Pamela A. Raymond - US grants

This proposal describes investigations into the addition of new neurons and the resultant reorganizations of synaptic connectivity which occur in the visual system during postembryonic growth of goldfish. Previous work has shown that rods are produced from special precursor cells and continuously inserted into the differentiated retina even in adults. The proposed studies seek to discover how the new rods are integrated into the preestablished retinal circuitry, what are the functional consequences of the delayed and prolonged development of rods, and what are the rules that govern neurogenesis in the normal and regenerating retina. There are five Specific Aims. 1. To describe synaptogenesis of photoreceptor cells (rods) in the larvae and juvenile retina of goldfish. 2. To examine the development of the retinomotor response and to determine the neural circuitry involved. 3. To examine the relationship between Muller glia and migrating neuronal precursors. 4. To determine whether mechanical stretch modulates the rate of cell proliferation in the retina and whether cell death is involved. 5. To examine the specificity of the rod precursors population using immunocytochemistry and to challenge the rod precursors to lose their specificity when the retina regenerates following destruction by metabolic toxins. These experiments are relevant to two central issues in developmental neurobiology. The first is: how do neurons adjust to changes in the relative numbers or proportions of other neurons with which they form connections? The second is: how do cellular interactions between neurons and glial cells influence the development of structure in the brain? The studies on retinal regeneration will also provide important information about repair and replacement of neurons in the central nervous system.

This award provides funds for the establishment of a Research Training Group in Development of the Nervous System. The faculty group is a mixture of outstanding senior and junior investigators who come from a variety of disciplinary backgrounds and work with a variety of organisms, but share a common interest in development of the nervous system. The research programs in which trainees will participate are aimed at three problems central to the development of all nervous systems: neurogenesis, axonal navigation, and synaptogenesis. The funds will provide stipends for graduate students and postdoctoral fellows, will support research participation by undergraduate students, will defray part of the cost of the trainees' research and will enable the trainees to attend scientific meetings. In addition, funds will be used to purchase specialized research equipment to be used by trainees, and to bring experts from other research and academic institutions to aid in a summer laboratory and lecture course for trainees. In recent years, remarkable advances in the use of genetics, biochemistry, and microscopy have led to significant new knowledge about the development of multicellular organisms. Despite this, much remains to be learned about development and, in particular, about the mechanisms of key problems posed by the development of many different tissues and structures. These include the differentiation of cell types, the basis of cell-cell recognition, and the migration of cells during development. In this respect, some of the most exciting opportunities and, at the same time, most difficult challenges are presented by developmental studies of animal nervous systems, which typically contain a large variety of cell types and extend throughout the animal. Successful research in this area requires a mixture of diverse intellectual and experimental skills not often taught in traditional neuroscience programs. This award will provide funds for the training of young neuroscientists who have the multiple skill needed to attack one of the last great frontiers of modern biology.

Normal function of the vertebrate nervous system depends on a precise pattern of distribution of the different cell types. For example, cone photoreceptors, which constitute a sheet of light-sensitive neurons in the retina of the eye, come in three main types, each type maximally sensitive to light of a certain color, red, green or blue. The different cone-cell types are arranged in a regular, repeating lattice or mosaic pattern. The developmental mechanisms that build the lattice, i.e. that control the spacing and determine the fate of the presumptive cone cells, are not well understood. Most biologists agree that the immature cells send signals to their neighbors that determine the fate of the neighboring cells. To begin to understand this process, the earliest stages in the development of the cone mosaic will be examined. Molecular markers will used to identify the early developing cone types. The hypothesis to be tested is that one cone type acts as the "founder" to establish a blueprint of the mosaic pattern, and the other cone types develop later, filling in the pattern. This study will give new insight into how the distribution of different neuron types are established and will provide new information about the way cells communicate with each other during development.

