Charles R. Dearolf

Identification of a Stat Gene That Functions in Drosophila Development

A Drosophila Stat gene (D-Stat) with a zygotic segmental expression pattern was identified. This protein becomes phosphorylated on Tyr-704 when coexpressed in Schneider cells with a Drosophila janus kinase (JAK), Hopscotch (HOP). The phosphorylated protein binds specifically to the consensus sequence TTCCCGGAA. Suppressor mutations of hopTum-I, a dominant hyperactive allele of hop whose phenotype is hematocyte overproduction and tumor formation, were selected. One of these mutants, statHJ, mapped to the same chromosomal region (92E) as does D-Stat, had an incompletely penetrant pair rule phenotype, and exhibited aberrant expression of the pair rule gene even skipped (eve) at the cellular blastoderm stage. Two D-STAT-binding sites were identified within the eve stripe 3 enhancer region. Mutations in either of the STAT-binding sites greatly decreased the stripe 3 expression in transgenic flies. Clearly, the JAK-STAT pathway is connected to Drosophila early development.

The Drosophila Tumorous-lethal hematopoietic oncogene is a dominant mutation in the hopscotch locus

The Drosophila Tumorous-lethal (Tum-l) mutation acts as an activated oncogene, causing hematopoietic neoplasms, overproliferation, and premature differentiation. Tum-l is a dominant mutation in the hopscotch (hop) locus, which is required for cell division and for proper embryonic segmentation. The Tum-l temperature-sensitive period for melanotic tumor formation includes most of larval and pupal development.

Viking : Identification and characterization of a second type IV collagen in drosophila

We have taken an enhancer trap approach to identify genes that are expressed in hematopoietic cells and tissues of Drosophila. We conducted a molecular analysis of two P-element insertion strains that have reporter gene expression in embryonic hemocytes, strain 197 and vikingICO. This analysis has determined that viking encodes a collagen type IV gene, alpha2(IV). The viking locus is located adjacent to the previously described DCg1, which encodes collagen alpha1(IV), and in the opposite orientation. The alpha2(IV) and alpha1(IV) collagens are structurally very similar to one another, and to vertebrate type IV collagens. In early development, viking and DCg1 are transcribed in the same tissue-specific pattern, primarily in the hemocytes and fat body cells. Our results suggest that both the alpha1 and alpha2 collagen IV chains may contribute to basement membranes in Drosophila. This work also provides the foundation for a more complete genetic dissection of collagen type IV molecules and their developmental function in Drosophila.

Hemolymph-dependent and -independent responses in Drosophila immune tissue.

Insects possess an antimicrobial defense response that is similar to the mammalian innate immune response. The innate immune system is designed to recognize conserved components of microorganisms called pathogen‐associated molecular patterns (PAMPs). How host receptors detect PAMPs and transmit the signals to mount the immune response is being elucidated. Using GFP‐Dorsal, ‐Dif, and ‐Relish reporter proteins in ex vivo assays, we demonstrate that Drosophila fat bodies, a major immune tissue, have both hemolymph‐dependent and ‐independent responses. Microbial preparations such as lipoteichoic acid (LTA) and peptidoglycan (PGN) can stimulate some responses from dissected and rinsed larval fat bodies. Therefore, at least some aspects of recognition can occur on fat body cell surfaces, bypassing the requirement of hemolymph. Our results also show that supernatants from bacterial cultures can stimulate the nuclear translocation of Dorsal in dissected fat bodies, but this stimulation is strictly hemolymph‐dependent. Various biochemical assays suggest that the factors from bacterial supernatants that stimulate the hemolymph‐dependent nuclear translocation are likely made up of proteins. We further show that Dorsal mutant larvae have much lower phenoloxidase activity, consistent with a more important role of Dorsal in innate immunity than previously shown.

The Hopscotch Jak kinase requires the Raf pathway to promote blood cell activation and differentiation in Drosophila.

Abstract. Cytokines regulate the development and differentiated functions of hematopoietic cells by activating multiple signaling pathways, including the Jak-Stat pathway, the PI3-kinase pathway, and the Ras/Raf pathway. While the Jak-Stat interaction has been extensively studied, the relationship between this pathway and other cytokine-induced signaling pathways is not fully understood. In Drosophila melanogaster, mutations that result in hyperactivity of the Jak kinase Hopscotch (Hop) cause an activation of the larval blood cell encapsulation response, including blood cell aggregation and differentiation of plasmatocytes into apparent lamellocytes. Here, we demonstrate that Hop requires the activity of the Raf pathway to promote the activation response of larval plasmatocytes, and provide evidence to suggest that the Hop and D-Raf proteins physically interact. We also show that basal level activity of the Raf pathway is required for the accumulation of circulating blood cells.

Hyperactivation of the Drosophila Hop jak kinase causes the preferential overexpression of eIF1A transcripts in larval blood cells.

Jak kinase-Stat protein pathways play a critical role in the response of blood cells to a range of cytokines and growth factors. We are using the fruit fly, Drosophila melanogaster, as a model system to elucidate additional components of Jak-Stat pathways, and to determine how abnormalities in this pathway lead to hematopoietic leukemia-like defects. To identify downstream targets, we conducted a molecular screen for genes whose transcripts are overexpressed in response to activation of the Drosophila Hop Jak kinase. We identified a Drosophila homolog of eIF1A, a eukaryotic initiation factor found in humans and other eukaryotes. D-eIF1A is highly overexpressed in the hemocytes and lymph glands of third instar larvae carrying the dominant, gain-of-function mutation hop(Tum-l). A quantitative comparison of poly(A)(+) RNA levels between D-eIF1A and other known Drosophila translation initiation factors indicates that D-eIF1A transcripts preferentially overaccumulate in response to the hyperactive Hop pathway. Our results support the model that D-eIF1A is one of the target genes through which the Drosophila Jak kinase pathway regulates hemocyte development.

A reverse genetic screen in Drosophila using a deletion-inducing mutagen

We report the use of the cross-linking drug hexamethylphosphoramide (HMPA), which introduces small deletions, as a mutagen suitable for reverse genetics in the model organism Drosophila melanogaster. A compatible mutation-detection method based on resolution of PCR fragment-length polymorphisms on standard DNA sequencers is implemented. As the spectrum of HMPA-induced mutations is similar in a variety of organisms, it should be possible to transfer this mutagenesis and detection procedure to other model systems.