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.
1. Project Outcomes A. Major Findings Addressing Intellectual Merit: The studies supported by this grant provided important new knowledge about the genes and tissue interactions that establish this important ectodermal domain, which is required for the development of important cranial sensory structures, such as the olfactory epithelium, lens and inner ear. 1) The expression of neural crest (NC) and pre-placodal ectoderm (PPE) genes in the lateral zone that borders the neural plate causes the withdrawal of epidermal gene expression. Two Six1 mutants that cause some cases of the human BO/BOR syndrome also repress these genes, showing that the mutants are gain-of-function. These experiments demonstrate that the repression/withdrawal of epidermal genes allows the neural crest and PPE to form medial to the epidermis. 2) Increased levels of neural plate border genes repress PPE genes, particularly at early neural plate stages when the boundaries between ectodermal domains are being stabilized. 3) Expression of PPE genes in the neural plate expands that domain, which in turn pushes the NC domain more laterally and reduces the epidermal domain. Thus, the transcription factors that specify each domain reciprocally repress adjacent domains. This gene interaction can explain how domains are regulated during development. 4) Six1 does not regulate the expression of later-expressed Six genes in the placodes, its regulation of Sox11 in the PPE is dose dependent; and it represses the onset of Xiro1 expression, which is required for placode neuron differentiation. Cooperativity between Six1 and Eya1 appears to maintain the PPE in a progenitor state and repress genes that are required for later neural differentiation. These data allow us to build a gene regulatory network for the initial formation and differentiation of the PPE. 5) We identified 11 new Six1 putative co-factor genes that could influence the function of Six1 in placode development. 6) In collaboration with the laboratory of Sharon Milgram (UNC Chapel Hill and then NIH) we showed that the YAP transcriptional co-factor promotes an undifferentiated, progenitor state in both the neural crest and PPE. 7) We found that a neural plate gene (FoxD5) represses neural crest and PPE genes. It regulates some neural plate and border genes by transcriptional activation and others by transcriptional repression. Some of its actions are regulated by Notch signaling. Using microarray technology, we identified a large number of downstream targets and described their developmental expression patterns. We made several mutations in the FoxD5 protein and showed that elements in the N-terminus are responsible for transcriptional activation and elements in the C-terminus are responsible for transcriptional repression. B. Major Broader Impact Activities: General Scientific Impact: The results from this research elucidate the gene regulatory network that defines placodal fate, and provide new knowledge about boundary formation that can be applied to a wide range of tissues across species. This information is expected to be applicable across vertebrates and therefore be useful for understanding genetic and evolutionary changes that have occurred in these structures. Training Experiences: Dr. Moody sponsored nine undergraduate students to work in her laboratory on aspects of this project. Dr. Moody sponsored five high school students to work in her laboratory on aspects of this project. One was African-American and received a minority support grant. Dr. Moody sponsored one Full Professor from a primarily undergraduate institution to work in her laboratory during his sabbatical leave from teaching. Drs. Moody and Neilson engaged in undergraduate teaching at GWU and other undergraduate institutions to encourage students to incorporate research into their curricula. Outreach: The Moody laboratory provided all the Xenopus embryos displayed at the Society for Developmental Biology’s booth at the 2010 SciFest (Science and Engineering Festival) held on the Washington DC Mall, and at the 2012 SciFest held in the Washington DC Convention Center. At each festival there was an estimated attendance of over 500,000 people each day (2 days in 2010, 3 days in 2012). She and several members of her lab, including two undergraduate students, manned the Society for Developmental Biology’s booth and explained embryonic development to parents and children. Drs. Moody and Neilson visited local elementary and middle schools to demonstrate living embryos with microscopes, to explain embryonic development and show how exciting a career in science can be. Members of the Moody laboratory gave tours of the laboratory to various groups of undergraduate students to explain embryonic development, developmental genetics and encourage them to participate in research. 2. Other Products: The results of the FoxD5 microarray experiments (Section 1.A.7) were deposited in GEO (Accession number GSE11143). 3. Additional Information: none