The long range objectives of this project are to determine the role of cellular interactions in regulating neuronal commitment and differentiation during development and regeneration of the teleost neural retina, with special emphasis on photoreceptors. The focus of the research plan is on specialized cell-cell junctions and the calcium- dependent adhesion molecules, cadherins, as mediators of inductive signalling events. Fusion proteins produced from zebrafish N-cadherin cDNA will be used to generate polyclonal antibodies capable of blocking cadherin-mediated cell-cell adhesion. The hypothesis is that cadherins/adherens junctions play a role in mediating choice of photoreceptor fate. Cadherin-specific blocking antibodies, fusion proteins, N-terminal fragments, or synthetic peptides, are applied to whole embryonic eyes in organ culture, and production of photoreceptors is monitored with rod-specific and cone-specific monoclonal antibodies and with in situ hybridization using isotype-specific rod and cone opsin riboprobes. Since abnormal cadherin function has been implicated in cellular transformation and metaplasia, a better understanding of the role of cadherins in regulating normal cell phenotype is of interest.

The overall goal of this ongoing research program is to understand the molecular basis of cell-cell interactions that regulate retinal neurogenesis during development and regeneration. The zebrafish (Danio rerio) provides a powerful genetic model in which to define the causal relationships between retinal stem cells and the in vivo microenvironment that support neuronal regeneration and restoration of functional neural circuits in the retina of an adult organism. The concept that retinal neurons and MOiler glia derive from a common progenitor in the developing retina is widely accepted, and it is known that late stage retinal progenitors can generate both rod photoreceptors and MOiler glia. More surprising is the recent discovery that differentiated MOiler glia continue to function as neuronal progenitors, producing rod photoreceptors in the uninjured, adult teleost retina. In response to loss of retinal neurons, MOiler glia in the teleost retina dedifferentiate, reenter the cell cycle and generate multipotent retinal progenitors that generate neurons to repair the damage. A latent and abortive neurogenic capacity of MOiler glia in adult mammalian retinas has been demonstrated, however, almost nothing is known about the molecular regulation that switches MOiler glia from a state of reactive gliosis to neurogenesis. The proposed studies take advantage of zebrafish genetics to discover the cellular and molecular mechanisms that promote the endogenous neurogenic potential of MOiler glia. The specific aims of the proposed research are to evaluate whether candidate regulatory signaling pathways (Wntlp-catenin, Fgf and Notch) acting upstream of the proneural transcription factor ascl1a are required to activate a neurogenic program in MOiler glia, and to evaluate the hypothesis that epithelial characteristics related to apical-basal polarity regulate the neurogenic response of MOiler glia.

9413211 Oakley This award renews support of a joint training effort of 15 faculty from six departments in the Schools of Arts and Sciences, of Medicine and of Dentistry at the University of Michigan, Ann Arbor. The theme of this Research Training Group (RTG) is developmental neurobiology. Faculty and student research emphasize three aspects of the development of the nervous system: neurogenesis, axonal navigation, and synaptogenesis. The research addresses these issues in a variety of organisms, including invertebrates, fish, birds and mammals using a variety of techniques from cell and molecular biology. The RTG sponsors training at all three post-secondary levels. Besides opportunities for student research, RTG faculty have created new courses at both the undergraduate and graduate level. Graduate students are selected from students who apply to one of two departmental Ph.D. programs or an interdepartmental Neuroscience Ph.D. program, and receive support for up to three years. Postdoctoral fellows are selected for one year of support from among candidates proposed by participating faculty. Undergraduates are sponsored during both the academic year and summer term. Participants in the summer research program are recruited nationally. The level of involvement of undergraduates in the RTG is particularly noteworthy; over 80 such students carried out research under the auspices of the RTG during its first four years. ***

