William James McGinnis - US grants

Many Drosophila homeotic and segmentation genes contain a conserved protein-coding sequence - the homeo box. These genes function in the morphogenetic programming of early Drosophila development and genes homologous to the homeo box have been found in a wide variety of higher animals including mouse and human. A very realistic possibility is that the homeo box may enable the identification and cloning of genes controlling the patterning of early mammalian development. Dr. McGinnis is attempting to determine whether the homeo box encodes part of the morphogenetic programming function of homeotic genes. He is also studying the nature of the genetic functions that are included in the Drosophila gene family with homeo box homology. In addition he will determine what role the homeo box genes of the mouse play in development. Dr. McGinnis has made substantial contributions to the field of Developmental Biology by his discovery of the homeo box. The Program recommends support for this outstanding young investigator.

Deformed (Dfd) is a homeobox-containing homeotic selector gene that determines head-specific fates in the Drosophila embryo. In order to understand how the Dfd protein accomplishes its developmental programming role, we wish to understand how Dfd regulates specific downstream genes at specific times and in specific cells, and we also wish to understand the biochemical and biological functions of the downstream genes that are regulated in the Drosophila embryo. Since Dfd has mouse and human homologs that also putatively assign anterior-specific fates in the developing vertebrate body plan, many of the lessons we learn about Dfd-specific regulatory elements promise to increase our insight into mechanisms of vertebrate as well as fly development. We initially concentrate our efforts on the further characterization of the regulatory element of a recently identified candidate downstream gene, Distalless (Dll, also known as Brista). We plan to mutagenize the Dfd-dependent enhancer at Dll to define the size of the enhancer, and to test whether Dfd protein binding to the 3' element is required to mediate the Dfd-dependent regulatory effect. In addition, we plan to identify other regulatory factors that may be required for the Dfd-specificity of the Dll enhancer, and which may also restrict the activity of the Dll 3' enhancer to a subset of Dfd expressing cells in the maxillary segment. Finally, we plan to test whether other homeotic genes of the Antennapedia and Bithorax complexes might regulate patterns of Dll expression through the same element, and how this regulation is integrated with Dfd's regulation of the same gene. To identify and study new Dfd downstream genes, we intend to use both genetic and biochemical methods. One approach involves the identification of genes whose mutation results in phenotypic reversion of the homeotic transformations caused by ectopic expression of Dfd in the embryo. In addition, we plan to use subtractive methods to isolate genes that are expressed at higher levels in embryos in which Dfd is ectopically expressed. Finally, we have developed very stringent DNA binding assays that identify DNA fragments that bind Dfd protein very specifically, which should allow us to directly isolate some of the downstream regulatory regions targeted by Dfd.

Homeobox genes are an ancient family of developmental regulators that pattern animal embryos. We are fascinated by the genetic circuitry involving homeobox genes that assigns developmental fates to different cells in embryos, and how the redeployment of such circuits has helped to evolve morphological diversity during animal evolution. One aim is to test the idea that a function of microRNAs is to dampen the deleterious effects of sporadic transcription of developmental regulatory genes, such as those of the homeobox family. Another aim is based on our finding that genes of the core proximodistal appendage- patterning network of arthropods are expressed in the anterior neuroectoderm of Drosophila embryos, as well as in the anterior neuroectoderm of chordate embryos in an overlapping anteroposterior order. These genes encode the transcription factors Distal-less, apterous, dachshund, hemothorax, and buttonhead/Sp8. These results, in concert with existing expression data from a variety of other animals, including mammals, suggest that a pre-existing gene network for anterior head patterning, which eventually results in different regional specializations of the brain, was co-opted to pattern the proximodistal axis of bilateral appendages in animals. This model needs more experimental tests, and such tests are outlined in this proposal. These include testing for conserved cross-regulatory relationships among these genes in the anterior neuroectoderm of Drosophila embryos, and testing whether the same set of genes are expressed in the heads of animals that represent the common ancestors of present day arthropods and vertebrates. If validated, this model would represent an amazing example of how a gene network has been redeployed to innovate new morphological features in animal body plans. Another aim is to use high resolution in situ hybridization to study how certain homeobox genes are transcriptionally activated by long-range DNA enhancers at the level of individual genes in individual embryonic nuclei. RELEVANCE (See Instructions): This research will teach us more about how environmental influences alter activities of developmental control genes, which may underlie sporadic birth defects. This research will also increase our knowledge of the genetic circuitry that controls which cells in the developing head of embryos become different regions of the brain.

