Funding opportunities

Skeletogenic Neural Crest Cells in Embryonic Development and Adult Regeneration of the Jaw

Funding Type: 
New Faculty I
Grant Number: 
Funds requested: 
$2 374 911
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
The goal of this proposal is to develop cell-based therapies that lead to the better healing of traumatic head injuries. Our first strategy will be to use genetics and embryology in zebrafish to identify factors that can convert human embryonic stem cells into replacement skeleton for the head and face. Remarkably, the genes and mechanisms that control the development of the head are nearly identical between fish and man. As zebrafish develop rapidly and can be grown in large numbers, a growing number of researchers are using zebrafish to study how and when cells decide to make a specific type of tissue – such as muscle, neurons, and skeleton - in the vertebrate embryo. Recently, we have isolated two new zebrafish mutants that completely lack the head skeleton. By studying these mutants, we hope to identify the cellular origins and genes that make head skeletal precursors in the embryo. These genes will then be tested for their ability to drive human embryonic stem cells along a head skeletal lineage. Our second strategy will be to test whether a population of cells, similar to the one that makes the head skeleton in the embryo, exists in the adult face. We have found that adult zebrafish have the extraordinary ability to regenerate most of their lower jaw following amputation. In this proposal, we use sophisticated imaging and transgenic approaches to identify potential adult stem cells that can give rise to new head skeleton in response to injury. Traumatic injuries to the face are common, and treatment typically involves grafting skeleton from other parts of the patient to the injury site. Unfortunately, the amount of skeleton available for grafts is in short supply, and surgeries often result in facial disfigurement that causes psychological suffering for the patient for years to come. Here we propose two better treatments that would lead to more efficient healing and less scarring. The first treatment would be to differentiate human embryonic stem cells, a potentially limitless resource, into skeletal precursors that can be grafted into the head injury site. By understanding the common pathways by which head skeletal cells are specified in the zebrafish embryo and human embryonic stem cells, we will be able to generate skeletal replacement cells in large quantities in cell culture. The second treatment would be to stimulate adult stem cells already in the face to regenerate the injured head skeleton. If successful, our experiments on zebrafish jaw regeneration will allow us to devise strategies to augment the natural skeletal repair mechanisms of humans.
Statement of Benefit to California: 
Traumatic injuries to the head, such as those caused by car accidents and gunshot wounds, are commonly seen in emergency rooms throughout California. Current treatments for severe injuries of the head skeleton involve either grafting skeleton from another part of the body to the injury site or, in cases where there is not sufficient skeleton available for grafts, implanting metal plates. Although these operations save lives, they often result in facial disfigurement that causes psychological suffering for the patient for years to come. For this reason, there is enormous interest in cell-based skeletal replacement therapies that will heal the face without leaving disfiguring scars. Remarkably, the genes and mechanisms that control the development of the head are nearly identical between fish and man. Thus, we are using the zebrafish embryo to rapidly identify factors that can make head skeletal precursors, and then asking if these same factors can push human embryonic stem cells along a skeletal lineage. In addition, we have found that adult zebrafish have the extraordinary ability to regenerate most of their lower jaw following amputation, and we will use sophisticated imaging and transgenic approaches to identify potential adult stem cells that can regenerate the face. The successful completion of these experiments would allow us to both generate unlimited amounts of head skeletal precursors for facial repair and stimulate latent skeletal repair mechanisms. The combination of these approaches will lead to therapies that promote a more natural healing of the face, thus allowing Californians to eventually resume normal lives after catastrophic accidents.
