James Mahaffey
Bio
My research objective is to understand the genetic control of body patterning in animals. A conserved group of genes encoding homeodomain-class transcription factors (the hox genes) is responsible for establishing the anterior-posterior body pattern of most if not all animals. The encoded proteins specify regional identity by selectively activating necessary battery of “target” genes to establish segment-specific cell fates. However, since the proteins encoded by each hox gene have similar DNA recognition and binding properties, it is not clear how these proteins lead to very specific target gene activation and developmental fates. Clearly, the Hox proteins do not function alone but in concert with other factors.
Publications
- New Components of Drosophila Leg Development Identified through Genome Wide Association Studies , PLOS ONE (2013)
- The evolving role of the orphan nuclear receptor ftz-f1, a pair-rule segmentation gene , EVOLUTION & DEVELOPMENT (2013)
- Inference on treatment effects from a randomized clinical trial in the presence of premature treatment discontinuation: the SYNERGY trial , BIOSTATISTICS (2011)
- Genomic Consequences of Background Effects on scalloped Mutant Expressivity in the Wing of Drosophila melanogaster , GENETICS (2009)
- The drosophila gap gene giant has an anterior segment identity function mediated through disconnected and teashirt , GENETICS (2008)
- The appendage role of insect disco genes and possible implications on the evolution of the maggot larval form , DEVELOPMENTAL BIOLOGY (2007)
- Imaginal Discs, the Genetic and Cellular Logic of Pattern Formation. Lewis I Held, Jr. Cambridge University Press. 2005. 461 pages. ISBN 0 521 01835 8. Price £38. (paperback). (ISBN 0521 58445 0. Price £120. (hardback) published 2002) , Genetical Research (2006)
- Assisting Hox proteins in controlling body form: are there new lessons from flies (and mammals)? , CURRENT OPINION IN GENETICS & DEVELOPMENT (2005)
- An interactive network of zinc-finger proteins contributes to regionalization of the Drosophila embryo and establishes the domains of HOM-C protein function , DEVELOPMENT (2004)
- Expression of the Drosophila gene disconnected using the UAS/GAL4 system , GENESIS (2002)
Grants
Understanding the genetic control of animal body patterning has been a major goal of developmental geneticists, and his is the long-term goal of the Mahaffey lab. Drosophila has proved to be an excellent model system to study development. Genes controlling many various aspects of animal development have been identified first from studies of the fly. Now, one major goal is to assemble these genes into pathways and networks to understand how they work together to bring into being a complete organism. Animal development is an interactive process. Recent results from the Mahaffey lab have led them to a new hypothesis integrating Hox- specification segment type and the proximal/distal (P/D) genetic network of segment development. All segments of an insect have a proximal/distal axis, most notable on segments that have appendages, though the centerline of the body outward must be included, yet each segment and appendage is different along the anterior to posterior axis Appendages can be tailored for feeding, walking, mating etc, a process controlled by the Hox genes. From a developmental standpoint, there must be an integration of the Hox and P/D developmental pathways, and this is true from a molecular standpoint as well. Many of the known and proposed ?Hox cofactors? are members of the P/D network. One hypothesis is that the Hox proteins are, in fact, the real cofactors. The P/D transcription network establishes the gene expression necessary to produce a (somewhat) generic segment and appendage, but the Hox proteins modify (tweak) this expression yielding the differences observed in each segment and appendage. Since many segments and their appendages are quite similar in overall design, similar genes would be activated, but the Hox genes would modify this to generate differences. The critical point is, that targets of the Hox genes (which have been elusive) are in fact genes regulated by the P/D transcription network. There are several predictions from this hypotheis that will be addressed. Ectopic co-expression will be used to test whether different combinations of P/D and Hox factors will have different but predictable consequences on development. Clonal analysis and ectopic expression will be used to examine where the Drosophila Disconnected and Disco-related proteins fit into the P/D network. Though functionally redundant, these proteins are quite different outside of the conserved zinc finger region. Since it is unlikely that these proteins function alone, we will take advantage of their differences to identify interacting factors. Finally, to identify genes that are responding to (targets of) the P/D network, a genome-wide population-based assay will be used to identify genes that cause variation in second leg morphology. These will then be assayed to determine if their expression is modified under different Hox conditions. Hox genes control body patterning in all metazoans, and the indicating conserved mechanisms of animal pattern formation. Many of the patterning genes are also implicated in cancers and developmental defects, so understanding these mechanisms will provide new clues to human health issues. This study provides an opportunity to learn about the interactive natures of transcription factors and the evolution of gene networks. The proposed experiments provide an opportunity for students at many levels, from high school to graduate students) to participate in an interactive research program. Results obtained from this study will be disseminated through publications and presentations at local and national meetings.
