One of the most exciting possibilities in stem cell biology is the potential to replace damaged or diseased neural tissues affected by neurodegenerative disorders. Stem-cell-derived neurons provide a potentially limitless supply of replacement cells to repair damaged or diseased neurons. Typically, only one or a very few types of neurons are affected in most neurodegenerative diseases, and simply transplanting stem cells directly into a degenerating or damaged brain will not guarantee that the stem cells will differentiate into the specific neurons types needed. In fact, they may instead cause tumor formation. Thus, we must learn how to guide stem cells, cultured in a laboratory, toward a specific differentiation pathway that will produce neurons of the specified type. These cells would then provide a safe, effective way to treat neurodegenerative diseases and central nervous system injuries.
Since there are hundreds or thousands of types of neurons in the cerebral cortex, functionally repairing damaged neurons in the cortex will require a detailed understanding of the mechanisms controlling differentiation, survival, and connectivity of specific neuronal subtypes. In this proposal, I propose to investigate the molecular mechanisms that guide the neural stem cells in developing embryonic brains to generate two specific types of neurons – corticospinal motor neurons (CSMNs) and corticothalamic projection neurons (CTNs).
Our first goal is to understand what regulates the development of CSMNs. CSMNs are clinically important neurons that degenerate in Amyotrophic Lateral Sclerosis (ALS), and are damaged in spinal cord injuries. With our current technology, replacing damaged CSMNs has been impossible, due largely to a lack of understanding of what signals regulate their development. Our second goal is to identify genes that direct the neural stem cells to generate the CTNs. Despite their essential importance in sensory processing and involvement in epilepsy, mechanisms governing the development of CTNs have not yet been revealed. CSMNs and CTNs express many identical genes, and are generated from common neural stem cells in the embryonic brains. Yet it is unclear how they are specified from common stem cells. Our third goal is to identify transcription factor codes that neural stem cells employ to specifically generate either CSMNs or CTNs.
Currently, there is no cure for neurodegenerative diseases. Understanding how CSMNs and CTNs are generated during development provides the opportunity to design procedures to direct the stem cells cultured in a laboratory to specifically produce CSMNs or CTNs, which can then be used to replaced damaged or diseased neurons, such as those affected by ALS, or spinal cord injuries.
Neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), affect tens of thousands of Californians. There are no cures for these devastating diseases, nor effective treatments that consistently slow or stop them. The research proposed in this application may provide the basis for a novel, cost-effective, cell replacement therapy for ALS, thereby benefiting the State of California and its citizens.
Stem cells offer a potential renewable source of a wide range of cell types that could be used to replace damaged cells involved in neurodegenerative diseases or in spinal cord injuries. At present, transplanting stem cells directly into patients is problematic, because this approach may instead cause tumor growth. To support safe and effective cell transplants, it is important to differentiate stem cells prior to the therapy into the specific cell types affected by the diseases. Understanding how different types of neurons are generated during development provides an opportunity to develop new methods to guide the differentiation of stem cells into the proper neuron types.
In this application, we propose to uncover the mechanisms that regulate the neural stem cells in developing mouse brains to generate different neuronal types in the cerebral cortex, including the corticospinal motor neurons (CSMNs) and the corticothalamic neurons (CTNs). CSMNs are the neurons that degenerate in ALS and are affected in spinal cord injuries. Dysfunction of CTNs has been implicated in epilepsy. Understanding the mechanisms regulating neural stem cells to generate CSMNs and CTNs in vivo will help scientists and physicians to direct stems cells to produce CSMNs or CTNs to replace damaged neurons in patients with neurodegenerative conditions.
SYNOPSIS: This applicant proposes to analyze the function of candidate genes in regulating the differentiation of embryonic neurons. The goal of the experiments in this proposal is to understand cell fate decisions that regulate the formation of two related neural subtypes in the cortex: cortical spinal motor neurons (CSMNs) that reside in layer five and cortical thalamic neurons (CTNs) that reside in layer six. CSMNs are clinically important neurons that have been shown to degenerate in Amyotrophic Lateral Sclerosis (ALS) and are damaged in spinal cord injury. CTNs play important roles in sensory processing and their dysfunction has been implicated in epilepsy.