Review Summary: 
SYNOPSIS: This application proposes to investigate the cellular origins and molecular mechanisms underlying skeletal regeneration in the head using zebrafish and human embryonic stem cell (hESC) model systems. This work follows on the investigator's previous work identifying zebrafish mutants that show loss of cranial neural crest-derived skeleton, and will use many of the unique resources available to him. There are two Aims, focused first on the embryonic function of these mutated genes, and then on regeneration of the adult jaw. Findings in zebrafish will be extended to analyze skeletogenesis from hESC. First, in Aim 1, Dr. Crump will make use of 2 novel zebrafish mutants (lpy and myx) that he isolated and that show specific losses of components of the neural crest (NC)-derived head skeleton to identify molecularly distinct populations of crest precursor cells. Both genes will be positionally cloned, and tested for their abiltiy to induce skeletal fate by RNA injection into zebrafish embryos. He also will perform fate-mapping studies, using NC promoters to drive expression of a photoconvertible fluorescent protein in transgenic fish, in order to identify the cellular origins of skeletal precursors within the neural crest. Finally, in what could be a separate Aim, discoveries made in the zebrafish system will be translated to hESC, by assaying whether tet-inducible expression of Sox9, lpy, or myx in hESC can drive neural crest or skeletal differentiation. As a related but not interdependent line of investigation, in Aim 2 the applicant will study skeletogenic populations in adult zebrafish, which he has found possess a remarkable ability to regenerate their jaws. The cellular source of adult regenerative cells will be determined by labeling distinct populations of cells by photoconversion, and then following their contributions to the regenerated jaw by live imaging. These early progenitor cells will also be isolated and transplanted into wild type or mutant fish to assess their multipotency, and will be ablated, using nitroreductase (NTR) expression, to assess their role during regeneration. Finally, the molecular requirements of adult jaw regeneration will be compared to those for embryonic skeletogenesis by assaying jaw regeneration in adult lpy heterozygotes, which are viable as adults and show variable NC defects. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The concept of skeletal face regeneration is a complicated problem that will clearly benefit from a better understanding of the NC-derived progenitors and how they can be specified towards defined lineages. The zebrafish system provides clear strengths in terms of isolating and manipulating these progenitors in embryos, and the identification of the mutant genes could lead to novel insight. If successful, this project will molecularly identify novel regulators of skeleton formation and provide a high-resolution fate map for vertebrate NC-derived cells. It also will yield new insight into skeletal regeneration, by direct imaging and functional analysis of jaw regeneration in the adult zebrafish. These experiments are innovative in that they exploit unique models and approaches developed by the PI, and will use the discoveries made in the zebrafish model to create rational strategies for generating hESC-derived NC skeletal precursors. The induction of specific NC derivatives from hESCs is an important concept that is not currently well studied, and the proposed use of both zebrafish and hESC research is also innovative, with both scientific and medically relevant promise. Therefore, the project has the potential to generate important new information that could be translated into cell-based and regenerative medicine. The overall experimental design is ambitious but logical, and balances straightforward genetic analysis with more high-risk exploratory approaches. In most cases, the feasibility of the proposed experiments is well-supported by the PI's previous experience and preliminary data, although additional data in support of certain aspects of the project, for instance the kikumeGR protein, would have strengthened the application. The strongest and best-supported studies are those in zebrafish embryos related to fate mapping NC-derived progenitors. On the other hand, molecular cloning of the myx and particularly the lpy gene may present difficulties, particularly as lpy mutants show semi-dominance. Nevertheless, the PI does propose multiple approaches and appropriate alternatives to the identification of this gene, and so it is reasonably likely that he will succeed. However, if these genes cannot be identified, or are slow to yield to identification, then subsequent studies proposed in the application (e.g. overexpression to determine the sufficiency of lpy or myx for skeletogenesis, identification of human lpy and myx homologs, and testing of these in the hESC system) may be prevented or significantly delayed. Overall, the studies tracing embryonic lineages and cloning the mutants are solid, but it remains too early to know if this can be developed into a stem cell project. The studies in hESCs and adult jaw regeneration are relatively preliminary in nature. The hypothesis that lpy and/or myx are “master regulators” of cell lineage fate is a leap of faith, and success in these experiments would require that one or more of these factors is sufficient for skeletogenesis. There is no data to support this or reason to presume that forced expression in hESCs or zebrafish embryos would be sufficient to direct cell fates. The genes might encode rather downstream components of the pathway. This would still be important to discover, but would make most of the project difficult to evaluate for feasibility. What if they encode an ECM component, MMP, or cell survival factor? In addition, the studies with lpy and myx depend on the existence of human homologs of these genes, which may or may not be easy to identify. The applicant acknowledges these risks, and provides some alternatives (e.g. similar analysis of other NC-expressed genes). In addition, assessment of hESC differentiation to the skeletal lineage will rely solely on marker expression analysis - no functional tests are proposed. Functional analysis will be needed to confirm differentiation of hESC to skeletal crest precursors. The second aim of the proposal will pursue exciting new data generated in the applicant's lab that demonstrates a striking capacity for regeneration in the adult zebrafish jaw. The studies proposed to better characterize this process and to identify the cellular mediators of jaw regeneration are clearly important. However, the description of the experiments lack detail. It is proposed that the amount of amputated jaw and subsequent regeneration will be correlated, but no specific description of how the degree of amputation will be defined and/or performed is provided. Jaw regeneration will be assessed at weekly intervals – what is the survival rate of amputated embryos? Analysis of jaw regeneration in additional zebrafish mutants that display defective blastema formation and fin regeneration is intriguing. The use of photoconversion-mediated lineage tracing to track the contributions of various cell types is an interesting and powerful approach. Similarly, using CFP-NTR transgenes to ablate cell populations, and thereby test the requirement of sox10- or fli1-expressing cells, is an attractive approach. However, whether these promoters will target only discrete defined progenitors is uncertain. Fli1 for example is expressed in all the endothelium and so the use of fli1:CFP-NTR to target NC progenitors for ablation would seem improbable. Furthermore, it is not clear whether the sox10 or fli1 promoters are active in the putative adult NC stem cell (NCSC) populations. The rationale for the use of the bactin2 promoter as an alternative, in combination with fli1:GFP as a reference, is not clear. The applicant also acknowledges a potential problem with the sensitivity of the labeling approach for rapidly proliferating precursors. It is also not clear whether skeletal progenitors of photoconverted single NC cells will still fluoresce, and therefore be traceable in the proposed studies. Furthermore, the PI will attempt to identify and characterize putative adult NCSC present in the jaw, which might contribute to regeneration. If the sox10 and fli1 promoters perform as hoped, and NC cells from adult jaws from fli1:GFP and sox10:mCherry double transgenic lines can be FACS sorted, the investigator proposes to transplant them back into wild type and lpy late gastrula staged embryos. However, problems may be encountered with the heterochronic transplants, as it is possible that adult cells will not engraft well or respond appropriately in the embryonic environment. No preliminary data demonstrating the feasibility of these experiments is presented. Notwithstanding these specific comments, the reviewers were very enthusiastic about this proposal. If successful, this project will identify novel regulators of skeleton formation and provide a high-resolution fate map for vertebrate neural crest-derived cells. It also will yield new insight into skeletal regeneration, by direct imaging and functional analysis of jaw regeneration in the adult zebrafish. The experiments are innovative, and success in these endeavors could have important implications for advancing treatment options for facial injury. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Crump is a new Assistant Professor (2006) at the Center for Stem Cell and Regenerative Medicine (CSCRM) and in the Department of Cell and Neurobiology at USC. Dr. Crump trained for his PhD with Cori Bargmann at UCSF (2 first author publications) and then had a very successful postdoc with Chuck Kimmel in Oregon, with 3 strong 1st author publications on craniofacial and skeletal patterning. He has received several awards including the Howard Hughes Medical Institute Pre-doctoral Fellowship, the Chancellor’s Award Fellowship at UCSF, and the O’Donnell Postdoctoral Fellow of Life Sciences Research Foundation Award. Dr. Crump has consistently published in top-level peer reviewed journals, including Neuron, Development, Science and Nature Neuroscience, and currently holds an NIH/NICRD RO1 (9/01/07- 08/31/12) to study epithelial-mesenchymal interactions in facial patterning. He has currently no experience with hESC, and is just developing the project on regeneration. Dr. Crump is clearly an expert in zebrafish genetics and embryology, and he and his research team are well qualified to pursue the proposed studies, and the environment is excellent Dr. Crump outlines a well thought-out and comprehensive career development plan, and describes an excellent plan for interdisciplinary research including his role in the Neurogenetic Institute, the Stem Cell Center and interactions with developmental biologists, dentists, and craniofacial geneticists. He is guided by a senior faculty committee headed by the Chair, Dr. Mike Snow. They will make formal evaluations of Dr. Crump’s progress towards tenure, including an early evaluation at the end of his third year. Formal tenure will be assessed at the end of year 6. His senior mentor is Rob Maxson, a craniofacial expert and director of the CIRM-funded Training Grant, which is funding Dr. Crump’s post doctoral fellow Dr. Sam Cox. Dr. Crump participates in bi-weekly CSCRM and Developmental Biology research meetings, where students and Post-Doctoral Fellows present their work on stem cell research. He will seek help in developing hESC projects with Martin Pera, who heads the stem cell center, and Dr. Lien, who works in zebrafish regeneration. He clearly plans to translate the principles learned in the visually accessible and genetically tractable zebrafish to inform studies of hESC differentiation. Dr. Crump states that “The CIRM new Faculty Award would be invaluable in my successful transition to two new fields: hESC differentiation and adult stem cell-mediated skeletal regeneration.” The 5 year award would allow time and financial support to build up stem cell-trained personnel, and master new techniques and expertise. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: USC has demonstrated a clear commitment to advance the career of this young investigator. Dr. Crump is fully independent, and he is cross-appointed into the stem cell center and a basic science department. The institution has provided ample laboratory space, administrative assistance, and start up funds to support Dr. Crump’s research, and he has full access free of charge to core equipment and facilities, including their CIRM-funded stem cell facility, confocal and light microscopes, FACS sorter and analyzer, DNA sequencing facility, microarray facility, computing, administrative support, and access to graduate students. The scientific environment at USC is stellar, and provides unique and necessary collaborative interactions that support the proposed studies. In particular, Dr. Crump will benefit from collaboration with Dr. Martin Pera, an expert in hESC culture and analysis who runs a CIRM-funded stem cell core, with Dr. Ching-Ling Lien at CHLA, whose lab focuses on zebrafish heart and fin regeneration, and with Dr. Marianne Bronner-Fraser, an expert in neural crest biology. USC has a long and successful history of promoting the successful development of research faculty. In addition, USC is clearly committed to ESC and hESC research, with the recent establishment of the Center for Stem Cell and Regenerative Medicine under the direction of Dr. Martin Pera, a recognized leader in hESC research. USC also maintains a CIRM-supported graduate training grant and a facility grant. One of Dr. Crumps postdocs is already funded by the CIRM training grant. DISCUSSION: There was great enthusiasm for this PI, who has an excellent track record in both his PhD and post-doctoral work, and who is an expert in zebrafish genetics and embryology. The proposal is well-written and exciting, and strikes a good balance between straightforward genetics and risky hESC work. His research is innovative, using an adult regeneration model in zebrafish which could be very powerful, and is not highly investigated. The PI is making good use of the zebrafish as a model organism. The reviewers reiterated that the mutated genes may not turn out be master regulators (but the PI is aware of this possibility and provides alternatives if genes turn out not to be master regulators), that the mutated genes have not yet been identified (but that it was likely that PI would clone them), and that the transgenes had not yet been generated and therefore their expression pattern had not been verified. Whether the promoters will target appropriate progenitors in the adult is not yet known. The question was raised whether the observed repair after jaw injury is really regeneration, not just wound healing, and if Sox10 could be used in the context of hESC lines. However, based on the excellent track record of the PI, the reviewers were confident that even if some experiments fail, he will produce some very interesting findings.

© 2013 California Institute for Regenerative Medicine