Undertsanding how genes are regulated is an important topic, touching on development, stem cell biology, wound healing, and the immune and inflammatory responses. Indeed, improper gene expression can lead to serious disorders and diseases, such as cancer. Genes are regulated by a class of proteins called transcription factors, which bind to specific DNA sequences and either promote or prohibit the expression of a nearby gene. While signaling through transcription factors is a well-studied phenomenon, how cells interpret these signals to make decisions is not yet fully understood. Some studies have suggested cells exhibit memory, effectively remembering the presence of a transcription factor long after it has been removed from the system, while other studies are more consistent with the notion that cells can only make decisions based on their present state. The conflict has arisen partly because we have previously lacked the tools to precisely manipulate transcription factor activity within a living organism. To address this, we propose to use photocaged DNA decoys to alter the activity of Dorsal, a transcription factor responsible for gene regulation along the dorsal-ventral (DV) axis of the fruit fly Drosophila melanogaster. Dorsal concentrations are highly dynamic, raising the question of whether these dynamics are crucial to the gene expression decisions made by the cells. Specifically, we will test whether the early dynamics of Dorsal signaling is necessary for the final pattern of the dorsal/ventral axis.
Small Interfering Proteins (SIPs) are proteins engineered from hyperthermophilic archaea and bacteria that bind to target proteins in a sequence-specific interaction. Specific exposed residues of the template proteins are randomly mutagenized to generate a super-library of SIPs. The library can be screened for SIPs that bind with high affinity and specificity to small organic or large protein targets. Here, the targets will be two proteins from Drosophila melanogaster. In a nutshell, these SIPs act like antibodies in that they specifically bind to the targeted proteins. However, they are much smaller (<10 kDa), and have properties that make them more stable and tighter binding than antibodies. SIPs can be selected to bind to specific domains of a protein. They are very sensitive to amino acid sequence or posttranslational modifications. Therefore, they can be selected to bind to a specific member of closely related protein family, or to a specifically modified form of a protein. Further, by selectively choosing the targeted region of a protein, SIPs can block protein function completely or only with respect to one domain of a multifunctional protein. To the best of our knowledge, these and similar non-antibody proteins have not been tested for use in protein targeting in whole organisms. This proposal is in response to the recent Dear Colleague Letter of March 5th, 2010, Enabling Partnerships to Enable Science ("Tools") NSF 10-028. The Mahaffey and Rao labs will identify SIPs that bind to specific regions of two target proteins from Drosophila melanogaster (act88F and the Zinc finger region of Disconnected and Disco-related). They will construct transgenic Drosophila containing UAS-SIP expression transgenes, activate the expression of these by crossing these lines with those containing specific Gal4 drivers, and then determine if expression of the SIP causes developmental defects similar to those observed in mutants that lack functional copies of the genes encoding these target proteins. In this manner, they will determine whether or not SIPs can be used to modulate protein function in a whole organism.
In 2008, Prof. Fred Gould led an initiative at North Carolina State University to establish a critical mass of researchers working on genetic pest management (GPM). As part of this initiative, Assistant Professor Marc??A? Lorenzen was recruited to the Department of Entomology in 2009. More recently, Assoc. Prof. Max Scott and Ms. Esther Belikoff joined the genetics and entomology departments respectively. Ms Belikoff will be running the core transgenesis facility that is an integral part of the GPM program. Here, we seek funding to equip the transgenesis facility. Historically GPM has been successfully applied to eradicate or suppress populations of a limited number of pest species (Krafsur 1998; Klassen and Curtis 2005). The screwworm fly (Cochliomyia hominivorax) has been eradicated from North and Central America by releasing insects that had been sterilized by irradiation. This type of GPM is known as the sterile insect technique or SIT. SIT has also been successfully applied to control populations of a number of fruit fly species such as the Mediterranean fruit fly (Ceratitis capitata). As the population densities of fruit fly species is typically much higher than screwworm fly, genetic strains were developed that improved the efficiency of SIT. For the medfly, a so-called genetic sexing strain enables the mass separation of males and females such that only sterile male flies are released in the field (Franz 2005). However, such genetic sexing strains are difficult to make and tend to be unstable under mass rearing conditions. With the development of methods for making transgenic insects it is now possible to engineer strains that are ideal for a GPM program (Handler 2002; Robinson et al. 2004). For example, tetracycline-repressible female lethal systems have been developed using female-specific gene promoters or introns that are alternatively spliced in males and females (Heinrich and Scott 2000; Thomas et al. 2000; Fu et al. 2007). These systems were initially tested in the model genetic organism Drosophila melanogaster as it is easy