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

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.

The JAK/STAT pathway and Drosophila development.

The JAK/STAT signal transduction pathway plays a critical role in mammalian cells, particularly in hematopoiesis and immune responses. Several components of this pathway have been identified and characterized in Drosophila. Mutational analyses of these components have revealed a number of interesting developmental roles, and provide a mechanism to identify other interacting molecules and pathways. Hence, the JAK/STAT pathway in Drosophila serves as an attractive model for in vivo functional analyses of JAK/STAT signaling.

Targeted recovery of mutations in Drosophila.

Reverse genetic techniques will be necessary to take full advantage of the genomic sequence data for Drosophila and other experimental organisms. To develop a method for the targeted recovery of mutations, we combined an EMS chemical mutagenesis regimen with mutation detection by denaturing high performance liquid chromatography (DHPLC). We recovered mutant strains at the high rate of approximately 4.8 mutations/kb for every 1000 mutagenized chromosomes from a screen for new mutations in the Drosophila awd gene. Furthermore, we observed that the EMS mutational spectrum in Drosophila germ cells shows a strong preference for 5'-PuG-3' sites, and for G/C within a stretch of three or more G/C base pairs. Our method should prove useful for targeted mutagenesis screens in Drosophila and other genetically tractable organisms and for more precise studies of mutagenesis and DNA repair mechanisms.

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.

JAKs and STATs in invertebrate model organisms.

Invertebrate organisms provide systems to elucidate the developmental roles of Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathways, thereby complementing research conducted with mammalian cells and animals. Components of the JAK/STAT protein pathway have been identified and characterized in the fruit fly Drosophila melanogaster and the cellular slime mold Dictyostelium discoideum. This review summarizes the molecular and genetic data obtained from these model organisms. In particular, a Drosophila JAK/STAT pathway regulates normal segmentation, cell proliferation, and differentiation, and hyperactivation of the pathway leads to tumor formation and leukemia-like defects. A Dictyostelium STAT regulates the development of stalk cells during the multicellular part of the life cycle. Future research utilizing these organisms should continue to provide insights into the roles and regulation of these proteins and their signaling pathways.

The Drosophila Jak kinase hopscotch is required for multiple developmental processes in the eye.

Jak kinases are critical signaling components in hematopoiesis. While a large number of studies have been conducted on the roles of Jak kinases in the hematopoietic cells, much less is known about the requirements for these tyrosine kinases in other tissues. We have used loss of function mutations in the Drosophila Jak kinase Hopscotch (Hop) to determine the role of Hop in eye development. We find that Hop is required for cell proliferation/survival in the eye imaginal disc, for the differentiation of photoreceptor cells, and for the establishment of the equator and of ommatidial polarity. These results indicate that hop activity is required for multiple developmental processes in the eye, both cell-autonomously and nonautonomously.

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.

Mutation in the Jak kinase JH2 domain hyperactivates Drosophila and mammalian Jak-Stat pathways.

The Jak (Janus) family of nonreceptor tyrosine kinases plays a critical role in cytokine signal transduction pathways. In Drosophila melanogaster, the dominant hop(Tum-l) mutation in the Hop Jak kinase causes leukemia-like and other developmental defects. Previous studies have suggested that the Hop(Tum-l) protein might be a hyperactive kinase. Here, we report on the new dominant mutation hop(T42), which causes abnormalities that are similar to but more extreme than those caused by hop(Tum-l). We determined that Hop(T42) contains a glutamic acid-to-lysine substitution at amino acid residue 695 (E695K). This residue occurs in the JH2 (kinase-like) domain and is conserved among all Jak family members. We determined that Hop(Tum-1) and Hop(T42) both hyperphosphorylated and hyperactivated D-Stat when overexpressed in Drosophila cells. Moreover, we found that the hop(T42) phenotype was partially rescued by a reduction of wild-type D-stat activity. Finally, generation of the corresponding E695K mutation in murine Jak2 resulted in increased autophosphorylation and increased activation of Stat5 in COS cells. These results demonstrate that the mutant Hop proteins do indeed have increased tyrosine kinase activity, that the mutations hyperactivate the Hop-D-Stat pathway, and that Drosophila is a relevant system for the functional dissection of mammalian Jak-Stat pathways. Finally, we propose a model for the role of the Hop-D-Stat pathway in Drosophila hematopoiesis.

A JAK-STAT pathway regulates wing vein formation in Drosophila.

