in human CNS disease and injury will require a careful understanding of the signaling factors required for neuronal differentiation of stem cells. One suspects that a great deal of exciting information can be gleaned from this creative proposal utilizing an intriguing and surprisingly-unexplored model organism. While the proposed experiments use an invertebrate system, it is reasonable to hope that genes identified in the planarian regenerative response may be important in human neurogenesis and CNS repair. Overall, this is a beautifully written and illustrated proposal with strong preliminary data. The model organism is rather easy to manipulate genetically, and genomic information is readily available. The flatworm is a simple organism yet it expresses genes homologous to the human genes. Also, the ability to regenerate, which may be related to the generation of stem cell-like cells by dedifferentiation, is well established. The generation of an antibody library is likely to produce neuronal-specific and neuronal subtype-specific antibodies that will be of value in future investigations, including the ones proposed here. The proposal is risky in the sense that it remains uncertain what the impact of knowledge about neurogenesis in planarians will be on the use of human stem cells. Nevertheless, it appears that the risk is worth taking considering the extraordinary repair capabilities of planarians. Freshwater planarian flatworms have the capacity to: carry out adult neurogenesis, restore new body parts (i.e., regenerating complete organisms from very small body pieces), and rapidly repair and regenerate the CNS from a population of adult pluripotent stem cells called neoblasts. These organisms provide a powerful model to investigate stem cell-based regeneration and remodeling of the CNS in order to accrue new information with respect to stem cell biology and tissue replacement. In addition to their extraordinary repair mechanisms, planarians are attractive organisms to study because large collections of planarian ESTs are available and the complete genome is known.. In addition planarians are amenable to RNAi screens: they are simple, they have bilateral symmetry, and gene expression can be examined by high-throughput in situ hybridization and silenced by RNAi. The applicant also points out that neoblasts are the only dividing cells in planaria and thus they can be labeled with BrdU. There has been an extraordinary amount of information obtained through the study of other primitive organisms such as C. elegans and Drosophila. Surprisingly, little work has been done related to molecular and genetic aspects of planarians. A major strength of the proposal is the applicant’s training in one of the pioneer labs in planaria biology, and his technical abilities. For instance, the PI has experience using microarrays, having demonstrated CNS expression of >200 genes in a previous study involving screening of 500 ESTs. In addition, the PI has access to a cDNA library from Dr. Phillip Newmark’s lab (where he pursued his post-doctoral fellowship) that represents the majority of the sequences spotted on the microarrays. Minor weaknesses of the proposed research are that it is a fishing expedition, and that there are potential problems in feasibility that require troubleshooting. For instance, the applicant indicates that the head of the flatworm is rich in neurons, and proposes that analyzing gene expression changes in the entire head will allow them to identify genes involved in the neurogenesis. However, the whole head tissue sample collected for the microarray contains many other types of cells, which may express entirely different sets of genes or which may regulate the same genes in different ways following regeneration. One might ague that they will be able to analyze only the genes that are known to be expressed in neuronal cell lineage. If that is the case, there is no value to performing a microarray and this could be a problem since the study is totally dependent on the microarray. In addition, identifying the primary genes initiating neuronal differentiation could be very difficult. Transgenesis approaches to visualize the neuronally-committed cell population might yield only qualitative results, since transfection efficacy in vivo could be very low and a quantitative analysis will require most of the cells in the area to be transfected. Antibody generation against MC540, which selectively stains cells that have electrically excitable membranes, could be useful to identify the neurons by immunohistochemistry. However, if FACS give 90-95% neurons form the tissue sample from the head then why don’t they use this method to detect cell population changes in the head? Furthermore, it is not clear that the cells that dedifferentiate in the flatworm at the beginning of the regeneration have all the characteristic properties of stem cells. A final potential weakness is that the proposal is a very ambitious one, involving the generation and characterization of mAbs for identification of CNS cells, the identification of genes important in regeneration through microarrays studies, and the use of transgenesis and RNAi knock-down to explore the function of these genes. It sounds like the PI is going to do it all. Although the preliminary data suggest that all of this is feasible, it still is a bit too ambitious. One reviewer was a bit concerned that the MAb production and hybridization were going to be carried out by companies and not in-house. One would have hoped that there were some on-site Cores that could have performed these studies under PI supervision. In summary, this is a well-written and creative proposal. This field is outside the main directions taken in stem cell biology but the approaches and planned experiments are logical and hang together well, and will provide information valuable for cell transplant treatment approaches to injuries and human CNS disease. