Kate F. Barald - US grants

This project, which will begin its tenth year of support from the NSF, addresses one of the critical questions in developmental biology: how do aa relatively small number of embryonic cells give rise to the multitude of cell types that make up the individual? An ideal model system in which this question can be addressed is the neural crest, which appears very early in the development in all vertebrate species. Although the neural crest is only recognizable for a few days during the course of development, it gives rise to a myriad of important structures in the body. Among the hundreds of neural crest derivatives is the entire peripheral nervous system, all of the pigment cells in the skin and the cells that make the dentin in the teeth. How are such an enormous number of diverse cell types made from a seemingly homogeneous population of cells in a normal individual? With previous NSF support, this laboratory devised sophisticated cell sorting techniques for isolating and studying subpopulations of neural crest cells. Furthermore, they can transplant neural crest cells and follow the transplanted cells throughout development. The current studies will specifically examine how individual neuronal cell types arise from the neural crest by examining the development of two identified subpopulations following transplantation. The events that direct apparently homogeneous neural crest cells to become specific neurons will be examined, and the specific control genes that are associated with these events will be identified. These studies are at the interface of cellular and molecular biology, where, for the first time, it is now possible to address one of the most fundamental questions in developmental neuroscience.

This proposal examines the potential role of the neurofibromatosis type 1 (NF1) gene and its protein product in development and differentiation of neuronal lineages from the neural crest (NC), a cell population that appears transiently during embryonic development of all vertebrates. The long-range objectives are to determine how the NC gives rise to the myriad of differentiated cells which arise from it, and most importantly, how neuronal lineages arise from the NC. Understanding the role of NF1 in normal NC development and differentiation will augment information on how the alteration or disruption of the gene results in the aberrant proliferation of certain crest derivatives that characterizes the disease Neurofibromatosis 1 in humans. The experiments outlined in this proposal are designed to determine if NF1 is an important gene in crest development by following its early expression pattern in the premigratory and migratory avian neural crest and in the derivatives of the crest that are involved in NF1. The avian neurofibromatosis 1 (alphaNF1) cDNA which has been cloned and partially sequenced in this lab is highly (at least 86%) homologous to the human NF1 cDNA at the nucleic acid level. Using an avian system as a model has unique advantages for neural crest studies. Not only is the avian system the best studied of all the NC systems and that which has produced the most information about crest development, it is the only system that is accessible and manipulable at very early developmental time points. These experiments are at he interface of molecular and cellular biology and address important questions of stem cell differentiation both in normal development and pathologies such as neurofibromatosis. The specific questions addressed include: 1. How does differentiation by retinoic acid (RA) upregulate alphaNF1 message levels in a src-transformed immortalized quail NC cell line that become neuronal upon differentiation? 2. Do primary avian NC cells express the gene and is it upregulated upon differentiation of NC into a variety of neurons? 3. What is the expression pattern of the gene and the protein product during differentiation of the NC in vivo? 4. If anti-sense constructs of the alphaNFI cDNA or gene are introduce into the src-transformed quail NC cell line, is differentiation and/or alphaNF1 upregulation blocked? 5. Does transplantation of the src-transformed quail NC cell line, onto chick NC pathways that do not lead to neuronal differentiation, lead to upregulation of the alphaNF1 gene?

9509088 McKitrick Birds possess more hindlimb muscles than their closest living relatives, the crocodiles and alligators. In addition, some muscles primitively present in birds have been lost numerous times within the avian lineage during the course of history. If historical relationships among crocodilians and birds (archosaurs) are to be understood, hypotheses must be constructed about the sequence of appearance and loss of specific muscles or muscle parts. Prior to such analysis, however, it is crucial to develop hypotheses about which muscles in birds and crocodilians are homologous in the historical sense, i.e., which muscles correspond in the two groups. Identifying these homologies has not been possible with traditional techniques such as gross anatomical study of muscle form. In this proposal, a new approach will be taken to analyze homologies in archosaur hindlimb muscles by the use of developmental studies of muscle proteins using avian and crocodilian embryos. Muscle proteins have different types, termed isoforms, at different stages of organismal development,and these are highly conserved among species. The developmental shifts in isoforms of contractile proteins such as actins, myosins, tropomyosins and troponins, as well as other related proteins follow specific patterns. The earliest forms of these proteins are replaced by embryonic versions and later by neonatal and adult forms. Since individual muscle groups bear "signature" sequential patterns, the PI will examine the protein isoforms and their messages in birds and crocodilians to see whether muscles of known homology in the two groups have retained the ancestral patterns. If so, such patterns can be used to hypothesize historical relationships between muscles of heretofore questionable homology. %%% This approach has the potential to solve important problems in comparative biology about the relationship among traits in related organisms where historical change has in effect obliterated the record. ***

