The NDNF-like factor Nord is a Hedgehog-induced extracellular BMP modulator that regulates Drosophila wing patterning and growth

Hedgehog (Hh) and bone morphogenetic proteins (BMPs) pattern the developing Drosophila wing by functioning as short- and long-range morphogens, respectively. Here, we show that a previously unknown Hh-dependent mechanism fine-tunes the activity of BMPs. Through genome-wide expression profiling of the Drosophila wing imaginal discs, we identify nord as a novel target gene of the Hh signaling pathway. Nord is related to the vertebrate Neuron Derived Neurotrophic Factor (NDNF) involved in Congenital Hypogonadotropic Hypogonadism and several types of cancer. Loss- and gain-of-function analyses implicate Nord in the regulation of wing growth and proper crossvein patterning. At the molecular level, we present biochemical evidence that Nord is a secreted BMP-binding protein and localizes to the extracellular matrix. Nord binds to Decapentaplegic (Dpp) or the heterodimer Dpp-Glass bottom boat (Gbb) to modulate their release and activity. Furthermore, we demonstrate that Nord is a dosage-depend biphasic BMP modulator, where low levels of Nord promote and high levels inhibit BMP signaling. Taken together, we propose that Hh-induced Nord expression fine tunes both the range and strength of BMP signaling in the developing Drosophila wing.

Modeling the mechanics of growing epithelia with a bilayer plate theory

Epithelia, which consists of cell sheets lying on a substrate, are prevalent structures of multi-cellular organisms. The physical basis of epithelial morphogenesis has been intensely investigated in recent years. However, as 2D mechanics focused most attention, we still lack a rigorous description of how the mechanical interactions between the cell layer and its substrate can lead to 3D distortions. This work provides a complete description of epithelial mechanics using the most straightforward model of an epithelium: a thin elastic bilayer. We first provide experimental evidence in Drosophila tissues that localized alterations of the cell-substrate (the extracellular matrix) can lead to profound 3D shape changes in epithelia. We then develop an analytical model modifying the Foppl-von Karman equation with growth for bilayers. We provide a complete description of all contributions from biophysical characteristics of epithelia. We show how any localized inhomogeneity of stiffness or thickness drastically changes the bending process when the two layers grow differently. Comparison with finite-element simulations and experiments performed on Drosophila wing imaginal discs validate this approach for thin epithelia.

Hherisomes, hedgehog specialized recycling endosomes, are required for high level hedgehog signaling and tissue growth

In metazoans, tissue growth and patterning is partly controlled by the Hedgehog (Hh) morphogen. Using immuno-electron microscopy on Drosophila wing imaginal discs, we identified a cellular structure, the Hherisomes that contain the majority of intracellular Hh. Hherisomes are recycling tubular endosomes and their formation is specifically boosted by overexpression of Hh. Expression of Rab11, a small GTPase involved in recycling endosomes, boosts the size of Hherisomes and their Hh concentration. Conversely, increased expression of the transporter Dispatched, a regulator of Hh secretion, leads to their clearance. We show that increasing Hh density in Hherisomes through Rab11 overexpression enhances both the level of Hh-signaling and disc pouch growth, whereas Dispatched overexpression decreases high level Hh-signaling and growth. We propose that upon secretion, a pool of Hh triggers low level signaling, whereas a second pool of Hh is endocytosed and recycled through Hherisomes to stimulate high level signaling and disc pouch growth. Altogether our data indicate that Hherisomes are required to sustain physiological Hh activity necessary for patterning and tissue growth in the wing disc.

The ESCRT Machinery regulates Retromer dependent Transcytosis of Septate Junction Components in Drosophila

Loss of ESCRT function in Drosophila imaginal discs is known to cause neoplastic overgrowth fuelled by mis-regulation of signalling pathways. Its impact on junctional integrity, however, remains obscure. To dissect the events leading to neoplasia, we used transmission electron microscopy (TEM) on wing imaginal discs temporally depleted of the ESCRT-III core component Shrub. We find a specific requirement for Shrub in maintaining Septate Junction (SJ) integrity by transporting the Claudin Megatrachea (Mega) to the SJ. In absence of Shrub function, Mega is lost from the SJ and becomes trapped on endosomes coated with the endosomal retrieval machinery Retromer. We show that ESCRT function is required for apical localization and mobility of Retromer positive carrier vesicles, which mediate the biosynthetic delivery of Mega to the SJ. Accordingly, loss of Retromer function impairs the anterograde transport of several SJ core components, revealing a novel physiological role for this ancient endosomal agent.

