Sally A. Moody - US grants

The primary goal of this research is to determine whether the pattern of peripheral innervation in the vertebrate embryo is regulated by intrinsic factors related to lineage. Specifically, the experiments are designed to test the hypothesis that clonal and compartmental lineage relationships may be involved in guiding the first developing sensory and motor axons (pioneer axons) to their targets. Peripheral tissue and the central nervous system of the frog are parcellated into compartments, each one of which is derived from a specific ancestral cell group in the 512-cell stage blastula. The proposed research will investigate whether pioneer axons grow along specific pathways to specific targets by recognizing nonneuronal cells belonging to the same compartment and/or clone. Lineage analysis will be performed by marking clones in the frog blastula. Individual blastomeres will be intracellularly injected with horseradish peroxidase. This marker does not spread to adjacent cells, is inherited by all progeny of the injected cell and is not toxic to normal development. Members of the marked clone will be identified by histochemical procedures that render the reaction product visual at both the light and electron microscopic levels. At the ultrastructural level, I will determine whether pioneer axons use clonally related peripheral cells as substrates during pathway formation, and whether pioneer axons preferentially synapse on clonally related targets cells. In addition, peripheral compartments will be ablated, or transplanted to ectopic locations, in order to determine whether pionner axons will grow ectopically to associate with compartmentally related tissues. Finally, the sensory pioneer neurons will be ablated to determine whether their presence is required for the observed confinement of motor pioneer axons to compartmentally related tissues in normal embryos. This research will enhance our understanding of peripheral axonal guidance, since the clone marking technique to be used will allow a lineage analysis of neuronal connectivity for the first time in a vertebrate embryo.

DESCRIPTION (provided by applicant): There is great excitement in the field of stem cell biology because it may be possible to produce specific kinds of neural cells from ES cells, adult brain stem cells or even bone marrow stem cells. There is a considerable base of knowledge regarding the normal in vivo processes of neural fate induction, stabilization and specification. This important information needs to be expanded in molecular detail so that it can be applied to the goal of obtaining large numbers of stem/progenitor cells that will express only a neural fate when transplanted into damaged or congenitally deficit tissue. The proposed experiments will study the function of several transcription factors that are expressed early in the neural plate and which expand the neural ectoderm when over-expressed. Because these genes are downstream of neural inductive signaling and upstream of neural differentiation genes, we call them neural fate-stabilizing genes. We posit it is important to understand: (1) how neural fate-stabilizing genes function during the earliest steps of neural specification; (2) how these genes interact with each other to expand the embryonic neural stem cell population called the neural plate, and 3) how these genes direct embryonic neural stem cells down the desired differentiation pathways. Once the precise functions of these genes are understood in the embryo, we may be able to regulate their expression to expand neural stem cells, define various stages of neural stem cell specification and put this information to clinical use.

DESCRIPTION (Verbatim from applicant's abstract): The acquisition of differentiated characteristics by individual cells is a critical problem of development. In many animals the program that transforms the fertilized egg into an organized multi-cellular animal involves several processes, including unequal distribution or expression of cytoplasmic factors from the oocytes, lineage-specified mechanisms, complex cascades of cellular interactions and region-, tissue- and cell-specific transcription of zygotic genes. Sorting out the relative importance of these steps during the development of the vertebrate retina is an important challenge because this information may provide the means for correcting congenital malformations and replacing populations lost during regenerative disease or trauma. We have focused on elucidating the mechanisms by which different subtypes of amacrine cell fates are determined. During the past application period, we demonstrated that: (a) vegetal embryonic lineages are repressed from making retina by maternal factors; (b) the D1.2.1 embryonic lineage is determined during cleavage stages to produce a specified subset of amacrine cells; (c) the descendants of one embryonic lineage are altered by genetically suppressing growth factor signaling; and (d) some embryonic lineages produce their specific retinal descendants in response to their position in a field of BMP/noggin signaling during neural induction. These findings lead us to investigate four critical issues regarding retinal fate determination. In specific aim 1, we will investigate whether the depletion of two maternal factors, which play roles in endotherm induction, will allow vegetal lineages to express a retinal fate. We will also investigate which elements of the genetic pathway upstream and downstream of the gene cerebus restore retinal fate competence to this lineage. In specific aim 2, we will test whether the temporally- and spatially-regulated expression of eye field transcription factors determine whether non-retinal blastomeres can be transformed to express a retinal fate. In specific aim 3, we will test whether the blockade of activin or BMP signaling affects specific amacrine cell lineages. In specific aim 4, we will test whether transcription factors that are expressed in the embryonic eye fields regulate the expression of specific amacrine subtypes and amacrine-based lineages. These studies will combine lineage, immunocytochemical and molecular genetic approaches to elucidate the fundamental steps in the early specification of retinal lineages. We will utilize the many experimental advantages of Xenopus and the abundant knowledge available regarding maternal factors, growth factor signaling and transcriptional factor expression to elucidate the cellular and molecular mechanisms by which different amacrine cell populations are determined.