We have cloned and sequenced a 414-bp avian neurofibromatosis 1 (aNF1) cDNA which is 82% identical to the human NF1 gene at the nucleic acid level and 93% identical at the predicted protein sequence. We have used this probe to establish the normal expression pattern of aNF1 in early chicken and quail embryos, and to examine the potential role of aNF1 in neural crest (NC) development. Northern blot analysis shows a large (12.6kb) aNF1 transcript expressed with increasing levels from day 2 to day 6 in ovo. Immunoprecipitation studies show a similar increase in the expression of NF1 protein product, neurofibromin, from day 2 to day 6 in ovo. In situ hybridization experiments show aNF1 is expressed no earlier than stage 8 (26-29 hours in ovo), with ubiquitous expression from stage 8 through stage 29 (6 days in ovo). Immunohistochemistry labels a subset of neurofibromin-positive neural crest cells at stages 13 (2 days in ovo) and 20 (3 days in ovo) and neural crest derivatives at stage 27 and 29 (5 and 6 days in ovo). To examine the role of neurofibromin in neural crest cells further, we have generated a quail neural crest cell line, sQNC-1, by infecting primary quail NC cultures with the PA101 temperature sensitive mutant of the Rous Sarcoma virus. When treated with 5x10-7M retinoic acid (RA), these cells take on a neuronal morphology: they extend long neuron-like processes, and begin to express a neuron-specific isoform of '-tubulin. Concomitant with RA- induced differentiation, we see an increase of at least ten-fold in both NF-1 message and protein levels, inviting speculation that NF1 plays a role in the differentiation of neurogenic cells in the neural crest.

The inner ear of vertebrates is a complex labyrinth of fluid-filled canals, pouches and ducts encased in bone. These organs contain sensory hair cells and supporting cells, and aid in balance and movement control, with some parts specialized for hearing, as in the cochlea of mammals. This complex three-dimensional structure arises during embryonic development from a thickened flat patch called a placode on the side of the head. It is unclear what genes are involved in the developmental program for these precisely 'engineered' tubes and pouches from this originally simple patch of skin. Preliminary results suggest that there are important interactions between two proteins, one called 'bone-morphogenetic protein #4' (BMP4) and the other called 'noggin' that may act as an antagonistic pair of regulators for patterning. This project utilizes anatomical, genetic and molecular biology along with novel microsurgical techniques to implant tiny beads containing the protein-expressing cells directly in the developing ear. Results will clarify how genetic expression of particular proteins can shape the morphology of a complex sensory organ. This work will have an impact in several areas of neuroscience, and the project also will provide multidisciplinary training for students.