0120728
Bier

A major challenge following the completion of genome sequencing is to determine the expression patterns of all genes during development and in the adult. Obtaining this data is critical if we are to unravel the complex regulatory networks that regulate genome expression. Although whole genome gene expression can be analyzed by current microarray techniques, this method lacks spacial discrimination and is relatively low resolution in time and magnitude, since by its nature, it measures average gene expression levels over large heterogeneous cell populations. To understand the regulatory interrelationships between genes, many of which are regulated in highly dynamic and spatially restricted patterns, one must ultimately know how the genome is expressed on a cell-by cell basis throughout development. The most obvious way to obtain fine scale gene expression data at single cell resolution is by performing genome scale in situ hybridization experiments.

Dr. Bier, Dr. McGinnis, and Dr. Pevzner will address this problem jointly by developing a multiplex in situ hybridization method that will greatly facilitate and enable the acquisition of genome expression data at single cell resolution. In addition, this collaborative team will validate the method by applying it to two well defined hypothesis driven questions. The specific goals of this proposal are to: 1) Develop a multiplex RNA in situ hybridization labeling technique, 2) Analyze Hox gene regulatory networks repressing limb development, and 3) Identify genes mediating cross-talk between signaling pathways.

Impact Statement: Because the same genetic systems create pattern during development in diverse metazoans, fine spatial and temporal scale analysis of these regulatory relationships in Drosophila will provide an essential framework for analyzing how these core genetic pathways have served as substrates for modification by natural selection during evolution to tailor body plans to different environments and ecological niches. This knowledge is essential for resolving deep structures of metazoan phylogeny and may reveal whether multicellular metazoans co-opted a polarity generating mechanism present in facultative colonial unicellular organisms to create metameric pattern along the A/P axis. In addition, the methodologies we develop and the understanding we gain of cellular responses to developmental signals will form the basis for creating detailed mathematical models of cellular states and will be critical for evaluating how adult organisms respond

We intend to characterize a novel epidermal barrier wound response pathway in Drosophila. The . epidermis of both insects and mammals erects a largely impermeable barrier that preventsdehydration, limits injury to soft tissue, and blocks microbial invasion. A major component of this barrier in insects is the cuticular layer, and a major component in mammals is the stratum corneum of the skin. Recent findings from our lab and others indicate that a conservedgenetic pathway, mediated by Grainy head transcription factors, is used in both insects and mammals to regulate the constructionand repair of the epidermal barrier. Thus, we now have the exciting prospect of applying the strengths of Drosophila genetics and molecular biology to the study of epidermal barrier repair in animals. To identify newgenes and molecules in the barrier wound repair pathway, we will perform genetic screens to identify the signaling molecules and receptorsthat inform epidermal cells of their proximity to barrier woundsites. We will also mutagenize barrier wound responsecis-regulatory elements to determine the DNA binding sites required to activate transcription in responseto aseptic wounds,and the factors that act throughthose binding sites. We will perform assays to identify the intracellular signals and biochemical mechanisms that instruct wound pathway transcription factors to activatewound responseenhancers after aseptic injury. A better understanding of this barrier wound repair pathway should improve human health, as the proposed researchwill identify new genes and molecules that can be tested for their influence on epidermal barrier healing in humans. The importance of the human epidermal barrier is documentedby the scoresof tragic and disfiguring human genetic diseases in which the barrier is compromised, by the need for rapid repair of the barrier in the prevention of sepsis and fluid loss in wounded humans, and by the need for properly regulated barrier replacement in the prevention of scarring.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

In this proposal, the investigators propose to automate the acquisition of gene expression data from individual cell nuclei in Drosophila embryos and use this new quantitative tool to analyze the formation of a sharp straight border between two domains of cells. Their goal in these studies is to create computational tools that will permit the automated acquisition and analysis of multiple fluorescent signals from single cells in the Drosophila embryo. These advances should allow for the quantitative analysis of 10-20 gene expression patterns at single nucleus resolution using a multi-probe based approach to in situ hybridization. The proposed automated multiplex methods should contribute substantially to enabling the creation of comprehensive gene expression atlases such as the Brain Atlases created by the Allen Institute, which are important for understanding basic biological processes such as organization of tissues and organs and changes in these structures during the evolution of developmental patterns. These methods should also make it possible to determine the exact relative expression patterns of different genes expressed in similar regions, which currently is not possible. Such refined gene expression maps will therefore make it possible to reconstruct transcription profiles for multiple genes simultaneously in individual cell types within an embryo. The software and protocols will be made available to the scientific community, and this project will include a graduate student and several undergraduates. Thus, it will also have important educational benefits for society.