Novel interplay between JNK and Egfr signaling in Drosophila dorsal closure.

Dorsal closure (DC) is a developmental process in which two contralateral epithelial sheets migrate to seal a large hole in the dorsal ectoderm of the Drosophila embryo. Two signaling pathways act sequentially to orchestrate this dynamic morphogenetic process. First, c-Jun N-terminal kinase (JNK) signaling activity in the dorsal-most leading edge (LE) cells of the epidermis induces expression of decapentaplegic (dpp). Second, Dpp, a secreted TGF-ß homolog, triggers cell shape changes in the adjacent, ventrally located lateral epidermis, that guide the morphogenetic movements and cell migration mandatory for DC. Here we uncover a cell non-autonomous requirement for the Epidermal growth factor receptor (Egfr) pathway in the lateral epidermis for sustained dpp expression in the LE. Specifically, we demonstrate that Egfr pathway activity in the lateral epidermis prevents expression of the gene scarface (scaf), encoding a secreted antagonist of JNK signaling. In embryos with compromised Egfr signaling, upregulated Scaf causes reduction of JNK activity in LE cells, thereby impeding completion of DC. Our results identify a new developmental role for Egfr signaling in regulating epithelial plasticity via crosstalk with the JNK pathway.

ESCRT-0 complex modulates Rbf-mutant cell survival by regulating Rhomboid endosomal trafficking and EGFR signaling.

The Rb tumor suppressor is conserved in Drosophila, and its inactivation can lead to cell proliferation or death depending on the specific cellular context. Therefore, identifying genes that affect the survival of Rb-mutant cells can potentially identify novel targets for therapeutic intervention in cancer. From a genetic screen in Drosophila, we identified synthetic lethal interactions between mutations of fly Rb (rbf) and the ESCRT-0 components stam and hrs We show that inactivation of ESCRT-0 sensitizes rbf-mutant cells to undergo apoptosis through inhibition of EGFR signaling and accumulation of Hid protein. Mutation of stam inhibits EGFR signaling upstream of secreted Spi and downstream of Rhomboid expression, and causes Rhomboid protein to accumulate in the abnormal endosomes labeled with both the early and late endosomal markers Rab5 and Rab7. These results reveal that ESCRT-0 mutants inhibit EGFR signaling by disrupting Rhomboid endosomal trafficking in the ligand-producing cells. Because ESCRT-0 also plays crucial roles in EGFR downregulation after ligand binding, this study provides new insights into how loss of ESCRT-0 function can either increase or decrease EGFR signaling.

Negative regulation of Egfr/Ras pathway by Ultrabithorax during haltere development in Drosophila.

In Drosophila, wings and halteres are the dorsal appendages of the second and third thoracic segments, respectively. In the third thoracic segment, homeotic selector gene Ultrabithorax (Ubx) suppresses wing development to mediate haltere development (E.B. Lewis, 1978. A gene complex controlling segmentation in Drosophila. Nature 276, 565-570). Halteres lack stout sensory bristles of the wing margin and veins that reticulate the wing blade. Furthermore, wing and haltere epithelia differ in the size, shape, spacing and number of cuticular hairs. The differential development of wing and haltere, thus, constitutes a good genetic system to study cell fate determination. Here, we report that down-regulation of Egfr/Ras pathway is critical for haltere fate specification: over-expression of positive components of this pathway causes significant haltere-to-wing transformations. RNA in situ, immunohistochemistry, and epistasis genetic experiments suggest that Ubx negatively regulates the expression of the ligand vein as well as the receptor Egf-r to down-regulate the signaling pathway. Electromobility shift assays further suggest that Egf-r is a potential direct target of Ubx. These results and other recent findings suggest that homeotic genes may regulate cell fate determination by directly regulating few steps at the top of the hierarchy of selected signal transduction pathways.

Receptor of activated C kinase 1 (RACK1) is necessary for the 20-hydroxyecdysone-induced expression of the transcription factor CHR3 in the spruce budworm Choristoneura fumiferana.

To initiate moulting and metamorphosis, 20-hydroxyecdysone (20E) binds to its nuclear receptors and the ligand-receptor complex then mediates changes in gene expression. Phosphorylation of the receptors is required for their function. The intracellular signal transduction pathway that is involved in receptor phosphorylation remains elusive. This study provides evidence that the receptor of activated C kinase 1 (RACK1) and protein kinase C (PKC) signal transduction cascade is involved in the 20E-induced expression of the moult-associated transcription factor CHR3. A cDNA clone encoding a receptor of activated C kinase 1 was isolated from Choristoneura fumiferana (CfRACK1). This single copy gene coded a 36 kDa protein and was expressed ubiquitously in all of the developmental stages and the tissues tested, including the midgut, epidermis, fat body, head, Malpighian tubules, ovary and testis of larvae. High levels of the transcripts were also detected in a midgut-derived CF-203 cell line. We noticed that the green fluorescence protein-fused CfRACK1 protein was distributed in the cytosol surrounding the nuclei in stably transformed cells. Interference of CfRACK1 mRNA suppressed the 20E-induced expression of the transcription factor CHR3. Dequalinium-14; 1,1'-decamethylenebis-4-aminoquinaldinium diiodide (DECA), an inhibitor of RACK1 binding to protein kinase C, blocked the 20E-induced expression of CHR3 and accumulation of the ecdysone receptor (EcR) in the nuclei. All of these data together suggest that 20E-induced expression of CHR3 may involve phosphorylation of the ecdysone receptor component through the PKC/RACK1 signal transduction cascade, which facilitates the import of the receptor into the nuclei of cells.

