William R. Jeffery - US grants

Current understanding of the physiological role of messenger ribonucleoprotein (mRNP) is hampered by the lack of suitable assays for the function of specific proteins bound to messenger RNA (mRNA) in these particles. In the proposed study, the slime mold, Physarum polycephalum, is employed as a unique biological system for the investigation of the structure and function of two divergent types of mRNP, translationally-active mRNP from the polysomes of vegetative plasmodia and stored mRNP from the post-ribosomal supernatant of a dormant stage. The specific objectives are to develop methods for the isolation of polysomal and stored mRNP using cell fractionation techniques and oligo(dT)-cellulose chromatography, characterization and comparison of the RNA and protein moieties of polysomal and stored mRNP, analysis of the formation of stored mRNP during dormancy and its activation during germination by radioactive labelling methods, comparison of the translational efficiency and relative metabolic stability of polysomal and stored mRNP, and the development of assay systems to test the function of mRNP proteins in translation and stability. It is envisioned that this study will ellucidate structural differences exhibited by mRNA in different cellular states and provide roles for at least some of the proteins associated with this molecule.

In this proposal we will continue our recent investigations of the pattern of mRNA distribution during early development and initiate studies on the mechanism of mRNA localization and the role of localized mRNA in embryonic determination. The eggs and embryos of the ascidian Styela, a classic organism for the study of embryonic determination which exhibits colored cytoplasmic regions of known morphogenetic fate, is used as a model system. The specific aims are as follows. 1.) Examination of the distribution of specific mRNA sequences, in particular actin mRNA and other yellow crescent mRNAs, in the egg. 2.) Determination of the oogenetic orgin and embryonic fate of specific mRNA localizations. The first two aims will be accomplished by in situ hybridization with cloned DNA probes and in vitro translation of mRNA prepared from mass isolated egg cytoplasmic regions. 3.) Investigation of the role of the cytoskeletal framework in specific mRNA localization. This will be accomplished by a combination of non-ionic detergent extraction and in situ hybridization. Finally, 4.) bioassays, based on UV irradiation and microinjection, will be developed to assess the role of localized mRNA molecules in embryonic determination. It is anticipated that these studies will provide insight into the spatial distribution of mRNA in embryos and the role of localized mRNA during embryogenesis.

0212110
Jeffery

Little is known about the origins of vertebrate features in the Phylum Chordata. Here, the focus is on aspects of urochordate development that are expected to shed new light on this important problem in evolution and development. The overall goal is to assess the origins of two key vertebrate features in the ascidian urochordates: (1) migratory neural crest cells and (2) larval musculature specified by inductive processes. The ascidian species used in contemporary developmental biology (e. g., Ciona, Molgula, Styela, and Halocynthia) exhibit rapidly developing, highly streamlined tadpole larvae. These derived larva appear to lack migratory neural crest cells and contain only about 40 tail muscle cells, which are primarily specified by localized cytoplasmic determinants. The streamlined tadpoles, which have evolved for rapid dispersal, are not representative of most ascidian larvae. Most ascidian species have larger, slowly developing tadpoles with more complex heads and robust tails, which are likely to represent the true ancestral state. Accordingly, Dr. Jeffery will introduce here the ascidian Ecteinascidia turbinata as an experimental system to study the origin of key vertebrate features in the urochordates. Ecteinascidia, a close relative of Ciona, has a giant tadpole larva exhibiting a head with preformed siphon rudiments, enlarged sensory organs, and pharyngeal gill slits, and a robust tail with 1134 muscle cells. The development of ascidians with highly differentiated tadpoles has been virtually ignored and is ripe for analysis using modern technologies. The Ecteinascidia system will permit him to combine a variety of experimental embryological and molecular approaches, including use of ongoing Ciona genomic and EST databases, to study the evolution of development. This proposal specifically addresses neural crest and muscle development in Ecteinascidia embryos. First, he will determine whether Ecteinascidia embryos have migratory cells homologous to vertebrate neural crest cells using a combination of vital dye and gene marking studies. He already has strong evidence from DiI marking experiments that migratory neural crest-like cells are present. He will determine the embryonic sources, regional migration patterns, and developmental fates of the neural crest-like cells, and compare their properties to vertebrate neural crest cells. Second, he will determine whether the development of tail muscle cells in Ecteinascidia is controlled by cytoplasmic determinants, the predominant means of tail muscle specification in ascidians with streamlined larvae, or by inductive processes, the method of muscle specification characteristic of vertebrates. These studies will be carried out by a combination of cell lineage tracing, in situ mRNA and protein localization, and blastomere isolation and recombination. The intellectual merit of these studies is that they will provide new information on the evolutionary history of neural crest cells and the mechanisms of tail muscle development in the urochordates. The broader impacts of the proposed activity is that it will address the origin of key vertebrate features in the Phylum Chordata and focus our future attention on particular chordate or non-chordate groups in order to chart the evolutionary beginnings of the complex vertebrate body plan. Finally, the research is planned to foster general education in research by incorporating undergraduate students into the investigative process.