DESCRIPTION (provided by applicant): The long-term goals of this work are to understand the molecular signaling pathways involved in the development and regeneration of inner ear sensory and neuronal populations. The studies done during the initial grant period were directed at discovering the upstream genes that regulate expression of the growth factor Bone Morphogenetic Protein 4 (BMP4) and its role in the formation of sensory hair cells (HC) in inner ear development. The work done to date has demonstrated that BMP4/antagonist cascades are critical for normal inner ear morphogenesis (Gerlach et al, 2000;Gerlach-Bank et al, 2002 and Gerlach-Bank et al, 2004) and has identified a novel BMP4 promoter, which is expressed in the inner ear and which is repressed by retinoic acid (RA), explaining why RA-treated inner ears resemble BMP4 antagonist-treated ears (Thompson et al, 2003). In this renewal application, attention is focused on the downstream genes, particularly the role of ZIC genes in inner ear development. The hypothesis that directs this work is that ZIC genes are critical transcription factors that serve as regulatory genes, channeling undifferentiated otocyst precursor cells either to a sensory neuron or sensory hair cell (HC)/supporting cell (SC) fate from a common precursor cell in the otocyst. This laboratory has shown that both ZIC1 and ZIC 2 are expressed at the right time and in the right cells to play critical regulatory roles in this cell lineage selection (Warner et al, 2003). In addition, studies of immortalized inner ear cell lines and ZIC2 knockdown mouse models in this laboratory (Gerlach-Bank et al, in prep.) have provided additional support for this hypothesis. The experiments in this proposal therefore investigate the role of ZIC genes and the atonal class downstream genes they control in sensory cell and sensory neuron lineage specification in the inner ear. For these experiments immortalized otocyst cell lines that represent neuron-like, hair cell- (HC) or supporting cell (SC)-like, and neuronal/sensory precursor-like IMO cell populations (precursor-like) will be used (Germiller et al, submitted). Findings from the cell line experiments will then be tested in functional experiments in "real" inner ears in embryos of chick and ZIC knockout and knockdown mice. The hypotheses to be tested include: ZIC1 genes control sensory neuron formation through regulation of Neurogeninl and NeuroD and ZIC2 genes control sensory hair cell formation in the inner ear by regulating Math1. ZIC1 is also known to downregulate MathI (Ebert et al, 2003) and in some cases in the CNS, BMP4 is thought to upregulate Mathl (Ebert et al, 2003;Alder et al, 1999) but whether this is through effects on ZIC genes is unknown. Gene profiling through microarrays and analyses of loss- and gain-of-function experiments in these model systems will allow tests of all these hypotheses.

DESCRIPTION (provided by applicant): Sensory hair cell (HC) loss is a major cause of human deafness, tinnitis and balance disorders. However, despite great progress in many laboratories, the molecular bases for both hair cell development and regeneration are still not understood. We propose to test the novel hypothesis that mammalian hair cell regeneration in vivo will be possible if we can induce HCs to re-express the critically important growth factor Bone Morphogenetic Protein 4 (BMP4). In the experiments proposed here, we will make a transgenic "knock-in" mouse, in which BIVIP4 can be expressed in mammalian hair cells under a dual control system. We will engineer constructs that place a full-length BMP4 under the control of the hair-cell specific brn3.1 promoter, in a tet-on inducible system, so that providing the transgenic mice with doxycycline (DOX) will turn on BMP4 expression specifically in terminally differentiated hair cells of the inner ear that normally do not express BIVIP4. BIVIP4 is an axial patterning morphogen, but more significantly, it is a major "plasticity" gene that keeps sensory and neuronal cells in a proliferative state, resembling stem cells. We will first express this tet-on construct in immortalized inner ear cell lines derived from day 9 Immortomouse otocysts and in mouse embryonic stem cells (ES) in culture. We have made constructs that drive luciferase, to test the proof of principle. We can also compare the genetic expression repertoire of the cells induced with DOX to those of cells that are not induced by RT-PCR and by gene array techniques. Next we will make a transgenic knock-in mouse that is capable of re-expressing BMP4 in all of the hair cells of the inner ear. We will study the cellular morphology and HC repair and regenerative capacity of these mice in the presence and absence of DOX; that is, when the hair cells are either capable of re-expressing BIVIP4 or not. We are testing the hypothesis that if BIVIP4 is re-expressed in hair cells of the inner ear, returning these cells to an earlier more 11 stem cell-like" state, we will be able to regenerate a sensory epithelium after either noise-induced trauma or ototoxic aminoglycoside exposure. By using focal noise exposure or precisely dosed and delivered aminoglycosides, we can vary the site and extent of the lesions, and determine the degree to which repair, regeneration and/or preservation play a role in restoring a functional sensory epithelium.

This project is creating a comprehensive research responsibility and ethics (RRE) training program for undergraduate, graduate and postdoctoral scholars and researchers at the University of Michigan in the basic and social sciences and engineering. By virtue of its size, this campus presents unique challenges and opportunities. This interdisciplinary program is based on a comprehensive library of web-based podcasts, which serve as the basis for discussion of case studies by interdisciplinary small groups of students and postdocs as they attempt to solve problems of research responsibility and ethics (RRE).

The discussion groups use the podcast library of lectures, panel discussions, mock IRB boards, and interviews with researchers and ethicists. Subjects range from ethical and moral reasoning and values presented by faculty from the Philosophy Department through issues of mentoring, fraud, fabrication and plagiarism to specific problems in social science research and professional ethics and regulatory issues. To reinforce the information discussed in the small groups, interdisciplinary "ethics slams" are held among course participants. Evaluation of the project is through periodic questionnaires, long-term follow up through self-reported behavioral data, and outside evaluation.