Sally A. Moody, Ph.D.
Proposal# IOS-0817902
"Molecular specification of the pre-placodal ectoderm."

The "ectoderm" of the vertebrate embryo is initially patterned into four domains: epidermis (skin), neural plate (the precursor of the central nervous system), neural crest (a precursor of the peripheral nervous system) and pre-placodal ectoderm (the precursor of several sensory organs in the head). In particular, the pre-placodal ectoderm gives rise to the olfactory and auditory systems, the lens of the eye, numerous sensory neurons in the head and the lateral line organ in fish and amphibians. Although the developmental mechanisms that regulate the formation of the neural plate, neural crest and epidermis have been studied extensively, very little is known about the development of the pre-placodal ectoderm. This proposal will elucidate the molecular interactions that: 1) induce the formation of the pre-placodal ectoderm; and 2) establish and maintain the boundaries between the four ectodermal domains as the head develops. Although these early steps of placode development have been overlooked in mutant mouse studies, perhaps because they occur very early when rodent embryos are especially fragile, they are particularly amenable to study in Xenopus frogs because of easy access to the early developmental stages and wealth of available molecular and embryological tools. The proposed studies will provide important new knowledge about the genes and tissue interactions that establish this important ectodermal domain, and is expected to elucidate the genetic and evolutionary changes that have occurred in these structures across vertebrates. In terms of Broader Impacts, because the Xenopus system allows one to visualize and manipulate gene expression without extensive technical expertise, the experimental approaches can be taught to high school, undergraduate and graduate students and thereby provide an outstanding learning experience for students interested in exploring a career in science.

DESCRIPTION (provided by applicant): The cranial sensory placodes are important embryonic precursors that give rise to critical structures in the vertebrate head, including olfactory epithelium, lens, acoustic and vestibular organs and the cranial sensory ganglia. In addition, migrating cranial neural crest require an interaction with placodes in order to form craniofacial cartilages properly. Despite the fact that the cranial placodes have been histologically recognized for over a century and the vital contributions to cranial sensory organs have been recognized for nearly as long, very little is known about the molecular mechanisms that specify the cranial sensory progenitor cells (SPCs) or lead to individual placode identity and differentiation. One goal of this proposal is to identify the gene regulatory network that regulates the specification of multipotent placode-derived SPCs to understand the genetic hierarchy underlying cranial sensory organ formation. Damage to placode-derived structures or congenital defects of the placodes or their common precursor tissue can have a devastating effect on an individual, profoundly impairing the sense of smell, sight, hearing, balance, taste and somatothesis of the face. Recent studies have connected mutations in genes that play a central role in placode development to several craniofacial disorders. A second goal of this proposal is to identify new candidate genes that underlie craniofacial birth defects that involve placode-derived sensory structures. To accomplish these goals, we shall focus our studies on the regulation of the transcription factor Six1 for two reasons. First, mutations of Six1 result in the branchio-otic syndrome 3 (BOS3, OMIM 608389), which is characterized by craniofacial defects and hearing loss. Second, Six1 knock-out in mouse and Six1 knock-down in Xenopus and chick result in severe defects in several placode-derived structures. To accomplish our two main goals, we shall determine: 1) what genes directly regulate Six1 expression using bioinformatics, in vivo and biochemical assays; 2) the epistatic relationships between Six1 and its downstream targets, relying on microarray data obtained from two different animal models; and 3) which of the genes that act down-stream of Six1 are direct targets. The proposed experiments represent a close collaboration between the Moody and Streit labs; our labs have worked in parallel on similar topics in placode development using two different animal models (Xenopus and chick, respectively). We now propose to combine our expertise to create a gene regulatory network that regulates cranial sensory precursor specification and placode development. Using two different, powerful animal models will provide the strongest information for conserved gene regulation across vertebrates that will have the most relevance to human craniofacial syndromes. This research project utilizes classical embryological approaches in the hands of two experts renowned for their studies of placode development and combines them with cutting edge technological advances. This combination of experimental strengths predict that important information regarding normal development as well as discovery of novel genetic causes of craniofacial birth defects will be forth coming from this project.