DESCRIPTION (provided by applicant): The development and growth of the vertebrate eye must be perfectly coordinated in order to transmit correct visual images to the brain. The lens is responsible in part for coordinating eye growth but the underlying molecular and cellular mechanisms are unknown. We will investigate this problem in Astyanax mexicanus, a teleost species consisting of an eyed surface dwelling form (surface fish) and a blind cave-dwelling form (cavefish). Eye primordia are initially formed during cavefish development but subsequently arrest and degenerate, resulting in a blind adult. The first tissue to degenerate is the lens. Remarkably, transplanting a surface fish embryonic lens into a cavefish optic cup can restore a complete eye in adult cavefish. We propose to study the molecular and cellular basis of lens regulation of eye growth and development in Astyanax, which is uniquely suited for lens manipulations. The first three specific aims are designed to determine the cellular events underlying the arrest of retinal, corneal, and scleral differentiation in cavefish and how the lens modulates these events. In these aims we will combine lens transplantation methods with specific molecular markers for retina, cornea, and sclera development. The fourth specific aim is to identify candidate genes encoding lens-signaling factors affecting the development of other eye parts. The genes will be obtained by a subtractive hybridization using cDNA libraries obtained from isolated surface fish and cavefish lenses. The fifth specific aim is to determine the role of the candidate genes in lens degeneration and lens-regulated development of other eye tissues. This will be done by loss-of-function and gain of function studies, as well as by capitalizing on specific properties of the candidate genes. We expect this research to provide new insights into lens signaling processes and fill a major gap in our understanding of both normal and abnormal eye development.

Little is known about the evolutionary origin of the neural crest, a population of migratory embryonic cells with an amazing variety of derivatives in vertebrate embryos. Here, Dr. Jeffery proposes to continue a research program that identified potential homologues of neural crest cells in the ascidian Ecteinascidia turbinata, a tunicate chordate representing a group of marine animals that may be the sister group of vertebrates. He previously obtained evidence for the existence of neural crest-like cells (NCLC) in this ascidian species based on following cell migration from the dorsal embryonic midline, expression of neural crest markers, association with structures resembling placodes, which collaborate with neural crest cells to form many vertebrate features, and differentiation into body pigment cells, one of the prime vertebrate neural crest derivatives. These studies were facilitated by the usually large size of Ecteinascidia embryos and larvae, which permitted a merger of molecular with classical approaches for identifying candidates for neural crest-like cells (NCLC). Here he proposes to continue these studies in Ecteinascidia and in the model ascidian Ciona intestinalis, in which he has also recently discovered NCLC. The overall objective is to evaluate the hypothesis that ascidian NCLC and vertebrate neural crest cells had a common ancestry by exploring their embryonic origin(s), the expression patterns of key vertebrate neural crest regulatory genes, their migration pathways, and their developmental fates. In addition, he will explore the diversity of NCLC in ascidians by comparing these features in Ecteinascidia and Ciona, related species with highly divergent morphologies and life histories. This investigation will continue to combine classical embryological approaches, such as cell lineage, cell migration, and developmental fate analysis by vital dye marking, with molecular approaches, including gene expression analysis. In Ciona the objectives will be facilitated by the existence of the genome project and cDNA databases as well as the opportunity of transgenesis. These studies will provide insights into the evolutionary origin and history of neural crest cells, furnish novel information on the fates and diversity of ascidian NCLC, identify the similarities and differences between ascidian NCLC and vertebrate neural crest cells, and help to understand the evolution of vertebrates from an invertebrate chordate ancestor.
A broad impact of this research is the continued integration of research and higher education, which is accomplished by involving undergraduate students in original research, by training pre-doctoral and post-doctoral investigators, and by our outreach to high school science programs. All of these activities include different genders and underrepresented minorities. The laboratory will continue to be a useful and highly appreciated local resource for teaching and illustrating evolutionary biology to high school students, and will also continue to serve as a resource for their short supervised science projects. The evolution of the neural crest is an excellent example of a morphological change that is experimentally tractable, has occurred within our own phylum, and is subject to interpretation as a novelty through conventional evolutionary theory. In addition to broad impacts directly related to the laboratory research, he will continue to participate in outreach activities related to primary and secondary school education and education of the lay public, including radio and TV broadcasts that explain the scientific evidence supporting evolution and highlight the seminal work of scientists working in this field, and to serve as advisor in the planning and writing of articles on evolution for the general public (Natural History Magazine) and for gifted children (Muse Magazine).