To broaden the impact of the program beyond the reach of its website, the materials are being shared with the NSF-sponsored Michigan AGEP alliance schools (Michigan State University, Wayne State University and Western Michigan University), the 400 institutions in the Inter-university Consortium for Political and Social Research (ICPSR), the University of Michigan ADVANCE program, the Office of Student Success (that sponsors the Summer Institute and Summer Research Opportunity Program for under-represented students in the graduate programs), the Undergraduate Research Opportunity Program (that sponsors 1000 undergraduate researchers per year), and the Bouchet Society, an honor society for underrepresented minority graduate students.

"This award is funded under the American Recovery and Reinvestment Act of 2009
(Public Law 111-5)."

In this project new technologies based on the emerging sciences of microfluidics and microelectro-poration have been developed in a partnership between neuroscientists and engineers to address fundamental questions of nervous system and sensory system development. Using a model organism, the tiny zebrafish, newly recognized factors "immune system cytokines" are being studied for their roles in the earliest stages of development of the nervous system and sensory systems (ear and eye). A zebrafish microfluidic bioreactor was developed to expose specific circumscribed parts of the growing embryo's nervous system or sensory systems to streams of morphogens (fundamental growth promoting molecules), in controlled concentrations. In the bioreactor, small areas of the developing nervous system, the ear or the eye can be bathed in such morphogens, including MIF (macrophage migration inhibitory factor), which is an "inflammatory" cytokine. Cytokines are vital for immune system development as well as triggers of damaging inflammatory reactions after the immune system forms. The primary investigator's laboratory has made the surprising discovery that immune system cytokines, and especially MIF, are also vital for the very earliest development of nervous system neurons (and pathfinding of neurites) and sensory cells in the eye and ear. MIF could also be involved in repair processes at concentrations orders of magnitude below those that cause inflammation. MIF and other cytokines therefore act as "neurotrophins" or directional nerve growth factors in these systems, a surprising outcome to scientists in a field that had long thought only classical (already identified) neurotrophins could play such a role. The use of microfluidic technologies as well as electroporation of molecules directly into the developing zebrafish inner ear were key to this new line of research. This project investigates the mechanism of action by which MIF exposure to circumscribed parts of the nervous system and developing sensory systems promotes their development and how blocking it alters development. Fundamental links between the developing nervous and sensory systems and the immune system can be studied in this model. Zebrafish auditory system development is, in many ways, the same as that in the human ear. The same molecules that are active in zebrafish are active in humans, but zebrafish are easier to study. Development takes place outside the mother and is extremely rapid (a scale of hours to a few days rather than weeks or months). This project will provide training opportunities for interdisciplinary teams of neuroscientists and engineers at all levels from undergraduate and graduate students (who developed the zebrafish bioreactor) to the postdoctoral and faculty level, who supervised the work.

DESCRIPTION (provided by applicant): A Postbaccalaureate Research Education Program (PREP) will be created at the University of Michigan to increase the ability of underrepresented minority students to be accepted into excellent doctoral (Ph.D.) programs in the biological and biomedical disciplines throughout the country. The University of Michigan has long had a strong history of encouraging undergraduates toward research careers and in recent years has gained national attention through its commitment to recruiting a diverse and talented student population. In the biosciences, there are extensive research opportunities for undergraduates from UM and elsewhere and strong and coordinated bioscience graduate programs that have been successful and recruiting and graduating a diverse student body. However, in our graduate applications and recruiting travels throughout the country we frequently encounter students who, for a variety of reasons, are not yet quite prepared to matriculate and succeed in strong graduate programs, but could be with specific types of further preparation. The creation of a PREP at Michigan would provide promising students from around the country with the needed preparation to both successfully apply to strong graduate programs and to succeed once they matriculate. The described project speaks directly to the objectives of the PREP program announcement from the NIH. Students from several ethnic and racial backgrounds are severely underrepresented in PhD-level research positions that address issues of human health. The Michigan PREP provides students with opportunities to prepare themselves for graduate school in a wide range of different disciplines in the Schools of Medicine, Public Health, Engineering (Biomedical), Pharmacy, Kinesiology, and Dentistry.