Dr. Chen has identified candidate genes that, by virtue of their expression patterns and activities, are likely to contribute to cortical neuron subtype determination and to participate in the transcription factor codes that regulate cortical projection neuron fates. She proposes to analyze the functions of these genes in regulating the in vivo differentiation of embryonic neural progenitors of CSMNs or CTNs by analyzing gene expression and axonal projections in existing mutant mice and by manipulating gene expression in neural progenitors in vivo using electroporation of siRNA or gain-of-function constructs. Collectively, these studies will enable her to explore the mechanisms of fate specification in cortical development.
The first specific aim plays off of preliminary studies by the applicant and others which show that 1) Fezf2 is required for normal development of cortical spinal motor neurons in mice; and 2) a second transcription factor, Ctip2, may function downstream of Fezf2. The broad goal of aim one is to identify downstream targets of Fezf2 in NSCs using Affymetrix cDNA expression arrays. The Fezf2-PLAP mutant allele will be used as a marker to purify Fezf2 +/- and Fezf2-/- progenitor cells by FACS at 3 embryonic stages (E10.5, E11.5 and E13.5).
The second specific aim is derived from preliminary studies wherein the applicant has discovered three transcription factors (Tbr1, Nfia and Nfib) that are expressed specifically in CTNs and their progenitors. Tbr1 is expressed in the subplate and in layer 6 (CTN) neurons immediately after they exit the cell cycle. Nfib is a gene that regulates the transition from neurogenesis to gliogenesis in the developing spinal cord. The goal of specific aim two is to identify the functions of these three transcription factors in the formation of CTNs. Loss of function mutations for all three of the transcription factors at issue have been generated by other investigators and are available to the applicant. The applicant proposes to carefully scrutinize the various knockout phenotypes.
The third specific aim is to derive the transcription factor "combinatorial code" that distinguishes CSMNs from CTNs. The applicant points out that these two neural subtypes share common patterns of gene expression, including expression of Fezf2. She wonders why these two neural subtypes project differentially to the spinal cord and to the thalamus. She speculates that expression levels of Fezf2 and Ctip2 may be the key. Another possibility is that genes highly expressed in layer 6 neurons - such as Crg4, Tbr1 or Nfia - may distinguish CTNs from CSMNs. She has devised a set of over-expression and mis-expression experiments to test these hypotheses.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This proposal deals with a fundamental problem in stem cell-based therapies for neurological disease: how can cultured NSCs be instructed to differentiate into specific neural subtypes? The proposed research is likely to make a major contribution to our understanding of how subtypes of cortical projection neurons are determined. The issues dealt with in this proposal could have an important impact on the treatment of human neurodegenerative diseases, and they could be applied to human embryonic stem cell (hESC) differentiation protocols for stem cell therapy applications.
Overall, the proposal is well-written, the study plan is carefully considered and well-organized, and the experiments will produce interpretable results. The directions used by the principal investigator (PI) are creative ones. The experimental approach and concepts are fairly standard, and use existing mouse mutants and technologies. There is an impressive use of cutting-edge technology within the expertise of the PI, as the PI builds on studies that she performed during her post-doctoral fellowship. The applicant has paid attention to potential pitfalls and devised alternative approaches where possible. The work proposed will probably succeed and become a useful contribution to the literature in the fullness of time. The preliminary data demonstrate that the PI can carry out the proposed studies, and provide reasonable evidence that the genes that she has chosen to study should be further pursued. An additional strength of the proposal is the PI. Although her publications are not great in number, they have been of excellent quality. She is committed to the stem cell field and has the potential to become a leader in the field. Of note, the PI is presently already funded by CIRM, which may be either a plus, minus or neutral issue with respect to consideration of the present proposal.
The major weakness of this proposal from the perspective of this particular granting mechanism is that the experimental approach is more solid than innovative: one does not sense any paradigm shift in the making. Apart from moderate innovation, the other drawback is the degree of relevance to the goals of CIRM, since she is studying embryonic neural progenitors (not stem cells as stated). In addition, since no human ES cells will be analyzed, this would be a strong RO1 proposal, raising the question of whether the proposal is it worthwhile to fund using CIRM funds. Finally, there is possible overlap of the present proposal with a funded 2 year CIRM grant that needs to be addressed. The funded CIRM proposal in many ways would fill in the present proposal into a more comprehensive one.