Project Summary

Intellectual Merit:

The evolutionary and developmental mechanisms responsible for the loss of vision in cave animals are not understood. This proposal continues Dr. Jeffery's research on eye degeneration in the teleost Astyanax mexicanus, which has an eyed surface-dwelling form (surface fish, SF) and many eyeless cave-dwelling forms (cavefish, CF). CF form eye primordia but they arrest in growth and degenerate due to lens apoptosis. The results of his previous grant showed that: (1) Hedgehog (Hh) signaling is enhanced along the CF anterior midline and controls eye degeneration by inducing lens apoptosis, (2) the molecular chaperone Hsp90< is specifically activated in the CF lens and required for lens apoptosis, (3) Hh expression along the embryonic midline is gradually restricted to developing taste buds, and (4) expression of genes positively regulated by Hh signals (e. g. pax2.1a in the optic vesicles and nkx2.1a/b in the neural plate) are expanded in CF. Constructive traits that may compensate for vision, including increases in taste buds, olfactory nerve tracts and bulbs, and the ventral telencephalon and hypothalamus, have also evolved in CF. This continuation proposal explores the hypothesis that eyes have degenerated due to enhanced Hh midline signaling though pleiotrophic positive and negative effects on downstream target genes resulting in developmental tradeoffs between constructive and regressive traits. The specific objectives are: (1) to determine whether Hh signaling controls lens apoptosis by affecting an antagonism between the Dlx5 (and/or Pitx3) and Pax6 transcription factors and/or by regulating Hsp90< expression and coordinately increasing olfactory development, (2 and 3) to determine whether enhanced Hh signaling is responsible for (2) the increased number of taste buds in CF and (3) for enlargement of the ventral telencephalon and hypothalamus in CF, and (4) to determine by genetic analysis whether gustatory, olfactory, and hypothalamic enhancement are coupled to eye degeneration as developmental tradeoffs. Most of the methodologies and reagents to be used in the continued work, including a panel of more than 100 Astyanax cDNAs, have been acquired in the previous grants, and Dr. Jeffery has preliminary results showing that his hypothesis is plausible and ready for more intensive investigation. The results of this study are expected to provide new insights into the mechanisms responsible for CF eye degeneration.

Broader Impacts:

Studies of cave animals advance discovery and understanding because they provide unique perspectives to research and education in evolutionary developmental biology. Two hypotheses, neutral mutation and natural selection, have been advanced to explain regressive changes such as eye loss in cave animals, but until recently there was little or no experimental support for either of them. These experiments on Astyanax CF are the first to suggest that eye loss is an indirect effect of natural selection on adaptive traits via the positive and negative effects of pleiotropic Hh signals. Pleiotropic effects may have general implications in the evolution of development. In addition to the Hh pathway, other critical developmental signaling pathways have negative and positive regulated targets and the potential to generate evolutionary changes by developmental tradeoffs. The generation of evolutionary changes via pleiotropy is largely underappreciated as a mechanism for directing microevolutionary changes in development, and these studies are designed to shed more light on this process. Another broad impact of this research is the continued integration of research and education, which is accomplished by involving undergraduate students in original research, by training pre-doctoral and post-doctoral investigators, and by the laboratory's outreach to high school science programs. All of these activities include different genders and underrepresented minorities. The cavefish laboratory and colony will continue to be a useful and highly appreciated local resource for teaching and understanding evolutionary biology to high school students, and will also continue to serve as a resource for their short supervised science projects. The degeneration of the cavefish eye is an excellent example of a morphological change that is experimentally tractable, has occurred very recently, and is subject to interpretation through evolutionary theory. Finally, the cavefish colony has an impact beyond this laboratory, serving to support other researchers and springboard the generation of similar colonies internationally.

A SGER award is made to the University of Maryland to conduct an urgent biodiversity survey of two cave systems in the El Abra Formation in Mexico. Due to direct mining and collateral impact by sinking water tables, these caves are predicted to be destroyed or uninhabitable for cave fauna in the near future. This study will document the endangered cave invertebrate fauna and preserve and disseminate specimens for biological research. The survey will cover aquatic and terrestrial cave habitats in pristine and impacted areas of both caves.