Morphological and molecular effects of 20-hydroxyecdysone and its agonist tebufenozide on CF-203, a midgut-derived cell line from the spruce budworm, Choristoneura fumiferana.

The morphological and molecular responses of a midgut-derived cell line of the spruce budworm, Choristoneura fumiferana, to 20-hydroxyecdysone (20E) and the nonsteroidal ecdysone agonist, tebufenozide (RH-5992), were investigated. The cells responded to these compounds by clumping, generating filamentous extensions, increased mortality and expression of the transcription factor, Choristoneura hormone receptor 3 (CHR3). This cell line can be used as a model system to study the mode of action of ecdysone and its agonists. With subsequent passaging in ecdysteroid-containing medium, the degree of clumping increased and the clumping could not be reversed by subculturing in ecdysteroid-free medium. Cell numbers of the adapted cell lines in 20E and RH-5992 containing media were not significantly decreased, compared to the control, but both cell lines accumulated less (14)C-labeled RH-5992 and lost the capability of expressing CHR3 in response to these compounds. Taken together, the cell lines appeared to develop a mechanism to adapt to the toxic effects of these compounds. Arch. Insect Biochem. Physiol. 55:68-78, 2004.

Measuring peptide secretion using the FMRFamide tagging technique.

Peptides are transmitters produced by a wide variety of neurons and neuroendocrine cells. They mediate a remarkable range of physiological processes. To better understand the roles played by peptides, a number of methods have been developed that can monitor their secretion. Although each has particular strengths, they cannot rapidly detect the secretion of chemically defined peptides. However, a recently developed approach termed "FMRFamide-tagging" may be useful in this regard. A genetically encoded electrophysiological tag is attached to the peptide prohormone of interest. The "tagged" prohormone together with an ionotropic receptor that binds the tag are expressed in the cell type under investigation. Secretion of the tag (and the co-secreted peptide of interest) are revealed by rapid inward membrane currents that are due to the activation of the tag receptor. In this manner, peptide secretion can be followed on a millisecond time scale. This protocol gives the details of the approach and its potential application to a range of cell types.

Argos and Spitz group genes function to regulate midline glial cell number in Drosophila embryos.

The midline glia of the Drosophila embryonic nerve cord undergo a reduction in cell number after facilitating commissural tract morphogenesis. The numbers of midline glia entering apoptosis at this stage can be increased by a loss or reduction of function in genes of the spitz group or Drosophila EGF receptor (DER) pathway. Argos, a secreted molecule with an atypical EGF motif, is postulated to function as a DER antagonist. In this work, we assess the role of argos in the determination of midline glia cell number. Although all midline glia express DER, argos expression is restricted to the midline glia which do not enter apoptosis. Fewer midline glia enter apoptosis in embryos lacking argos function. Ectopic expression of argos is sufficient to remove all DER-expressing midline glia from the nerve cord, even those that already express argos. DER expression is not terminated in the midline glia after spitz group signaling triggers changes in gene expression. It is therefore likely that an attenuation of DER signaling by Argos is integrated with the augmentation of DER signaling by Spitz throughout the period of reduction of midline glia number. We suggest that signaling by Spitz but not Argos is restricted to adhesive junctions. In this manner, midline glia not forming signaling junctions remain sensitive to juxtacrine Argos signaling, while an autocrine Argos signal is excluded by the adhesive junction.

Ectopic activation of torpedo/Egfr, a Drosophila receptor tyrosine kinase, dorsalizes both the eggshell and the embryo.

The Drosophila gene torpedo/Egfr (top/Egfr) encodes a homolog of the vertebrate Epidermal Growth Factor receptor. This receptor is required several times during the life cycle of the fly for the transmisson of developmental cues. During oogenesis, Top/Egfr activation is required for the establishment of the dorsal/ventral axis of the egg and the embryo. To examine how ectopic Top/Egfr activation affects cell fate determination, we constructed an activated version of the protein. Expression of this activated form (lambda top) in the follicle cells of the ovary induces dorsal cell fates in both the follicular epithelium and the embryo. Different levels of expression resulted in different dorsal follicle cell fates. These dorsal cell fates were expanded in the anterior, but not the posterior, of the egg, even in cases where all the follicle cells covering the oocyte expressed lambda top. The expression of genes known to respond to top/Egfr activation, argos (aos), kekkon1 (kek 1) and rhomboid (rho), was also expanded in the presence of the lambda top construct. When lambda top was expressed in all the follicle cells covering the oocyte, kek 1 and argos expression was induced in follicle cells all along the anterior/posterior axis of the egg chamber. In contrast, rho RNA expression was only activated in the anterior of the egg chamber. These data indicate that the response to Top/Egfr signaling is regulated by an anterior/posterior prepattern in the follicle cells. Expression of lambda top in the entire follicular epithelium resulted in an embryo dorsalized along the entire anterior/posterior axis. Expression of lambda top in anterior or posterior subpopulations of follicle cells resulted in regionally autonomous dorsalization of the embryos. This result indicates that subpopulations of follicle cells along the anterior/posterior axis can respond to Top/Egfr activation independently of one another.

Transcriptional expression of a putative tachykinin-like peptide receptor gene from stable fly.

STKR is a 4118 bp clone from a stable fly, Stomoxys calcitrans, cDNA library which encodes a protein with significant amino acid identity to tachykinin-like peptide receptors. Ribonuclease protection assays and RT-PCR were utilized to examine the transcriptional expression of STKR from various life stages of the stable fly. STKR expression was detectable in all stages, but was most abundant in isolated adult fly gut and lowest in developing embryos.