to genetically manipulate. It is more difficult to test such systems in pest species because efficient transformation methods have been developed for only a few species. Nevertheless, tetracycline-repressible female lethal strains of the medfly (Fu et al. 2007) and the mosquito disease vector Aedes aegypti (Fu et al. 2010) have been made and successfully tested in the laboratory. Rather than suppress a mosquito population, transgenic technology can also be used to replace a population with a strain that can no longer transmit disease (Terenius et al. 2008). While the focus of most users of the facility is on developing strains for a GPM program, transgenic insects can also be used for gene function studies and for production of proteins of commercial interest. One of the users of the facility will use transgenic technology to determine if potential targets for novel insecticides are essential for mosquito development or fertility. Further, another user (EntoGenetics) will use the facility to make transgenic silk moth that produce spider silk. Transgenesis facility: It will be advantageous for the users to share resources as the equipment for insect transgenesis is relatively expensive. It is often necessary to optimize several parameters to be able to routinely transform the species of interest. Scott and colleagues developed a method for transformation of blowfly species (Heinrich et al. 2002). Similarly, Lorenzen and colleagues developed a method for transformation of the red flour beetle (Lorenzen et al. 2003) and the Mahaffey lab has many years experience making transgenic Drosophila. Other users of the facility have considerable experience working with Lepidopterans. Such wide-ranging expertise will be invaluable in developing methods for species that have not yet been transformed. Ms. Belikoff has considerable experience in insect transgenesis. She will run the facility and provide training to new users.
Though it has been more than twenty years since the cloning of the Drosophila Hox complexes, scientists are still uncertain how they control animal body patterning. hox genes encode sequence-specific DNA-binding proteins that specify body pattern by controlling downstream or ?target? genes. Different Hox proteins (or combinations of them) are expressed in different segments, and, therefore, each segment expresses a specific set of target genes, which leads to segment specific morphological features. However, in vitro all Hox proteins bind to similar, relatively simple DNA sequences with a core consensus of TAAT. Surrounding bases can influence binding strength, but there appears to be little specificity or, more appropriately, selectivity, in the DNA binding properties of different Hox proteins. There is evidence that this is true in vivo as well, so it is difficult to resolve how individual Hox proteins can have such specific developmental roles while having similar, rather nonspecific DNA binding properties. Previously, genetic evidence was obtained that the partially redundant C2H2 zinc finger proteins Disconnected (Disco) and Disco-related (Disco-r) are cofactors for the Hox proteins Deformed (Dfd) and Sex combs reduced (Scr) during development of the embryonic maxillary and labial gnathal segments, respectively. As specific cofactors, the encoded zinc finger proteins establish the domain in which only certain Hox proteins can function. However, these genes appear to have another role. They establish the general gnathal (or post-oral) segment type. Since there are other zinc finger encoding genes, such as teashirt (tsh) that have similar properties, this model of zinc finger/Hox partnerships appears to govern development throughout the embryo. The work described here will evaluate this model further. Experiments described will: (1) determine the biochemical properties of the zinc finger?Hox partnership. The DNA binding and protein-protein interactions will be examined and it will be determined if the zinc finger and Hox proteins have cooperative interactions during DNA binding. (2) There are indications that the N-terminal arm of the homeodomain may be important for determining which Hox protein functions with which zinc finger protein (Disco or Tsh). This will be tested by altering the amino acid sequence of this domain and determining whether or not this changes the ability to function with Disco or Tsh. This question addresses Hox functional specificity. (3) The final goal examines the control of development by specific Hox?zinc finger identities. Ectopic expression of Hox and zinc finger proteins will be used to generate embryos that develop with approximately a single segment type (maxillary vs labial; gnathal vs trunk) throughout the whole embryo. The differences in gene expression will be compared using microarray gene expression profiling. Hox genes control body patterning in all metazoans, so understanding how this is accomplished is paramount to understanding how animal body patterns have arisen and changed throughout animal evolution. Further, genes encoding zinc finger transcription factors are the most abundant class in animal genomes. They have been implicated in many developmental and disease conditions. This study provides an opportunity to learn about the interactive nature of transcription factors during gene expression. Further, this will provide an opportunity for students at many levels to experience an interactive research program that relies on many different disciplines, genetics, cell biology, biochemistry and genomics.
James W. Mahaffey will be on IPA assignment intermitantly for three months with NSF. His duties wil be as a Program Officer in the IOB devision of Biology. The worktime will be for two months.