Unanticipated domain requirements for Drosophila Wnk kinase in vivo.

WNK (With no Lysine [K]) kinases have critical roles in the maintenance of ion homeostasis and the regulation of cell volume. Their overactivation leads to pseudohypoaldosteronism type II (Gordon syndrome) characterized by hyperkalemia and high blood pressure. More recently, WNK family members have been shown to be required for the development of the nervous system in mice, zebrafish, and flies, and the cardiovascular system of mice and fish. Furthermore, human WNK2 and Drosophila Wnk modulate canonical Wnt signaling. In addition to a well-conserved kinase domain, animal WNKs have a large, poorly conserved C-terminal domain whose function has been largely mysterious. In most but not all cases, WNKs bind and activate downstream kinases OSR1/SPAK, which in turn regulate the activity of various ion transporters and channels. Here, we show that Drosophila Wnk regulates Wnt signaling and cell size during the development of the wing in a manner dependent on Fray, the fly homolog of OSR1/SPAK. We show that the only canonical RF(X)V/I motif of Wnk, thought to be essential for WNK interactions with OSR1/SPAK, is required to interact with Fray in vitro. However, this motif is unexpectedly dispensable for Fray-dependent Wnk functions in vivo during fly development and fluid secretion in the Malpighian (renal) tubules. In contrast, a structure function analysis of Wnk revealed that the less-conserved C-terminus of Wnk, that recently has been shown to promote phase transitions in cell culture, is required for viability in vivo. Our data thus provide novel insights into unexpected in vivo roles of specific WNK domains.

Glypicans define unique roles for the Hedgehog co-receptors boi and ihog in cytoneme-mediated gradient formation.

The conserved family of Hedgehog (Hh) signaling proteins plays a key role in cell-cell communication in development, tissue repair, and cancer progression, inducing distinct concentration-dependent responses in target cells located at short and long distances. One simple mechanism for long distance dispersal of the lipid modified Hh is the direct contact between cell membranes through filopodia-like structures known as cytonemes. Here we have analyzed in Drosophila the interaction between the glypicans Dally and Dally-like protein, necessary for Hh signaling, and the adhesion molecules and Hh coreceptors Ihog and Boi. We describe that glypicans are required to maintain the levels of Ihog, but not of Boi. We also show that the overexpression of Ihog, but not of Boi, regulates cytoneme dynamics through their interaction with glypicans, the Ihog fibronectin III domains being essential for this interaction. Our data suggest that the regulation of glypicans over Hh signaling is specifically given by their interaction with Ihog in cytonemes. Contrary to previous data, we also show that there is no redundancy of Ihog and Boi functions in Hh gradient formation, being Ihog, but not of Boi, essential for the long-range gradient.

Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc.

Hedgehog (Hh) signal molecules play a fundamental role in development, adult stem cell maintenance and cancer. Hh can signal at a distance, and we have proposed that its graded distribution across Drosophila epithelia is mediated by filopodia-like structures called cytonemes. Hh reception by Patched (Ptc) happens at discrete sites along presenting and receiving cytonemes, reminiscent of synaptic processes. Here, we show that a vesicle fusion mechanism mediated by SNARE proteins is required for Ptc placement at contact sites. Transport of Ptc to these sites requires multivesicular bodies (MVBs) formation via ESCRT machinery, in a manner different to that regulating Ptc/Hh lysosomal degradation after reception. These MVBs include extracellular vesicle (EV) markers and, accordingly, Ptc is detected in the purified exosomal fraction from cultured cells. Blockage of Ptc trafficking and fusion to basolateral membranes result in low levels of Ptc presentation for reception, causing an extended and flattened Hh gradient.

From top to bottom: Cell polarity in Hedgehog and Wnt trafficking.

Spatial organization of membrane domains within cells and cells within tissues is key to the development of organisms and the maintenance of adult tissue. Cell polarization is crucial for correct cell-cell signalling, which, in turn, promotes cell differentiation and tissue patterning. However, the mechanisms linking internal cell polarity to intercellular signalling are just beginning to be unravelled. The Hedgehog (Hh) and Wnt pathways are major directors of development and their malfunction can cause severe disorders like cancer. Here we discuss parallel advances into understanding the mechanism of Hedgehog and Wnt signal dissemination and reception. We hypothesize that cell polarization of the signal-sending and signal-receiving cells is crucial for proper signal spreading and activation of the pathway and, thus, fundamental for development of multicellular organisms.

Unique and Overlapping Functions of Formins Frl and DAAM During Ommatidial Rotation and Neuronal Development in Drosophila.