The goal of this project is to contribute to the development of a national science and engineering academic workforce that includes the full participation of women at all levels of faculty and academic leadership, particularly at the senior academic ranks, through the transformation of institutional practices, policies, climate and culture. The University of Michigan proposes to undertake three different types of interventions to improve the opportunities and circumstances of tenure-track women faculty in basic science
and engineering fields. These include: (1) a campus climate initiative, which will focus on activities (e.g., workshops, focus groups, climate surveys, consultation on increasing pools of female applicants in searches) that have been identified, or will be created, and made available to any interested science or engineering unit (a department or college) throughout the University; (2) a gender equity resource fund, which will provide new types of direct support to individuals; and (3) a departmental transformation initiative, which will permit a sequenced program of activities to be developed and tailored to a small number of units on a competitive basis. This sequenced program (including internal review or self-study, goal-setting, and a series of targeted activities addressing recruitment, retention and/or climate issues) will enable a sustained, committed intervention within a single department, as well as provide a model of change for other institutional units.
All three sets of programs will be evaluated by independent researchers. Evaluations will be conducted throughout the course of the Award, using both qualitative and quantitative methods. Results of early evaluations will be used to revise
Programs.

This multi-level program is designed to improve the campus environment for women
faculty in science and engineering at the University of Michigan, and as a result to increase the successful recruitment, retention and promotion of tenure-track women faculty in basic science fields. The presence and success of these women faculty will in turn affect the expectations and attitudes of the many women and men who are graduate and undergraduate students in science and engineering fields. Many of these individuals will go on to have science and engineering careers themselves; because UM trains so many students, it is anticipated that the impact of this program will reach well beyond this university. Creation of a more equitable climate at UM will affect other campuses through the next generation of science and engineering faculty who will themselves train students, as well as non-academic work settings in which scientists and
engineers trained at UM are employed.

This project is supported by the NSF ADVANCE Program.
The overall mission of the ADVANCE Program is to increase the participation of women in the scientific and engineering workforce through the increased representation and advancement of women in academic science and engineering careers.

[unreadable] DESCRIPTION (provided by applicant): Retinal photoreceptors are highly specialized neurons with unique, differentiated features associated with detection of photons and transduction of neural signals. The distribution, spacing and spectral identity of individual photoreceptor subtypes is an important parameter that influences many properties of visual function, including sensitivity to low levels of light, visual acuity, color vision, etc. Most vertebrates, including zebrafish, have multiple spectral classes of cone photoreceptors as well as rod photoreceptors. However, the molecular control of photoreceptor determination and cell fate choice, especially the mechanisms that regulate choice of cone spectral classes, are poorly understood. In part, this is because the typical model system (rodents) have rod-dominated retinas. In contrast, teleost fish have abundant cone photoreceptors, and the zebrafish has emerged as a powerful model for forward genetic studies to discover developmental regulatory genes. The objective of the proposed mutagenesis screen is to characterize zebrafish mutants that selectively disrupt cell fate determination of cone photoreceptors in the developing retina and to identify the genes involved. [unreadable] [unreadable] The cone photoreceptors in zebrafish retina include four spectral types, each of which expresses a specific opsin gene that produces a visual pigment with a maximum absorption at wavelengths corresponding to red, green, blue or ultraviolet, respectively. The cones in the zebrafish retina form a precise mosaic pattern such that rows of red and green double cones alternate with rows of blue and ultraviolet single cones, superimposed on an intrinsic pattern of reiterative, mirror-image symmetry. The cone mosaic pattern is generated in a curvilinear wave of differentiation that sweeps across the presumptive photoreceptor layer from optic stalk to retinal margin. The mechanisms that control cell fate determination of cone photoreceptors to produce this highly ordered spatial array are not known. The precision of the spatial and temporal organization of the cone mosaic pattern makes this an ideal model system in which to identify genes that perturb the organization and cell type specification of cone photoreceptors. [unreadable] [unreadable] The rationale for studying cone photoreceptor cell fate determination and patterning is that alterations in the fundamental developmental processes that lead to neuronal specification are thought to be responsible for a number of congenital malformations of the human brain and retina, which impair neuronal function and can result in mortality, morbidity, physical or mental disabilities. Discovering the molecular mechanisms that pattern the cone photoreceptor array in the zebrafish retina will lead to a better understanding of the factors that regulate choice of retinal cell fate and mediate visual behaviors. [unreadable] [unreadable]