From a scientific perspective, caves offer a fertile study ground for a diversity of fields, including phylogenetics, taxonomy, biogeography, behavioral ecology, and developmental biology. Combined with the advances in molecular and genomics techniques, they are emerging as a promising model system for understanding the tempo and mode of evolution, and the convergence of structural, functional and behavioral changes across diverse taxonomic groups. Additionally, caves provide a multitude of ecological niches, whose relative stability and antiquity enable relict faunas to persist. Their presence often point to larger hydrological and geological events, and thus cave fauna can usefully inform disciplines other than biology.

DESCRIPTION (provided by applicant): Many vertebrates have robust capacities for tissue regeneration as embryos and juveniles but this power fades during development and aging. The mechanisms of age-related decline in regenerative capacity are unknown and an area in which new approaches and model systems are necessary. This proposal develops the tunicate Ciona intestinalis as a chordate model for studying the effects of aging on tissue regeneration. Although not previously used as an aging model, Ciona has many favorable attributes for this purpose: it has powerful capacities for regeneration, which decline during aging, a sequenced genome, a collection of cDNA and ESTs covering 80% of the total genes, stable transgenic lines with tagged gene expression in specific tissues, and a short life history in which age is directly related to size. As a member of a chordate invertebrate group that is inferred to be the sister taxon of vertebrates, information obtained about the relationship between aging and regeneration in tunicates may be relevant to humans. The five specific aims of this proposal are designed to obtain a molecular and cellular understanding of defective oral siphon regeneration during aging. We will focus on the sensory pigment organs of the oral siphon, possible photoreceptors that regenerate with high fidelity in young and middle age animals, but show specific defects in old animals. The first three aims will focus on the cellular events involved in producing age-related defects in pigment organ regeneration. The first aim will investigate the effects of aging on maintenance of the stem/progenitor cell niche for pigment organ precursors and precursor cell migration to the wound site during regeneration. The second aim will focus on age-related changes in cell death and proliferation in the siphon wound site, the stem/precursor niche, and the reforming pigment organs during regeneration. The third aim explores the role of siphon nerves and the CNS in regenerative aging using ablation techniques and a transgenic line with GFP staining throughout the nervous system. The fourth and fifth aims investigate the molecular mechanisms responsible for age-related defects in pigment organ regeneration. The fourth aim will identify and determine the expression patterns of genes involved in regeneration, including components of the Notch signaling system, which preliminary studies have implicated in the regenerative process. The fifth aim will use RNAi gene silencing to establish how functional changes in regeneration genes are involved in the cellular aging processes revealed in the first three aims. This research is expected to fill a major gap in understanding how regenerative capacity declines with aging in a model chordate representing the closest living relative of vertebrates. PUBLIC HEALTH RELEVANCE: The ability to replace injured tissues fades with aging in most vertebrates, including humans. This study will develop the tunicate Ciona intestinalis, a vertebrate relative with powerful regeneration abilities that also recede with age, as a model to study the relationship between aging and tissue regeneration.

? DESCRIPTION (provided by applicant): The sclera is the fibrous elastic coat surrounding the eyeball that provides the structural support and flexibility critical for focusing an image on the retina. Despite its importance in optic development and disease, the molecular mechanisms of sclera development and degeneration are poorly understood. This proposal will fill this gap by capitalizing on novel opportunities for studying sclera development and degeneration in the teleost Astyanax mexicanus, a single species consisting of eyed surface (surface fish) and blind cave (cavefish) forms. In this species, eye tissue manipulation methods, genomic tools, and - most importantly - the existence of a powerful genetic approach, are available. Eye primordia are initially formed in cavefish but subsequently arrest and degenerate. The first cavefish tissue to degenerate is the lens, which subsequently affects development of other optic tissues including the sclera. Transplantation of a surface fish embryonic lens into a cavefish optic cup restores the normal sclera phenotype, showing that the lens functions in sclera development. Other eye tissues, namely the retinal pigment epithelium (RPE) and cranial neural crest (CNC), may also control aspects of sclera development. The overall goal of this project is to determine the molecular mechanisms of sclera development and degeneration. The first specific aim is to investigate the roles of the lens, RPE, and CNC as organizers of sclera development. This aim will use a combination of physical and genetic manipulations to reveal the contributions of these eye tissues in organizing the developing sclera. The second specific aim will identify the genes expressed in the lens, RPE, CNC, and/or the sclera itself that mediate normal and abnormal sclera development. These genes will be filtered for specifically and reduced to a manageable number by mapping to a set of specific genomic regions (QTL) responsible for the abnormal sclera phenotype. The third specific aim will determine the function of the sclera related genes by manipulating their expression in surface fish and cavefish and determining the effects on sclera development. The fourth specific aim will identify the mutations in genes responsible for sclera defects. This study is designed to reveal the mechanisms, genes, and mutations responsible for visual decay through effects on development of the sclera.