The noncanonical Frizzled/planar cell polarity (PCP) pathway regulates establishment of polarity within the plane of an epithelium to generate diversity of cell fates, asymmetric, but highly aligned structures, or to orchestrate the directional migration of cells during convergent extension during vertebrate gastrulation. In Drosophila, PCP signaling is essential to orient actin wing hairs and to align ommatidia in the eye, in part by coordinating the movement of groups of photoreceptor cells during ommatidial rotation. Importantly, the coordination of PCP signaling with changes in the cytoskeleton is essential for proper epithelial polarity. Formins polymerize linear actin filaments and are key regulators of the actin cytoskeleton. Here, we show that the diaphanous-related formin, Frl, the single fly member of the FMNL (formin related in leukocytes/formin-like) formin subfamily affects ommatidial rotation in the Drosophila eye and is controlled by the Rho family GTPase Cdc42. Interestingly, we also found that frl mutants exhibit an axon growth phenotype in the mushroom body, a center for olfactory learning in the Drosophila brain, which is also affected in a subset of PCP genes. Significantly, Frl cooperates with Cdc42 and another formin, DAAM, during mushroom body formation. This study thus suggests that different formins can cooperate or act independently in distinct tissues, likely integrating various signaling inputs with the regulation of the cytoskeleton. It furthermore highlights the importance and complexity of formin-dependent cytoskeletal regulation in multiple organs and developmental contexts.

Balancing Hedgehog, a retention and release equilibrium given by Dally, Ihog, Boi and shifted/DmWif.

Hedgehog can signal both at a short and long-range, and acts as a morphogen during development in various systems. We studied the mechanisms of Hh release and spread using the Drosophila wing imaginal disc as a model system for polarized epithelium. We analyzed the cooperative role of the glypican Dally, the extracellular factor Shifted (Shf, also known as DmWif), and the Immunoglobulin-like (Ig-like) and Fibronectin III (FNNIII) domain-containing transmembrane proteins, Interference hedgehog (Ihog) and its related protein Brother of Ihog (Boi), in the stability, release and spread of Hh. We show that Dally and Boi are required to prevent apical dispersion of Hh; they also aid Hh recycling for its release along the basolateral part of the epithelium to form a long-range gradient. Shf/DmWif on the other hand facilitates Hh movement restrained by Ihog, Boi and Dally, establishing equilibrium between membrane attachment and release of Hh. Furthermore, this protein complex is part of thin filopodia-like structures or cytonemes, suggesting that the interaction between Dally, Ihog, Boi and Shf/DmWif is required for cytoneme-mediated Hh distribution during gradient formation.

The WIF domain of the human and Drosophila Wif-1 secreted factors confers specificity for Wnt or Hedgehog.

The Hedgehog (Hh) and Wnt signaling pathways are crucial for development as well as for adult stem cell maintenance in all organisms from Drosophila to humans. Aberrant activation of these pathways has been implicated in many types of human cancer. During evolution, organisms have developed numerous ways to fine-tune Wnt and Hh signaling. One way is through extracellular modulators that directly interact with Wnt or Hh, such as the Wnt inhibitory factor (Wif-1) family of secreted factors. Interestingly, Wif-1 family members have divergent functions in the Wnt and Hh pathways in different organisms. Whereas vertebrate Wif-1 blocks Wnt signaling, Drosophila Wif-1 [Shifted (Shf)] regulates only Hh distribution and spreading through the extracellular matrix. Here, we investigate which parts of the Shf and human Wif-1 (WIF1) proteins are responsible for functional divergence. We analyze the behavior of domain-swap (the Drosophila and human WIF domain and EGF repeats) chimeric constructs during wing development. We demonstrate that the WIF domain confers the specificity for Hh or Wg morphogen. The EGF repeats are important for the interaction of Wif-1 proteins with the extracellular matrix; Drosophila EGF repeats preferentially interact with the glypican Dally-like (Dlp) when the WIF domain belongs to human WIF1 and with Dally when the WIF domain comes from Shf. These results are important both from the evolutionary perspective and for understanding the mechanisms of morphogen distribution in a morphogenetic field.

Effect of pierced metallic objects on SAR distributions at 900 MHz.

A study of the interaction between mobile phone antennas and a human head in the presence of different types of metallic objects, attached and pierced to the compressed ear, is presented in this article. Computed and measured results have been performed by considering a quasi-half-wavelength dipole as the radiating source and measurements with the DASY4 dosimetric assessment system. Two different human head models have been implemented: a homogeneously shaped sphere and a three-level head model with four different kinds of tissue. Antenna input impedance, reflection coefficient, radiation patterns, SAR distribution, absorbed power, and peak SAR values have been computed and measured for diverse scenarios, electromagnetic simulators, and organs. Despite the measuring accuracy limitations of the study, both simulated and measured results suggest that special attention has to be paid to peak SAR averaged values when wearing metallic objects close to the radiation source, since some increment of peak SAR averaged values is expected.