Stephen H. Devoto - US grants

DESCRIPTION (provided by applicant): Multiple phases of myogenesis have been observed in all species. For example, in mammals, embryonic myogenesis is followed by fetal myogenesis, then neonatal myogenesis, finally myogenic repair. Although the basic transcriptional machinery for these different phases of myogenesis is similar and reasonably well understood, very little is known about the basic developmental biology of these different myogenic periods. In particular, it is not clear if there is a direct, lineal relationship between cells that underlie these different epochs. Is there one type of myogenic precursor that sequentially produces different types of muscle, or are there many types of precursors? Do the same signals regulate cell fate choices at each stage? Can we learn something relevant to stem cell biology by studying these different phases of muscle patterning? This application seeks to answer these long term questions, using the powerful combination of cellular embryology and genetics possible in the zebrafish. Inspired by lineage experiments, the earliest slow muscle precursors were proposed to be induced by Hedgehog signaling. After these embryonic slow muscle fibers develop, there are one or more phases of myogenesis in zebrafish, and the later, post-embryonic phase of myogenesis is only partially regulated by Hedgehog signaling. This application aims to use single-cell injections of lineage tracers to identify the precursors to this post-embryonic muscle growth. It also aims to characterize the role of Hedgehog signaling in post-embryonic myogenesis, using a combination of genetics, pharmacology, and experimental embryology. This research will answer questions of fundamental importance to the understanding of how cells develop into muscle fibers. This information is critical to understanding muscle diseases including heart disease and muscular dystrophy. When the signals regulating myogenesis are understood, it will be possible to guide stem cells towards a myogenic fate, to design therapies for devastating human diseases.

Abstract Naegele NSF 0070352


A Confocal Microscope for Research and Teaching in Biology and Neuroscience

A Zeiss 510 Confocal Microscope will be used for research and training in developmental biology and neuroscience at Wesleyan University. This new confocal microscope will enable the three primary faculty users and members of their research laboratories to study the dynamic movements of cells and proteins in living embryos and to identify intercellular junctions between cells in thick sections of brain tissue. In addition, training on the confocal microscope will be an important component of a Biology department graduate-level course in advanced microscopy, including confocal, immunofluorescence, and electron microscopy. The confocal microscope facility is part of an Advanced Instrumentation Facility in the Science Center at Wesleyan University. This facility already contains scanning and transmission electron microscopes, an adjacent wet laboratory, a room for tissue sectioning, and a computer room with associated digital scanners and other image processing equipment.

The projects to be carried out by major users of this confocal microscope, Drs. Devoto, Kirn, and Naegele, and members of their laboratories, each focus on vertebrate development. The Devoto laboratory will focus on the genetic and molecular guidance cues used by migrating muscle cells in living zebrafish embryos. The Kirn laboratory will address the mechanisms of neuronal replacement and neurogenesis in the brains of adult birds. The Naegele laboratory will study cellular and molecular signals regulating programmed cell death and engulfment of dying neurons in the rodent cerebral cortex and visual system. Occasional use of the confocal is also planned by five additional faculty in the Biology and Molecular Biology and Biochemistry Departments who study a variety of biological problems ranging from the role of transcription factors in embryo development to the cell cycle regulation in yeast. Additional minor use by one extramural group Pfizer, Inc. is planned for 2 days/month. Funds from this extramural group will be a significant source of revenue for long-term maintenance of the confocal, including service contracts.

We anticipate that this confocal microscope and the associated research programs will have a significant impact on research and training at Wesleyan University in the fields of developmental biology and neurobiology. The objective of our science training programs at Wesleyan University is to provide high-quality research experiences for undergraduate and graduate students, as well as postdoctoral fellows and visiting scientists. This hands-on training is of fundamental importance for careers in scientific research, technology, and education. Special initiatives at Wesleyan University advance women and minorities in science, as well as providing access for students with disabilities. It is anticipated that the new confocal microscope will contribute significantly to a basic understanding of how cells in the developing embryo migrate, form connections, and how some undergo programmed cell death as part of their normal developmental plan. These studies will ultimately lead to a better understanding of how the vertebrate brain and body are constructed during development and maintained throughout the life of the organism.

DESCRIPTION (provided by applicant): Our long-term goal is to understand how cell-cell interactions regulate patterning in the development of the fore-brain and the trunk musculoskeletal system. Our labs have demonstrated that Hedgehog signaling is required for both of these processes. Hedgehog signaling is also critical for regulating cell proliferation in humans, activation of Hh signaling is associated with several of the most common cancers. Hedgehog signaling is incompletely understood, and there are very few mutations in model vertebrates that activate Hh signaling. We propose to use a genetic selection protocol to identify large numbers of mutations in a variety of genes in the Hedgehog signaling pathway. We will do this by selecting for mutants in zebrafish that are resistant to low doses of cyclopamine, a drug that specifically inhibits Hedgehog signaling. We expect to find loss of function mutations in the many genes that encode inhibitory regulators of Hedgehog signaling. We also expect to find activating mutations in the many genes that encode activating components of the pathway. Some of these will be new mutations in previously identified components, while some mutations will identify previously unsuspected components in the Hh signaling pathway. All of the mutations will be useful tools for understanding not only the mechanism of Hedgehog signaling, but also the consequences of the activation of Hedgehog signaling on muscle and brain development. In addition, these mutations will identify genes that are likely to play important roles in the development of human cancer.

DESCRIPTION (provided by applicant): Diseases of the vertebrae include severe malformations such as spondylocostal dysostosis, and milder ones including scoliosis, or curvature of the spine. About 1 in 1000 live births has a congenital form of one of these diseases. The genes implicated in these diseases also affect segmentation of the embryonic mesoderm, strongly suggesting that embryonic segmentation defects underlie vertebral malformations. We propose to explore the link between embryonic segmentation and vertebral development, focusing on three genes that are at the center of the segmentation, and have not been extensively characterized for their role in spine development. One of these, mespb, is regulated by Notch signaling. The other two are tbx6 and ripply1, which together regulate mespb. We will do all of our work in zebrafish, because its advantages of external development, high fecundity, transparency, and genetic tools will allow for rapid advances in understanding normal and pathological vertebral development. We will use mutants and anti-sense morpholinos to reduce, and already established transgenic animals to increase, the dosage of each of the three genes. We will assay both embryonic segmentation and vertebral shapes, using a quantitative assay to map the location and number of defects. The quantitative approach will allow us to test for correlations between levels of genes and number of defects. We will also characterize the development of vertebrae following perturbations in embryonic segmentation, to probe the relationship between embryonic segments and adult vertebral shapes. We will use gene expression, histological observations, and live imaging to characterize the development of vertebrae and ribs in animals in which embryonic segmentation was previously characterized. Finally, we will modify gene expression and examine the expression of sclerotome genes to begin to understand the relationship between segmentation defects and vertebral defects our research will be in the areas of molecular, cell, and developmental biology of vertebral development. The research will be carried out by a Ph.D. graduate student who will receive training in these research areas. S/he will be assisted by two undergraduates at a time, who will receive one to two years of training each. The total number of participating undergraduate trainees will be six.

An award is made to Wesleyan University to support the acquisition of a confocal microscope for use by faculty and students across multiple departments and disciplines. The new instrument will profoundly enhance the training environment and be used during many highly interactive laboratory courses taught by Wesleyan faculty to promote STEM career advancement. The proposed microscope will be housed in an advanced imaging facility that includes scanning and transmission electron microscopes. To support dissemination of science to the general public, as exemplified by the large number of articles authored by Wesleyan faculty that are printed in the popular media, data generated by the new system will be used in outreach presentations, science camps, local high-schools, an annual scientific imaging student prize, and similar forums.

Wesleyan boasts an unusually broad portfolio of research topics that will benefit from the new system, including evolution and development of morphology, optogenetic-based control of behavior, cell biology, physiology, developmental neuroscience, and chromosomal dynamics. Each of these areas is experiencing an expansion of research into spatial relationships, driven by advances in microscopy, and by the realization that researchers must study three-dimensional shapes and how those change over time to understand the structure and function of biological systems. Several of the research projects will only be possible because the local availability of the system. Data and analyses from these studies will be published in peer-reviewed scientific journals, presented at scientific meetings, and used in both educational and public outreach activities.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.