James W. Mahaffey

Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects

Gene product distribution is often used to infer developmental similarities and differences in animals with evolutionarily diverse body plans. However, to address commonalties of developmental mechanisms, what is really needed is a method to assess and compare gene function in divergent organisms. This requires mutations eliminating gene function. Such mutations are often difficult to obtain, even in organisms amenable to genetic analysis. To address this issue we have investigated the use of double‐stranded RNA interference to phenocopy null mutations. We show that RNA interference can be used to phenocopy mutations of the Deformed orthologues in Drosophila and Tribolium. We discuss the possible use of this technique for comparisons of developmental mechanisms in organisms with differing ontogenies.

Genomic Consequences of Background Effects on scalloped Mutant Expressivity in the Wing of Drosophila melanogaster

Genetic background effects contribute to the phenotypic consequences of mutations and are pervasive across all domains of life that have been examined, yet little is known about how they modify genetic systems. In part this is due to the lack of tractable model systems that have been explicitly developed to study the genetic and evolutionary consequences of background effects. In this study we demonstrate that phenotypic expressivity of the scallopedE3 (sdE3) mutation of Drosophila melanogaster is background dependent and is the result of at least one major modifier segregating between two standard lab wild-type strains. We provide evidence that at least one of the modifiers is linked to the vestigial region and demonstrate that the background effects modify the spatial distribution of known sd target genes in a genotype-dependent manner. In addition, microarrays were used to examine the consequences of genetic background effects on the global transcriptome. Expression differences between wild-type strains were found to be as large as or larger than the effects of mutations with substantial phenotypic effects, and expression differences between wild type and mutant varied significantly between genetic backgrounds. Significantly, we demonstrate that the epistatic interaction between sdE3 and an optomotor blind mutation is background dependent. The results are discussed within the context of developing a complex but more realistic view of the consequences of genetic background effects with respect to mutational analysis and studies of epistasis and cryptic genetic variation segregating in natural populations.

A Drosophila muscle-specific gene related to the mouse quaking locus

We have characterized a novel muscle-specific gene of Drosophila melanogaster, defined by enhancer trap strain 24B of Brand and Perrimon (1993). We show that transcripts of the gene accumulate within presumptive mesoderm and persist within developing muscles, strongly suggesting that the encoded protein is involved in muscle cell determination and differentiation. cDNA sequences reveal that the Drosophila protein is similar to quaking (64% identity over 210 amino acids), a protein essential for mouse embryogenesis, and gld-1 (53% identity over 162 amino acids) a germ-line-specific tumor suppressing protein of the nematode, Caenorhabditis elegans. We demonstrate that the Drosophila gene resides within the 93F chromosome subdivision, and describe its physical map. Finally, we have used the gene, which we have named quaking-related 93F (qkr93F), to identify a family of closely related KH domains.

An interactive network of zinc-finger proteins contributes to regionalization of the Drosophila embryo and establishes the domains of HOM-C protein function

During animal development, the HOM-C/HOX proteins direct axial patterning by regulating region-specific expression of downstream target genes. Though much is known about these pathways, significant questions remain regarding the mechanisms of specific target gene recognition and regulation, and the role of co-factors. From our studies of the gnathal and trunk-specification proteins Disconnected (DISCO) and Teashirt (TSH), respectively, we present evidence for a network of zinc-finger transcription factors that regionalize the Drosophila embryo. Not only do these proteins establish specific regions within the embryo, but their distribution also establishes where specific HOM-C proteins can function. In this manner, these factors function in parallel to the HOM-C proteins during axial specification. We also show that in tsh mutants, disco is expressed in the trunk segments, probably explaining the partial trunk to head transformation reported in these mutants, but more importantly demonstrating interactions between members of this regionalization network. We conclude that a combination of regionalizing factors, in concert with the HOM-C proteins, promotes the specification of individual segment identity.

A family of Drosophila genes encoding quaking-related maxi-KH domains.

We recently identified a Drosophilagene, wings held out (who), that specifies a STAR(signal transduction and RNA activation) proteinexpressed within mesoderm and muscles. Genetic evidencesuggests that WHO regulates muscle development and functionin response to steroid hormone titer. who is related tothe mouse quaking gene, essential for embryogenesis andneural myelination, and gld-1, a nematode tumor suppressor gene necessary for oocytedifferentiation, both of which contain RNA binding“maxi-KH” domains presumed to link RNAmetabolism to cell signaling. To initiate a broaderstudy of Drosophila WHO related proteins we used degenerate primers encodingpeptides unique to maxi-KH domains to amplify thecorresponding genes. We recovered nine genes, allspecifying single maxi-KH domain proteins havingtripartite regions of similarity that extend over 200amino acids. One is located within the 54D chromosomesubdivision, and one within 58C, while the remainingseven are within the 58E subdivision. At least four of these STAR proteins are expressed in ageneral manner, suggesting that maxi-KH domains areemployed widely in Drosophila.

The evolving role of the orphan nuclear receptor ftz-f1, a pair-rule segmentation gene.