Animals begin their life by undergoing a remarkable process of self-organization: Starting from a tiny, single-celled egg, they develop into an incredibly complex organism. Moreover, they do so without centralized control. No master builder directs each cell to its correct position in the final body plan. How exactly living cells are able to collaborate to create precisely constructed tissues and organs is the central question of developmental biology. This project will study a particular example of such self-organization in the fish eye. It will use a combination of approaches, including observations of eye organization, perturbation of this organization by laser pulses, and computer simulations. These techniques will provide a deeper understanding of the fish eye system. This fundamental knowledge about biological self-organization has potential applications ranging from new disease treatments to the design of synthetic self-organizing systems inspired by the mechanisms at work in the eye. As a collaboration between a physicist and a biologist, the project will create opportunities for interdisciplinary education at many levels. The investigators will place a special emphasis on involving students from traditionally underrepresented backgrounds in research early in their undergraduate careers.

The project will combine biological experiments with mathematical modeling to study the emergence of the striking crystalline arrangement of cone photoreceptor cells in the zebrafish retina. The guiding hypothesis is that the formation of this ordered lattice depends on anisotropic mechanical stresses imposed on the retinal epithelium by the annular ligament, a rigid ring of tissue that surrounds the retinal margin. Prior work has shown that a mathematical model of the interaction between mechanical forces and planar cell polarity can reproduce many of the observed features of the regular arrangement of cones, and, in particular, the model correctly predicted the presence of strongly anisotropic interactions between cells in perturbed retina. The current project will test the guiding hypothesis directly. The investigators will use new transgenic strains generated on a pigment mutant background to perform the first imaging and laser microsurgery of retinas in living, adult fish. This will provide a fine-grained quantitative characterization of the degree of mosaic order in space and time. Then, the investigators will observe how this order is affected by ablation of the annular ligament and the photoreceptor cells, using microsurgery to measure stress anisotropy in the retina.

The Michigan Biology Academy Scholars program, from the Departments of Ecology and Evolutionary Biology (EEB) and Molecular, Cellular, and Development Biology (MCDB) in the University of Michigan's College of Literature, Science and the Arts, supports up to twenty scholarship recipients per entering cohort, for a total of up to 80 students over a four-year period. This effort is part of the University of Michigan (M) STEM Academy which recently implemented a new integrated, holistic curricular and co-curricular support system for students with high ability and potential in STEM disciplines, but who, for reasons of socioeconomic status, first generation college status, racial or gender bias, or lack of rigor of high school background, might not be successful at a highly competitive elite research university. The Academy provides academic support for first and second year undergraduates to increase the number, success, and diversity of STEM students. The biological sciences component of the M-STEM Academy is geared towards increasing the number and diversity of students entering biologically-related fields by two different yet interrelated methods: (1) increase the actual number of students pursuing degrees in biological sciences, and (2) increase the retention to degree and the academic success and experiences of diverse students. Increased financial support is critical for both of these aspects to reduce the debt burden and the time spent away from studies to earn money during the academic year. Assessment and evaluation of the Michigan Biology Academy Scholars program is being provided by the Center for the Study of Higher and Postsecondary Education.

A special aspect of this project is the extensive collaboration across University of Michigan college boundaries. In particular, critical STEM departments in the College of Literature, Science and the Arts and the College of Engineering have initiated curricular innovations and bring a myriad of academic support offices in order to provide comprehensive services to all M-STEM students including:

- the Science Learning Center, which supports student learning in the sciences by providing well-coordinated resources, vibrant learning communities and engaging co-curricular programs;
- the Comprehensive Studies Program, a learning community that offers a variety of academic support services designed to support, academically enrich, and retain students;
- the Undergraduate Research Opportunity Program, a nationally recognized program that enhances the undergraduate educational experience through the integration of teaching and research;
- the Women in Science and Engineering Program, a nationally recognized model for advocacy and research on issues for girls and women in STEM fields; and
- through collaborations with other educational pipeline programs on campus, including the Michigan Louis Stokes Alliance for Minority Participation and the Michigan Alliances for Graduate Education and the Professoriate.