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: This is a promising investigator with the appropriate background to make advances in a field that is unexplored and potentially very exciting to stem cell work. The PI was a Research Assistant in the Division of Endocrinology at Wayne State University School of Medicine. He obtained his PhD in 2003 in Biology at Tufts University, followed by a post-doctoral fellowship from 2002-2007 at University of Illinois at Urbana-Champaign, working on planarians in the lab of Phillip A Newmark, a leader in this field. He is about to join San Diego State University as Assistant Professor in the Department of Biology in the Cell and Molecular Biology Program. Currently there is no other funding, thus this new faculty award will help to establish his career in the stem cell research field. The productivity of the PI is marginal, although he has published in good journals. He published four papers related to his PhD, including one in Science on firefly flashing. He published two papers as first author in 2005 related to his post-doctoral fellowship studies on planarians, one in Mol. Biol. Evol. and one in PNAS. He published one paper as a middle author in PNAS in 2006 concerning regeneration in planarians. The applicant has expertise in technologies used in the current proposal. However development of his expertise in stem cell research may be needed. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: Dr. Zayas is about to assume a position as Assistant Professor in the Department of Biology at San Diego State University. SDSU has provided him a generous research package, sufficient time for research, appropriate core facilities, and mentoring.. He will be in a recently renovated lab of ~1000 sq. ft. with a start-up package of $350,000. There is a FACS facility, Microchemical Core facility, a vivarium including a BSL-2 facility, and access to a confocal microscope. Dr. Zayas will have a Visiting Scholar appointment at UCSD, providing access to the UCSD CIRM-funded Stem Cell Core facility and the opportunity to recruit into his lab. Dr. Zayas will have no teaching for one of his first two semesters and a reduced teaching load for the second semester. Dr. Zayas will receive formal yearly feedback from facutly mentors, and there is a tenure-track committee. These facts indicate that total commitment in the development of the applicant’s career in the research field. San Diego State University has the largest Biology Department compared to similar departments in the CSU system. SDSU is making a major investment in stem cell research, and recently opened up a BioScience Center whose mission is the relationship between infectious disease and cardiovascular disease. One wonders whether Dr. Zayas may be somewhat isolated in his Department and at SDSU since there appear to be relatively few individuals with expertise in SCs.Although SDSU may not be a powerhouse of the stem cell research in San Diego, the applicant will be close to some of the very best stem cell research in California, and he recognizes that. DISCUSSION: Reviewers were captivated by this creative proposal that uses an innovative model system to study neural regeneration. They commented that this is a very different sort of proposal: it could potentially be important for the stem cell field, although its impact is not assured so this is a risky investment. Reviewers commented on the PI’s unique experience with planaria, although they commented on his lack of experience in stem cells and his potential isolation at SDSU. The applicant appeared to be aware of these limitations, and addressed them. Several technical issues that could impact the project’s feasibility were discussed, and some reviewers cautioned that the proposal was overly ambitious and too interconnected. In particular, panelists had the following concerns: 1. Generating a library of neuron-specific antibodies for planaria is expensive. Panelists strongly recommended that the investigator screen existing antibodies raised against other species to determine whether some of these studies could be conducted without the need to generate this battery of antibodies. One reviewer asked why the PI was using antibodies to visualize neurons when he could use a dye that recognizes excitable cells (MC540), but other panelists commented that antibodies could be used to identify subtypes of neurons 2. There was little discussion of proposed analysis of the experiments in the proposal. For instance, which time point following regeneration will be analyzed? Only examining one would severely limit the analysis. 3. In situ hybridizations could yield qualitative results on hundreds or thousands of genes. It was unclear how these would be analyzed, but even automated analysis could take a long time. In addition, reviewers were concerned that the in situs were not being done on site. 4. It was unclear how well the in vivo electroporation works. If only a subset of neurons are transfected and only a subset of those are competent to express the selected promoter, will expression be sufficient to affect phenotype? 5. Some reviewers were concerned about the PIs intent to use the whole head to do microarrays: it could be difficult to filter out the noise and sort out irrelevant genes. It was unclear how the PI intends to decipher where any changes are coming from (i.e., neurons or other cell types). Reviewers commented that institutional support for the applicant is strong. Although SDSU is not a well-established research center for stem cells, they are investing heavily in their program. Furthermore, SDSU is well-situated and the applicant is reaching out to neighboring institutions for collaborations. Overall, the reviewers were very positive about this proposal as they found it innovative, creative, and well-written.

© 2013 California Institute for Regenerative Medicine