We present evidence that the JAK-STAT signal transduction pathway regulates multiple developmental processes in Drosophila. We screened for second-site mutations that suppress the phenotype of the hyperactive hopTum-1 Jak kinase, and recovered a mutation that meiotically maps to the known chromosomal position of D-Stat, a Drosophila stat gene. This hypomorphic mutation, termed statHJ contains a nucleotide substitution in the first D-Stat intron, resulting in a reduction in the number of correctly processed transcripts. Further, the abnormally processed mRNA encodes a truncated protein that has a dominant negative effect on transcriptional activation by the wild-type cDNA in cell culture. statHJ mutants exhibit patterning defects that include the formation of ectopic wing veins, similar to those seen in mutants of the epidermal growth factor/receptor pathway. Abnormalities in embryonic and adult segmentation and in tracheal development were also observed. The hopTum-1 and statHJ mutations can partially compensate for each other genetically, and Hop overexpression can increase D-Stat transcriptional activity in vitro, indicating that the gene products interact in a common regulatory pathway.

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.

An amino acid substitution in the Drosophila hopTum-l Jak kinase causes leukemia-like hematopoietic defects.

Proteins of the Jak family of non-receptor kinases play important roles in mammalian hematopoietic signal transduction. They mediate the cellular response to a wide range of cytokines and growth factors. A dominant mutation in a Drosophila Jak kinase, hopscotchTumorous-lethal (hopTum-l), causes hematopoietic defects. Here we conduct a molecular analysis of hopTum-l. We demonstrate that the hopTum-l hematopoietic phenotype is caused by a single amino acid substitution of glycine to glutamic acid at residue 341. We generate a true revertant of the hopTum-l mutation, in which both the molecular lesion and the mutant hematopoietic phenotype revert back to wild type. We also examine the effects of the G341E substitution in transgenic flies. The results indicate that a mutant Jak kinase can cause leukemia-like abnormalities.

Drosophila awdK-pn, a homologue of the metastasis suppressor gene nm23, suppresses the Tum-1 haematopoietic oncogene.

The human nm23-H1 gene is a suppressor of solid tumour metastasis in some types of cancer. It is known that nm23 genes encode nucleoside diphosphate kinase polypeptides, but the regulatory pathways involving Nm23 are unclear. One approach to understanding nm23 function is to identify loci which interact with nm23. The Drosophila awd gene, a homologue of nm23, provides a model system for this genetic analysis. We report that the dominant awdK-pn allele suppresses haematopoietic defects associated with the Tum-l oncogene. Premature differentiation and aggregation of Tum-l blood cells is reduced by awdK-pn, resulting in an increased survival of Tum-l hemizygotes. Tum-l lethality is also suppressed by pn mutations, indicating the existence of a haematopoietic regulatory pathway involving the Tum-l, AwdK-pn and Pn proteins.

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.

Synthetic oligonucleotides recreate Drosophila fushi tarazu zebra-stripe expression.

A complex array of activator and repressor elements located within 669 bp proximal to the fushi tarazu (ftz) transcriptional start site is sufficient to generate the "zebra-stripe" expression pattern characteristic of the ftz gene. P-element-mediated transformation and ftz promoter/lacZ fusion genes were used to characterize, in detail, several of these transcriptional control elements. By reconstructing promoters with synthetic oligonucleotides containing cis-regulators of stripe expression, we show that these regulatory sites can function as independent units to direct position-specific transcription in the Drosophila embryo. In particular, we demonstrate that multiple copies of a positive regulatory site can mediate expression in both the odd- and even-numbered parasegments throughout most of the germ band and that negative regulatory sites can transform a continuous pattern of gene expression into discrete stripes. The reconstructed promoter system presented provides an effective means of studying molecular mechanisms governing spatially restricted transcription in the early embryo.

Transcriptional regulation of the Drosophila segmentation gene fushi tarazu (ftz)

ftz is one of the 'pair rule' segmentation genes of Drosophila melanogaster, and is an important component of the segmentation process in the fruit fly. We discuss the transcriptional mechanism which causes ftz to be expressed in a seven stripe pattern during embryogenesis.

The caudal gene product is a direct activator of fushi tarazu transcription during Drosophila embryogenesis.

A drosophila pair-rule segmentation gene, fushi tarazu (ftz), encodes a protein which is expressed in a characteristic seven-stripe pattern. The promoter sequences that are sufficient for generating this spatially restricted pattern of expression are located within 669 base pairs upstream of the transcription start site. Multiple transcriptional activators and repressors interact with this 'zebra-stripe' promoter unit to bring about the positional specificity of ftz transcription. Here we report that the homoeodomain-containing protein encoded by caudal (cad) is one such regulator. The cad gene product can increase the level of ftz transcription in the posterior half of the embryo by interacting with multiple copies of a TTTATG consensus sequence located in the zebra-stripe unit. This result demonstrates one pathway by which the product of a maternally expressed segmentation gene, expressed in an antero-posterior concentration gradient, can directly regulate the expression of a pair-rule gene.

Transcriptional control of Drosophila fushi tarazu zebra stripe expression.

The Drosophila segmentation gene fushi tarazu (ftz) is expressed in a characteristic pattern of seven stripes during early embryogenesis. We have used ftz-lacZ fusion genes to determine the effects of deleting relatively small segments of the ftz promoter region necessary for this expression. We find that this regulatory region contains multiple activator and repressor elements. The deletion of one particular activator element results in a preferential loss of expression in the posterior stripes, whereas the deletion of other activator elements causes a general reduction in expression throughout the germ band. The removal of repressor elements results in a loss of repression in the odd-numbered parasegments. We also find that the ftz upstream enhancer element functions primarily in epidermal cells. Our results indicate that ftz transcription is activated in each parasegment through the 'zebra stripe' promoter region and is then inhibited selectively in the odd-numbered parasegments by repressors that bind directly to elements within this promoter region.

Molecular consequences of awdb3, a cell-autonomous lethal mutation of Drosophila induced by hybrid dysgenesis.

The abnormal wing disc locus, which is at salivary gland chromosome position 100C-D of the Drosophila melanogaster genome, has been identified by a recessive lethal mutation, awdb3, induced by hybrid dysgenesis. When homozygous, this mutation causes abnormal development of the brain, the ovaries, and the larger imaginal discs as described in the preceding paper (C.R. Dearolf, E. Hersperger, and A. Shearn, 1988, Dev. Biol. 129, 159-168). The DNA corresponding to this locus was isolated from a genomic library prepared from awdb3 heterozygotes by screening with a P-element probe. The awdb3 allele resulted from the insertion of a P-element fragment into a gene that encodes an 0.8-kb poly(A)+ RNA. In mutant larvae, that 0.8-kb transcript is replaced by two chimeric transcripts that are 0.7 and 1.3 kb in length, both of which contain P-element and awd sequences. The wild-type awd+ gene transcript is most abundant during the second and third larval instars but is found at a lower level during every developmental stage as well as in continuous cell lines. Thus the awd+ gene transcript can be detected in normal larvae at a developmental stage long before defects are expressed in mutant larvae. Moreover, some tissues, for example the salivary gland of nonmutant, third-instar larvae, contain high levels of this transcript, even though these tissues appear to develop normally in mutant larvae.

Developmental consequences of awdb3, a cell-autonomous lethal mutation of Drosophila induced by hybrid dysgenesis.

In order to recover mutations affecting imaginal discs in a way which would allow the relevant genes to be readily cloned, a hybrid dysgenic screen was performed for mutations causing late larval/early pupal lethality. This paper describes that mutagenesis procedure and the phenotypes caused by the mutations that were recovered. Of 81 late larval/pupal lethal mutations that were recovered, 20 cause imaginal disc defects. These 20 mutations define 12 different genes. This paper also includes a description of the developmental defects caused by a mutation in one of those 12 genes which we have named abnormal wing discs (awd); the following paper (C. Dearolf, N. Tripoulas, J. Biggs, and A. Shearn, 1988, Dev. Biol. 129, 169-178) describes the cloning of the awd gene and an analysis of its pattern of transcription. awdb3 homozygotes develop at a normal rate until the end of the second larval instar, when their rate of development is reduced. After an extended third larval instar, they form puparia and die. Mutant wing discs have an abnormal morphology and extensive cell death. These abnormal wing discs, and also the leg and eye-antenna discs which appear to be morphologically normal, differentiate poorly or not at all when injected into metamorphosing larvae. Analysis of genetic mosaics indicates that the awdb3 mutation is expressed in a cell-autonomous manner in wing, leg, and eye-antenna discs. The larval brain and proventriculus in awdb3 homozygous third-instar larvae appear to be vacuolated due to the accumulation of lipid droplets. Mutant ovaries are unable to develop when injected into wild-type larvae, although mutant germ cells are capable of producing normal eggs.