The PI was involved in the characterization of Fezf2, a transcription factor expressed in NSCs and subcortical projection neurons, including CSMNs; the PI generated Fezf2-/- mice which have no corticospinal tract. The PI has preliminary data that show that Fezf2 is required for the activation of Ctip2 expression, a transcription factor essential for CSMN development into postmitotic neurons. A confusing result is that electroporating either Fezf2 of Ctip2 cDNA into Fezf2 -/- neurons rescues development of CSMNs, but electoporating Fezf2 does not restore Ctip2 expression.
Specific Aim 1 is to investigate downstream targets of Fezf2 in NSCs using cDNA arrays. They will purify Fezf2+/- and Fezf2-/- progenitor cells by FACS using an antibody to human placental alkaline phosphatase (PLAP), a membrane protein knocked into the Fezf2 locus. Genes identified in the arrays will be either knocked down or in utero electroporated to further investigate their importance. The use of the heterozygote and knock-out transcriptional profiling should provide interesting information. The planned experiments are feasible and worth doing, although there may be difficulties in determining factors involved in CSMN development if post-transcriptional alterations play an important role. A question not addressed is whether the PI will identify direct versus indirect targets of Fezf2. Also, when is Fezf2 first expressed in forebrain? Conditional ablation and analysis within a day would get closer to the question of direct targets.
Specific Aim 2 is to determine the functions of three transcription factors that the PI found are expressed specifically in CTNs and/or their progenitors: Tbr1 (which is expressed in the subplate and in layer 6 neurons immediately after they exit the cell cycle and is required for subplate neuron development), and Nfia and Nfib (which regulate the transition from neurogenesis to gliogenesis in the developing spinal cord). In order to carry out these studies, the PI will study mice that carry mutations in these genes (that have been prepared in other labs). In order to mark the CTN and their axons in Tbr1-/-, Nfia-/- and Nfib-/-mice, the PI will cross them with mice that carry a Grg4-LP allele that allows specific labeling of CTN cell bodies and axons with beta-galactosidase and PLAP respectively. One difficulty that she notes is that these genes affect subplate development and it is possible that pioneering axons from subplate neurons guide corticothalamic projections and therefore may affect CTN development in a non-cell autonomous way. For this reason, the PI will generate aggregation chimeras (between Tbr1-/-; Grg4-LP and wild type); these experiments should clarify whether PLAP-labeled axons are absent from the thalamus (suggesting Tbr1 regulates target selection of CTNs) or project normally to the thalamus (which would suggest that targeting of the corticothalamic axons depends on subplate development). These experiments are complex, but should clarify the non-cell autonomous issue and the function of the three genes in regulating layer 6 CTN development. The experiments are time- and money-consuming, as only 1/8 of the embryos are the right genotype. A complication in analyzing Tbr1-/- mice is that Tbr1 is required for subplate development. It is possible that pioneering axons from subplate neurons guide corticothalamic projections. Thus, absence of corticothalamic axons in Tbr1-/- mice may be due to defective subplate or to a cell-automomous defect in CTNs. To distinguish between these two possibilities the PI will restore normal subplate neurons and pioneering axons by generating aggregation chimeras. There seems to be some confusion as to how chimeras are made (mentions aggregating E1.5 embryos). To genotype chimeras between Tbr1-/- and Grg4-LP cells, mutant layer 6 CTNs should be stained with beta-galactosidase antibodies, but not with Tbr1 antibodies. One consideration is that the chimeras will only test cell autonomous functions of Tbr1.