Widely Used Mutants of eiger, Encoding the Drosophila Tumor Necrosis Factor, Carry Additional Mutations in the NimrodC1 Phagocytosis Receptor

The process of apoptosis in epithelia involves activation of caspases, delamination of cells, and degradation of cellular components. Corpses and cellular debris are then rapidly cleared from the tissue by phagocytic blood cells. In studies of the Drosophila TNF, Eiger (Egr) and cell death in wing imaginal discs, the epithelial primordia of fly wings, we noticed that dying cells appeared to transiently accumulate in egr3 mutant wing discs, raising the possibility that their phagocytic engulfment by hemocytes was impaired. Further investigation revealed that lymph glands and circulating hemocytes from egr3 mutant larvae were completely devoid of NimC1 staining, a marker of phagocytic hemocytes. Genome sequencing uncovered mutations in the NimC1 coding region that are predicted to truncate the NimC1 protein before its transmembrane domain, and provide an explanation for the lack of NimC staining. The work that we report here demonstrates the presence of these NimC1 mutations in the widely used egr3 mutant, its sister allele, egr1, and its parental strain, Regg1GS9830. As the egr3 and egr1 alleles have been used in numerous studies of immunity and cell death, it may be advisable to re-evaluate their associated phenotypes.

Drosophila hedgehog can act as a morphogen in the absence of regulated Ci processing

Extracellular Hedgehog (Hh) proteins induce transcriptional changes in target cells by inhibiting the proteolytic processing of full-length Drosophila Ci or mammalian Gli proteins to nuclear transcriptional repressors and by activating the full-length Ci or Gli proteins. We used Ci variants expressed at physiological levels to investigate the contributions of these mechanisms to dose-dependent Hh signaling in Drosophila wing imaginal discs. Ci variants that cannot be processed supported a normal pattern of graded target gene activation and the development of adults with normal wing morphology, when supplemented by constitutive Ci repressor, showing that Hh can signal normally in the absence of regulated processing. The processing-resistant Ci variants were also significantly activated in the absence of Hh by elimination of Cos2, likely acting through binding the CORD domain of Ci, or PKA, revealing separate inhibitory roles of these two components in addition to their well-established roles in promoting Ci processing.

Widely used mutants of eiger, encoding the Drosophila Tumor Necrosis factor, carry additional mutations in the NimrodC1 phagocytosis receptor

The process of apoptosis in epithelia involves activation of caspases, delamination of cells, and degradation of cellular components. Corpses and cellular debris are then rapidly cleared from the tissue by phagocytic blood cells. In studies of the Drosophila TNF, Eiger (Egr) and cell death in wing imaginal discs, the epithelial primordia of fly wings, we noticed that dying cells persisted longer in egr3 mutant wing discs than in wild type discs, raising the possibility that their phagocytic engulfment by hemocytes was impaired. Further investigation revealed that lymph glands and circulating hemocytes from egr3 mutant larvae were completely devoid of NimC1 staining, a marker of phagocytic hemocytes. Genome sequencing uncovered mutations in the NimC1 coding region that are predicted to truncate the NimC1 protein before its transmembrane domain, and provide an explanation for the lack of NimC staining. The work that we report here demonstrates the presence of these NimC1 mutations in the widely used egr3 mutant, its sister allele, egr1, and its parental strain, Regg1GS9830. As the egr3 and egr1 alleles have been used in numerous studies of immunity and cell death, it may be advisable to re-evaluate their associated phenotypes.

Genotoxic analysis of nickel–iron oxide in Drosophila

It is known that nickel–iron oxide nanocomposite (NiFe2O4NP) is used in many important areas such as modern industry, biomedical applications, magnetic resonance imaging, construction of sensors, targeted drug treatment, and photoelectric devices in our life. In this study, we have carried out a genotoxic evaluation of NiFe2O4NP (30 nm) in Drosophila melanogaster by using the wing somatic mutation and recombination assay. For this purpose, third instar larvae carrying the recessive genes ( flr3) and multiple wing hairs ( mwh) in their third chromosomes were used. The larvae were fed at concentrations ranging from 25 µg/mL to 200 µg/mL. The genotoxic effects of NiFe2O4NPs were evaluated according to mutant trichomes resulting from genetic changes (mitotic recombination, deletion, point mutation, nondisjunction) on development of the wing imaginal discs. Mutant clone evaluations were performed based on small single spots, large single spots, and twin spots classifications. The results showed that significant increases were observed in the frequency of all spots, indicating that the highest concentration of nanoparticles was able to induce genotoxic activity in the wing spot assay of D. melanogaster.