New Faculty II
$2 396 871
Statement of Benefit to California:
Injuries to the craniofacial structures are an important clinical problem, as are birth defects that affect these structures, and regenerative medicine approaches for these problems are under-studied. Relatively little is known about the fate choice mechanisms that lead to development (and potentially regeneration) of the skeletal structures of the face from neural crest stem cells. The goal of the work is to identify genes involved in neural crest-derived generation of skeletal structures. The approach is to take zebrafish genes which have been identified to be important in this pathway and overexpress them in human embryonic stem cells (hESC). These genes will then be used to generate preskeletal neural crest cells from hESC. The applicant will take advantage of two zebrafish mutants that were identified during the applicant’s training. In both of these mutants, early neural crest markers are lost. Combined with other fate-specific genes for neural crest cells, experiments will be performed to determine if the same gene repertoire will lead to neural crest and craniofacial skeletal fate determination by hESC. The final aim will probe the role of adult skeletal progenitors and neural crest stem cells in adult zebrafish jaw regeneration toward the long-term clinical goal of stimulating regeneration of damaged craniofacial structures in adult human. The reviewers felt that the grant was very ambitious and exciting. The proposal was described in the reviews as innovative and bold, not the least derivative, and also of considerable practical importance. The potential impact on regenerative medicine was thought to be very high. Reviewers also appreciated the care with which the proposal was written, and the scientific issues were well-articulated. Thoughtful consideration was given to potential experimental pitfalls and alternative strategies. The impressive body of useful preliminary data was also considered a strength. The proposal was unusual in planning a relevant use of human embryonic stem cells as a compliment to the work in a genetically accessible model organism (the zebrafish). One reviewer was particularly struck by the last part of this proposal based on data showing that the principal investigator (PI) has identified a putative adult stem cell population in the adult jaw that contributes to the remarkable ability of zebrafish to regenerate mandibular structures after surgical excision. Aim 3 includes studies to determine if these cells are essential for adult jaw regeneration, and expansion of this part of the grant into a larger part of the research plan would have strengthened the grant. The major concern raised by reviewers was that neither of the genes to be used for these studies has been cloned, and though the applicant is the one who identified the mutations in fish, the lack of the cloned genes (as the crucial reagent) may be a problem in getting the work started. One candidate gene has been narrowed down to a 6-gene region and one of the genes in this region has no human homolog. If one of the operative ‘genes’ that was identified in fish is actually an enhancer sequence, it could be completely unrelated to neural crest development, and the identification of the elements could also be a long process, delaying the application of the work to hES cells. Also one gene is dominant making the cloning potentially more difficult. The applicant also does not present convincing evidence that expression of only the two genes of interest will be sufficient to drive hESCs to a neural crest cell fate. In addition, if the operative gene products in these mutations turn out to be secreted factors that are not even expressed in crest, this would greatly alter the strategy that the PI would need to use in the hESC system. Enthusiasm was reduced because so much of the grant was based on contingency, and reviewers were concerned that this represented a general problem with the thought processes underlying the research proposal. The study plan was not, then, impeccably crafted, with pitfalls and alternatives adequately addressed. Nonetheless, because the applicant and institutional support were so strong, and there are many ways to solve the cloning problem, these concerns were balanced to generate considerable support for the application. Furthermore, a reasonable Plan B is to continue more intensive work on jaw development in the zebrafish model. The applicant brings a great educational pedigree to the work, and has a superb recent publication record including papers in Development and Neuron. The applicant’s graduate and post-doctoral education were all in first-rate programs and labs. The applicant’s focus has been systems development, most recently craniofacial, and so this application is also made in the context of current NIH, March of Dimes, and CIRM Seed grant funding. The applicant is an Assistant Professor in a new stem cell center. The thoughtful and detailed career development plan focuses on the applicant’s interest in developing an academic career in hESC biology. The applicant has carefully selected several strong mentors. It was the strength of the applicant that was the major factor in determining the enthusiasm for this proposal, given the high risk of developing the entire proposal based on the risky plan to clone the relevant zebrafish genes in a short period of time. The stem cell biology group at the applicant’s institution and the intellectual environment surrounding the applicant is similarly strong, with the newly set-up core lab being an especially important feature. The mentoring plan was thoughtful, and included a detailed career development plan taking advantage of the strengths of the institution. The institution has provided a substantial start-up package for the applicant, and the letters from institutional officials made it clear that the institution is invested in the career development of the applicant.