Segmentation is a critical developmental process that occurs by different mechanisms in diverse taxa. In insects, there are three common modes of embryogenesis—short‐, intermediate‐, and long‐germ development—which differ in the number of segments specified at the blastoderm stage. While genes involved in segmentation have been extensively studied in the long‐germ insect Drosophila melanogaster (Dm), it has been found that their expression and function in segmentation in short‐ and intermediate‐germ insects often differ. Drosophila ftz‐f1 encodes an orphan nuclear receptor that functions as a maternally expressed pair‐rule segmentation gene, responsible for the formation of alternate body segments during Drosophila embryogenesis. Here we investigated the expression and function of ftz‐f1 in the short‐germ beetle, Tribolium castaneum (Tc). We found that Tc‐ftz‐f1 is expressed in stripes in Tribolium embryos. These stripes overlap alternate Tc‐Engrailed (Tc‐En) stripes, indicative of a pair‐rule expression pattern. To test whether Tc‐ftz‐f1 has pair‐rule function, we utilized embryonic RNAi, injecting double‐stranded RNA corresponding to Tc‐ftz‐f1 coding or non‐coding regions into early Tribolium embryos. Knockdown of Tc‐ftz‐f1 produced pair‐rule segmentation defects, evidenced by loss of expression of alternate En stripes. In addition, a later role for Tc‐ftz‐f1 in cuticle formation was revealed. These results identify a new pair‐rule gene in Tribolium and suggest that its role in segmentation may be shared among holometabolous insects. Interestingly, while Tc‐ftz‐f1 is expressed in pair‐rule stripes, the gene is ubiquitously expressed in Drosophila embryos. Thus, the pair‐rule function of ftz‐f1 is conserved despite differences in expression patterns of ftz‐f1 genes in different lineages. This suggests that ftz‐f1 expression changed after the divergence of lineages leading to extant beetles and flies, likely due to differences in cis‐regulatory sequences. We propose that the dependence of Dm‐Ftz‐F1 on interaction with the homeodomain protein Ftz which is expressed in stripes in Drosophila, loosened constraints on Dm‐ftz‐f1 expression, allowing for ubiquitous expression of this pair‐rule gene in Drosophila.

New components of Drosophila leg development identified through genome wide association studies.

The adult Drosophila melanogaster body develops from imaginal discs, groups of cells set-aside during embryogenesis and expanded in number during larval stages. Specification and development of Drosophila imaginal discs have been studied for many years as models of morphogenesis. These studies are often based on mutations with large developmental effects, mutations that are often lethal in embryos when homozygous. Such forward genetic screens can be limited by factors such as early lethality and genetic redundancy. To identify additional genes and genetic pathways involved in leg imaginal disc development, we employed a Genome Wide Association Study utilizing the natural genetic variation in leg proportionality found in the Drosophila Genetic Reference Panel fly lines. In addition to identifying genes already known to be involved in leg development, we identified several genes involved in pathways that had not previously been linked with leg development. Several of the genes appear to be involved in signaling activities, while others have no known roles at this time. Many of these uncharacterized genes are conserved in mammals, so we can now begin to place these genes into developmental contexts. Interestingly, we identified five genes which, when their function is reduced by RNAi, cause an antenna-to-leg transformation. Our results demonstrate the utility of this approach, integrating the tools of quantitative and molecular genetics to study developmental processes, and provide new insights into the pathways and networks involved in Drosophila leg development.

The Drosophila gap gene giant has an anterior segment identity function mediated through disconnected and teashirt.

The C2H2 zinc-finger-containing transcription factors encoded by the disconnected (disco) and teashirt (tsh) genes contribute to the regionalization of the Drosophila embryo by establishing fields in which specific Homeotic complex (Hom-C) proteins can function. In Drosophila embryos, disco and the paralogous disco-related (disco-r) are expressed throughout most of the epidermis of the head segments, but only in small patches in the trunk segments. Conversely, tsh is expressed extensively in the trunk segments, with little or no accumulation in the head segments. Little is known about the regulation of these genes; for example, what limits their expression to these domains? Here, we report the regulatory effects of gap genes on the spatial expression of disco, disco-r, and tsh during Drosophila embryogenesis. The data shed new light on how mutations in giant (gt) affect patterning within the anterior gt domain, demonstrating homeotic function in this domain. However, the homeosis does not occur through altered expression of the Hom-C genes but through changes in the regulation of disco and tsh.

1.8 – Insect Homeotic Complex Genes and Development, Lessons from Drosophila and Beyond

Abstract The homeotic genes have enticed developmental biologists ever since their discovery during studies of Drosophila. That mutation of a single gene could have such profound effects on development, and that related Hox genes were present in all metazoans, brought hope of understanding “master regulators” of development. Yet 33 years after cloning the Drosophila Hox genes we still have much to learn. In this article and its update we present a review of Hox genes concentrating on the perspective of insect studies. We cover genetic and molecular studies and update recent results from genomic techniques. The Hox genes still present many mysteries.

Molecular Genetic Mechanisms in Oxidative Damage and Aging

A paradox of aerobic metabolism is that oxygen, the molecule upon which aerobic life depends, is toxic. Toxic oxygen compounds arise due to the production of highly reactive oxygen byproducts during aerobic metabolism (Fridovich, 1977). During normal aerobic metabolism oxygen is the final electron acceptor. Total reduction of 02 is a tetravalent process, one requiring four electrons. The majority of molecular oxygen consumed by aerobic organisms is reduced in a manner that will not produce toxic byproducts. This involves metal-containing enzymes capable of multivalent reduction of 02. However, significant amounts of 02 are reduced by adding a single electron at a time. This monovalent reduction of molecular oxygen can produce toxic products as follows: