JAK/STAT signaling promotes regional specification by negatively regulating wingless expression in Drosophila

During development, a small number of conserved signaling molecules regulate regional specification, in which uniform populations of cells acquire differences and ultimately give rise to distinct organs. In the Drosophila eye imaginal disc, Wingless (Wg) signaling defines the region that gives rise to head tissue. JAK/STAT signaling was thought to regulate growth of the eye disc but not pattern formation. However, we show that the JAK/STAT pathway plays an important role in patterning the eye disc: it promotes formation of the eye field through repression of the wg gene. Overexpression of the JAK/STAT activating ligand Unpaired in the eye leads to loss of wg expression and ectopic morphogenetic furrow initiation from the lateral margins. Conversely, tissue lacking stat92E, which cannot transduce JAK/STAT signals, is transformed from retinal tissue into head cuticle, a phenotype that is also observed with ectopic Wg signaling. Consistent with this, cells lacking stat92E exhibit ectopic wg expression. Conversely, wg is autonomously repressed in cells with hyperactivated Stat92E. Furthermore, we show that the JAK/STAT pathway regulates a small enhancer in the wg 3' cis genomic region. As this enhancer is devoid of Stat92E-binding elements, we conclude that Stat92E represses wg through another, as yet unidentified factor that is probably a direct target of Stat92E. Taken together, our study is the first to demonstrate a role for the JAK/STAT pathway in regional specification by acting antagonistically to wg.     

IL-4-dependent CD86 expression requires JAK/STAT6 activation and is negatively regulated by PKCdelta

CD86 expression is up-regulated in activated monocytes and macrophages by a mechanism that is not clearly defined. Here, we report that IL-4-dependent CD86 expression requires activation of ERK1/2 and JAK/STAT6 but is negatively regulated by PKCdelta. PMA differentiated U937 monocytic cells when stimulated with IL-4 increased CD11b and CD86 expression by 52- and 98-fold, respectively. PMA+IL-4 treatment also induced a synergistic enhancement of ERK1/2 activation when compared to the effects of PMA and IL-4 alone. Use of the mitogen or extracellular kinase (MEK) inhibitor, PD98059, completely blocked up-regulation of CD11b and CD86 demonstrating the importance of MEK-activated ERK1/2. JAK inhibition with WHI-P154-abrogated IL-4-dependent CD11b and CD86 up-regulation and inhibited STAT6 tyrosine phosphorylation. Importantly, CD11b and CD86 expression were not reliant on IL-4-dependent activation of phosphatidylinositol 3'-kinase (PI 3-kinase). Blockade of PKCdelta activation with rottlerin prevented CD11b expression but lead to a 75- and 213-fold increase in PMA and PMA+IL-4-dependent CD86 expression, respectively. As anticipated, increasing PKCdelta activity with anti-sense reduction of CD45 increased CD11b expression and reduced CD86 expression. Likewise, rottlerin prevented nuclear localization of activated PKCdelta. We conclude from these data that IL-4-dependent CD11b expression relies predominantly on enhanced activation of ERK1/2, while IL-4-dependent CD86 expression utilizes the JAK/STAT6 pathway.     

Tufted is a gain-of-function allele that promotes ectopic expression of the proneural gene amos in Drosophila

The Tufted(1) (Tft(1)) dominant mutation promotes the generation of ectopic bristles (macrochaetae) in the dorsal mesothorax of Drosophila. Here we show that Tft(1) corresponds to a gain-of-function allele of the proneural gene amos that is associated with a chromosomal aberration at 36F-37A. This causes ectopic expression of amos in large domains of the lateral-dorsal embryonic ectoderm, which results in supernumerary neurons of the PNS, and in the notum region of the third instar imaginal wing, which gives rise to the mesothoracic extra bristles. Revertants of Tft(1), which lack ectopic neurons and bristles, do not show ectopic expression of amos. One revertant is a loss-of-function allele of amos and has a recessive phenotype in the embryonic PNS. Our results suggest that both normal and ectopic Tft(1) bristles are generated following similar rules, and both are subjected to Notch-mediated lateral inhibition. The ability of Tft(1) bristles to appear close together may be due to amos having a stronger proneural capacity than that of other proneural genes like asense and scute. This ability might be related to the wild-type function of amos in promoting development of large clusters of closely spaced olfactory sensilla.     

Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe

Human urine contains a large number of proteins and peptides (the urinary proteome). Global analysis of the human urinary proteome is important for understanding urinary tract diseases. Bladder cancer is the most common urological cancer with higher incidence rates in endemic areas of Blackfoot disease (BFD) in southern Taiwan. The aim of this study was to use the proteomic approach to establish urinary protein biomarkers of bladder cancer. ADAM28, identified by proteomic approaches and confirmed by Western blotting, showed significant differences compared with normal individuals, so it may be a biomarker of bladder cancer.     

Not4 enhances JAK/STAT pathway-dependent gene expression in Drosophila and in human cells

The JAK/STAT pathway is essential for organogenesis, innate immunity, and stress responses in Drosophila melanogaster. The JAK/STAT pathway and its associated regulators have been highly conserved in evolution from flies to humans. We have used a genome-wide RNAi screen in Drosophila S2 cells to identify regulators of the JAK/STAT pathway, and here we report the characterization of Not4 as a positive regulator of the JAK/STAT pathway. Overexpression of Not4 enhanced Stat92E-mediated gene responses in vitro and in vivo in Drosophila. Specifically, Not4 increased Stat92E-mediated reporter gene activation in S2 cells; and in flies, Not4 overexpression resulted in an 8-fold increase in Turandot M (TotM) and in a 4-fold increase in Turandot A (TotA) stress gene activation when compared to wild-type flies. Drosophila Not4 is structurally related to human CNOT4, which was found to regulate interferon-γ- and interleukin-4-induced STAT-mediated gene responses in human HeLa cells. Not4 was found to coimmunoprecipitate with Stat92E but not to affect tyrosine phosphorylation of Stat92E in Drosophila cells. However, Not4 is required for binding of Stat92E to its DNA recognition sequence in the TotM gene promoter. In summary, Not4/CNOT4 is a novel positive regulator of the JAK/STAT pathway in Drosophila and in humans.     

Stem cell regulation by JAK/STAT signaling in Drosophila

Stem cells have become one of the "buzz" topics in the last decade or so. One of the best systems to study adult stem cells in vivo is in the model organism, Drosophila melanogaster. One hundred years of genetic analysis, a sequenced and highly annotated genome and genomics makes this a difficult organism to avoid. The JAK/STAT pathway has been shown to regulate stem cells during haematopoiesis and gametogenesis in Drosophila. In this review we cover the current literature and contrast each group of stem cells with respect to JAK/STAT signaling.     

Drosophila tufted is a gain-of-function allele of the proneural gene amos

Tufted is a classical Drosophila mutant characterized by a large number of ectopic mechanosensory bristles on the dorsal mesothorax. Unlike other ectopic bristle mutants, Tufted is epistatic to achaete and scute, the proneural genes that normally control the development of these sensory organs. In this report, I present genetic and molecular evidence that Tufted is a gain-of-function allele of the proneural gene amos that ectopically activates mechanosensory neurogenesis. I also systematically examine the ability of the various proneural bHLH proteins to cross-activate each other and find that their ability to do so is in general relatively limited, despite their common ability to induce the formation of mechanosensory bristles. This phenomenon seems instead to be related to their shared ability to activate Asense and Senseless.     

The egghead gene is required for compartmentalization in Drosophila optic lobe development

The correct targeting of photoreceptor neurons (R-cells) in the developing Drosophila visual system requires multiple guidance systems in the eye-brain complex as well as the precise organization of the target area. Here, we report that the egghead (egh) gene, encoding a glycosyltransferase, is required for a compartment boundary between lamina glia and lobula cortex, which is necessary for appropriate R1-R6 innervation of the lamina. In the absence of egh, R1-R6 axons form a disorganized lamina plexus and some R1-R6 axons project abnormally to the medulla instead of the lamina. Mosaic analysis demonstrates that this is not due to a loss of egh function in the eye or in the neurons and glia of the lamina. Rather, as indicated by clonal analysis and cell-specific genetic rescue experiments, egh is required in cells of the lobula complex primordium which transiently abuts the lamina and medulla in the developing larval brain. In the absence of egh, perturbation of sheath-like glial processes occurs at the boundary region delimiting lamina glia and lobula cortex, and inappropriate invasion of lobula cortex cells across this boundary region disrupts the pattern of lamina glia resulting in inappropriate R1-R6 innervation. This finding underscores the importance of the lamina/lobula compartment boundary in R1-R6 axon targeting.     

Vascular-specific expression of the bean GRP 1.8 gene is negatively regulated.

In French bean, the glycine-rich cell wall protein GRP 1.8 is specifically synthesized in the vascular tissue. To identify cis-acting sequences required for cell type-specific synthesis of GRP 1.8, expression patterns of fusion gene constructs were analyzed in transgenic tobacco. In these constructs, the uidA (beta-glucuronidase) gene was placed under control of 5' upstream deletions as well as internal deletions of the GRP 1.8 promoter. Four different cis-acting regulatory regions, SE1 and SE2 (stem elements), a negative regulatory element, and a root-specific element, were found to control the tissue-specific expression. Deletion of the negative regulatory element resulted in expression of the uidA gene in cell types other than vascular cells. The SE1 region was essential for expression in several cell types in the absence of further upstream regulatory sequences. Full-length promoters having insertions between the negative regulatory element and SE1 strongly expressed the gene in nonvascular cell types in stems and leaves. Thus, vascular-specific expression of the GRP 1.8 promoter is controlled by a complex set of positive and negative interactions between cis-acting regulatory regions. The disturbance of these interactions results in expression in additional cell types.     

Akt is negatively regulated by Hippo signaling for growth inhibition in Drosophila

Tissue growth is achieved through coordinated cellular growth, cell division and apoptosis. Hippo signaling is critical for monitoring tissue growth during animal development. Loss of Hippo signaling leads to tissue overgrowth due to continuous cell proliferation and block of apoptosis. As cells lacking Hippo signaling are similar in size compared to normal cells, cellular growth must be properly maintained in Hippo signaling-deficient cells. However, it is not clear how Hippo signaling might regulate cellular growth. Here we show that loss of Hippo signaling increased Akt (also called Protein Kinase B, PKB) expression and activity, whereas activation of Hippo signaling reduced Akt expression in developing tissues in Drosophila. While yorkie (yki) is sufficient to increase Akt expression, Akt up-regulation caused by the loss of Hippo signaling is strongly dependent on yki, indicating that Hippo signaling negatively regulates Akt expression through Yki inhibition. Consistently, genetic analysis revealed that Akt plays a critical role in facilitating growth of Hippo signaling-defective tissues. Thus, Hippo signaling not only blocks cell division and promotes apoptosis, but also regulates cellular growth by inhibiting the Akt pathway activity.     

Characterisation of Upd2, a Drosophila JAK/STAT pathway ligand

The characterisation of ligands that activate the JAK/STAT pathway has the potential to throw light onto a comparatively poorly understood aspect of this important signal transduction cascade. Here, we describe our analysis of the only invertebrate JAK/STAT pathway ligands identified to date, the Drosophila unpaired-like family. We show that upd2 is expressed in a pattern essentially identical to that of upd and demonstrate that the proteins encoded by this region activate JAK/STAT pathway signalling. Mutational analysis demonstrates a mutual semi-redundancy that can be visualised in multiple tissues known to require JAK/STAT signalling. In order to better characterise the in vivo function of these ligands, we developed a reporter based on a natural JAK/STAT pathway responsive enhancer and show that ectopic upd2 expression can effectively activate the JAK/STAT pathway. While both Upd and Upd2 are secreted JAK/STAT pathway agonists, tissue culture assays show that the signal-sequences of Upd and Upd2 confer distinct properties, with Upd associated primarily with the extracellular matrix and Upd2 secreted into the media. The differing biophysical characteristics identified for Upd-like molecules have implications for their function in vivo and adds another aspect to our understanding of cytokine signalling in Drosophila.     

Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe

During Drosophila optic lobe development, proliferation and differentiation must be tightly modulated to reach its normal size for proper functioning. The JAK/STAT pathway plays pleiotropic roles in Drosophila development and in the larval brain, has been shown to inhibit medulla neuroblast formation. In this study, we find that JAK/STAT activity is required for the maintenance and proliferation of the neuroepithelial stem cells in the optic lobe. In loss-of-function JAK/STAT mutant brains, the neuroepithelial cells lose epithelial cell characters and differentiate prematurely while ectopic activation of this pathway is sufficient to induce neuroepithelial overgrowth in the optic lobe. We further show that Notch signaling acts downstream of JAK/STAT to control the maintenance and growth of the optic lobe neuroepithelium. Thus, in addition to its role in suppression of neuroblast formation, the JAK/STAT pathway is necessary and sufficient for optic lobe neuroepithelial growth.