Specific Aim 3 is to identify the combinatorial transcription factor code that distinguishes the fate of CSMN and CTN and determine the effect of manipulating the expression of these factors. The hypothesis that is being tested is whether different expression levels of Fezf2 and Ctip2 proteins (or Grg4, Tbr1 and Nfia) determine whether CTNs project to the thalamus and CSMNs to the spinal cord. The fact that Ctip2 protein is expressed at a higher level in layer 5 CSMNs than in layer 6 CTNs (preliminary data) suggests that the level of this protein is important in the projection target. The PI will test whether Fezf2 is important in determining cell fate by raising an antibody to Fezf2 for immunohistochemical studies and determining the effect of increasing the level of expression of Fezf2 by in utero electroporation into Grg4-LP mice; for example, there may be redirection of PLAP-labeled axons to the corticospinal tract. In addition, the PI will test whether Grg4, Tbr1 and Nfia are part of the combinatorial transcription code by electroporating one or more of these genes into embryos that have already completed layer 6 neurogenesis but not yet layer 5. However, it was not clear how she will show the electroporated cell bodies are in layer 5.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant received her undergraduate education at one of the top tier colleges in China (Beijing University). She did her Ph.D. at SUNY Stony Brook with Sid Strickland studying fly oogenesis (Genetics and Development paper), and she conducted postdoctoral research with Susan McConnell at Stanford University studying Fezf2 in the cortex. She has published a total of five first-author papers, and two of these papers have appeared in very respectable journals. Her graduate work resulted in a publication in Development which led to receipt of the Larry Sandler Award, which is given annually for the most outstanding dissertation work in Drosophila. As a post-doc, she published an important paper in PNAS showing that Fezf2is required for layer 5 neuron (CSMNs) differentiation and axon projections. In 2006, Dr. Chen joined the Department of Molecular, Cellular and Developmental Biology at UC Santa Cruz, and she has since recruited a post-doctoral fellow and graduate student to her lab. She is presently funded for two years for a CIRM proposal which began in 8/07 entitled "In vitro differentiation of hESCs into CSMNs." This proposal aims to identify a marker for CSMNs, differentiate hESCs into CSMNs in vitro, and test whether the CSMNs can form functional circuits in vivo. The possibility of overlap of this SEED grant with the present proposal should be examined.
Dr. Chen has written a carefully-considered plan for career development. She states that receiving a CIRM grant would allow her to turn much of her energy from grant writing to developing her research program. In addition, should she receive a CIRM New Faculty Award, her department has agreed to allow her to buy one year of teaching relief, further enabling her to concentrate on generating the data necessary to obtain future federal funding. She has a number of excellent mentors at UCSB (David Feldheim), Stanford (Theo Palmer) and UCSF (John Rubenstein) who will guide her in cortical development and stem cell concepts, and Susan McConnell will continue to be supportive. This grant will give her secure funding for 5 years. The milestones are realistic and achievable.
In conclusion, Dr. Chen has a solid research record and has been well trained in genetics and neurobiology. She is likely to contribute original papers to the field of cortical development and has the potential to be a leader in the stem cell field.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The candidate’s department chair has provided a letter documenting the standard institutional commitments to junior faculty. In particular, the department has provided adequate space to the candidate and an opportunity to move into new space in 2007. All necessary core facilities are available for conduct of the research. Importantly, the applicant has access to a core microarray and transgenic mouse facility as well as a communal facility for FACS. The institutional committment regarding Dr. Chen's start-up is not detailed, but the candidate will have one full year of teaching relief - apparently if the present application is funded. Her appointment is tenure-track. At UC Santa Cruz, tenure is typically conferred within five to seven years of the initial appointment at the rank of Assistant Professor.
The scientific environment for stem cell research at UC Santa Cruz is good. The Department anticipates "hiring 3-4 new stem cell researchers in the near future" including one this fall. In addition, the Dean is seeking approval for the appointment of a senior level stem cell researcher. A stem cell research facility is due to begin construction in the Fall of '07. The PI is a member of a CIRM stem cell research group at UCSC that consists of over 10 individuals and organizes symposia, hosts outside speakers, etc. In addition, the candidate has a mentoring committee to guide her. Grants from junior faculty are reviewed by senior faculty. There are also collaborative interactions with individuals at UCSC, UCSF, and Stanford.
DISCUSSION: Reviewers commented that this is a strong, well-trained candidate in a good environment for stem cell research. The proposal is a solid basic science project that is interesting if not particularly innovative, although it does make use of the latest technology for gain-of-function and loss-of-function analyses. The PI has a foothold in a field that not many people are in, although she is studying neural progenitors rather than stem cells. The topic of the proposal has great relevance to diseases such as ALS involving cortical spinal motor neurons or cortical thalamic neurons. Overall, this is a talented investigator studying an important field at a good institution.