Intellectual Merit: Early in development, when embryos are composed of a simple ball of cells, some cells (animal blastomeres) receive information from the fertilized egg that will later direct them to form the nervous system. Very little is known about which molecules in the fertilized egg cause these cells to be more inclined than their neighbors to form neural tissue, a process which is called neural fate bias. The goals of this research project are to: 1) identify which molecules in animal blastomeres bias their descendant cells to form neural tissue; and 2) elucidate the molecular mechanism(s) by which these molecules act to accomplish neural fate bias. The project will utilize a novel system to culture animal blastomeres and assay their ability to produce neural tissue when exposed to molecules predicted to be involved in neural fate bias. The research will also test whether blastomeres that do not normally make neural tissue can be induced to do so by the molecules responsible for neural fate bias. Additional experiments will test whether these molecules instruct cells to express a neural fate by altering how embryonic cells respond to signaling within the embryo. Together, these experiments will enable discovery of novel molecules and novel mechanisms by which embryonic cells are instructed by the fertilized egg to form the nervous system.

Broader Impacts: This project will provide opportunities for scientific education and training at several levels. The experimental approaches will be taught to high school and undergraduate students, providing an outstanding learning experience for students interested in exploring a career in science. Under-represented high school students will be recruited from the Washington, DC public "School Without Walls" high school. Graduate level students will be trained in molecular biology, embryology, genomics, and bioinformatic approaches, providing an integrative, cutting edge research experience. Information collected in this project will be presented at local, regional, national and international meetings, including meetings specific for undergraduates, e.g., "Posters on the Hill" program. This will disseminate the research findings and will train students in public communication. This project will raise the scientific literacy of the general public, because members of the research team will organize hands-on presentations at local elementary and middle schools, and make presentations at upcoming Science and Engineering Festivals (SciFests), the next to be held in Washington, DC in April 2012.

An award is made to the George Washington University to develop a device that will be able to measure the production of broad types of biomolecules in multiple, individual cells of the developing embryo. This project will enhance education by integrating biology and chemistry, and provide new investigative tools to raise creative research opportunities in basic and applied research. Development of the single-cell mass spectrometer will require regular interactions between analytical chemists, biologists, mass spectrometrists, and curators of data repositories, essentially creating an interdisciplinary environment for students and researchers to accomplish training beyond the classical curriculum in these disciplines. By demonstrating the device at the George Washington University and discussing its design, performance, and use at national conferences and publications, this work will broaden scientific literacy and inform of the availability of the device to a broader base of users. Data resulting from measurements on the production of biomolecules during embryogenesis will be disseminated in publicly accessible data repositories, providing a larger number of users with access to facilitate research and education in cell and developmental biology and neuroscience. Notably, the combination of these scientific and outreach elements, including participation of local high-school students in the project, will enhance research and education at the interface of biology, instrument development, and analytical chemistry, providing interdisciplinary solutions to current and future challenges in science and education.

Characterization of biomolecular expression in single cells of the embryo will provide new insights into basic biochemical mechanisms that orchestrate embryonic development, the complex suite of processes by which a fertilized egg gives rise to an entire, fully functioning organism such as the frog, fish, or human. Although it has been technologically feasible to measure genes and transcripts in single embryonic cells, a lack of analytical technology has so far hindered the characterization of proteins, peptides, and metabolites in single embryonic cells. This project will provide one such technological innovation, a single-cell mass spectrometer, by combining traditional tools in cell and developmental biology and neuroscience with next-generation instrumentation from bioanalytical chemistry. Specifically, optical microscopy, microinjection, microcapillary electrophoresis, and nanoelectrospray ionization will be adapted to high-resolution tandem mass spectrometry to determine the production of biomolecules, proteins to metabolites, in multiple cells of the embryo using the frog Xenopus laevis and zebrafish as models. The instrument will be developed in collaboration with leading mass spectrometric industry, biologists, and students, and will be tested by biologists and students working with these important developmental models.

Project Summary The neural crest and cranial placodes are embryonic structures that contribute to a wide variety of cell types during development, including the peripheral nervous system, pigment cells, the craniofacial skeleton, teeth and peripheral sense organs. Neural crest and cranial placodes are derived from adjacent domains within the early ectoderm and their initial patterning is influenced by coordinated signals. Neural crest cells are highly migratory, arising at the dorsal aspect of the neural tube and invading the periphery before their final differentiation. Because of their migratory properties, study of the neural crest has pioneered the understanding of fundamental properties of epithelial-mesenchymal transitions, as well as single cell and collective cell migrations. Study of the pluripotent nature of neural crest has been instrumental in defining general stem cell properties across numerous tissue populations. The cranial placodes are ectodermal thickenings in the head that undergo complex morphogenesis to form sensory structures such as the olfactory sensory epithelium, lens, lateral line system and inner ear. In addition, neuronal precursors delaminate from the cranial placodes to form most of the neurons in cranial sensory ganglia, while the adenohypophyseal placode gives rise to the anterior pituitary gland. The emergence of the neural crest and cranial placodes is thought to have been instrumental in the evolutionary ascension of the vertebrates. The use of systems biology methods to construct the gene regulatory networks that control vertebrate development has also been pioneered in the study of neural crest and cranial placodes. The goal of the Neural Crest and Cranial Placodes Gordon Research Conference (NC&CP GRC) is to bring researchers together to exchange ideas, form collaborations, and mentor young scientists studying in this area of basic and biomedical research. This conference will facilitate shared insights and fuel advances in our understanding of two developmentally, evolutionarily, and clinically important populations of cells. The ultimate goal of the conference will be to accelerate the exchange of information across different model systems, and to promote technological innovations and a genome scale understanding of the mechanisms that govern the formation, and subsequent differentiation, of the neural crest and cranial placodes.

Branchiootorenal spectrum disorders (BOS) are characterized by craniofacial defects that include malformation of branchial arches (BAs), external ear, middle ear, and inner ear; a subset of patients also has kidney defects. Two causative genes are associated with BOS diagnoses, but these genes account for fewer than half of patient cases: the SIX1 transcription factor and a co-factor protein, EYA1, which binds to SIX1 and modifies its transcriptional activity. Thus, the causative genes for over half of BOS patients are yet to be identified. We hypothesize that there are other key co-factor proteins that bind to SIX1 to regulate its activity, and that mutations in these co-factors contribute to the unknown causes of BOS. The goal of this research program is to identify, in tractable model systems, additional genes whose altered functions contribute to the craniofacial malformations of BOS so that these genes can ultimately be included in human genetic screening. Using the Drosophila interactome data for the fly homologue of Six1, we identified 11 novel putative co-factors in Xenopus and showed that most of these are expressed in the developing BAs, ear and kidney, and therefore are potentially relevant to BOS. These proteins are highly conserved in humans, and our preliminary data show that five of them (Sobp, Zmym2, Zmym4, 2G4, Mcrs1) are required for development of the embryonic precursors of the branchial arches (neural crest [NC]), middle ear (NC) and inner ear (otic placode). In Aim 1, we will use gain- and loss-of-function approaches to determine whether these candidate cofactors play a role NC formation or migration, branchial arch cartilages or inner ear gene expression and formation. In Aim 2, we will evaluate the biochemical interactions of these gene products with Six1 and whether they affect Six1 transcriptional function. In Aim 3, we will determine whether the known BOS mutations in SIX1 affect candidate co-factor binding or function, and map what regions of the protein-protein interaction domains of Six1 and of each co-factor mediate binding and transcriptional activity. Our previous work and established model systems uniquely position us to validate whether these candidates are bone fide Six1 co-factors, and elucidate how they contribute to normal and dysmorphic craniofacial development. These analyses will provide important information that cannot be obtained from the limited patient material available. They also have the future potential to explain the phenotypic variability in BOS patients and provide a rationale for including new causative genes in BOS gene panels.