The Extracellular Regulation of Bone Morphogenetic Proteins in Drosophila and Sawfly Wing

Evolutionarily conserved signaling pathways mediate cell-cell communications during development. While the extracellular signal is precisely regulated to achieve dynamic morphogenetic events at the species level, it must be also flexible to generate the diversified morphologies through evolution. However, little is known about the mechanisms behind the precision and flexibility. The insect wing vein pattern can provide an excellent model to address this fundamental question, since species-specific wing vein patterns have been diversified through evolution. In this thesis, I study how evolutionarily conserved bone morphogenetic protein (BMP)-type ligand specifies diversified insect wing vein patterns using Dipteran Drosophila melanogaster and Hymenopteran sawfly Athalia rosae as models. In Drosophila, BMP-type ligand Decapentaplegic (Dpp) is expressed in the longitudinal veins (LVs) to maintain LVs and induce crossveins (CVs) fates during pupal stages. However, the distribution of Dpp remained largely unknown. Using GFP-tagged Dpp, I demonstrated that Dpp is directionally transported from LVs into the posterior crossvein (PCV) region by two extracellular BMP-binding proteins, Short gastrulation (Sog) and Crossveinless (Cv). In contrast, most of Dpp did not diffuse from LVs by Type I BMP receptor and a positive feedback mechanism. Thus the active transport and retention mechanisms allow diffusible Dpp to draw the complex wing vein patterns in Drosophila. To investigate how BMP signal instructs wing vein morphogenesis that involves apposition and cell shape changes between two wing epithelial layers, I then focused on the function of RhoGAP Crossveinless-C (Cv-C) during the PCV morphogenesis. I found that cv-c mediates PCV morphogenesis downstream of BMP signal by inactivating various Rho-type small GTPases. Interestingly, I found that cv-c is also required for Dpp transport, while Sog/Cv mediated BMP signal is guided at the ectopic wing veins caused by loss of Rho-type small GTPases. These observations identified a feed-forward mechanism coupling Dpp transport and PCV morphogenesis. To address how BMP signal specifies diversified insect wing vein patterns, I then introduced sawfly as a new model. I found that dpp is ubiquitously expressed but BMP signal reflects distinct fore- and hindwing vein patterns in sawfly. To address if Dpp transport mechanism is involved in wing vein formation, Cv/Tsg was identified from sawfly. Loss of dpp or cv/tsg by RNAi affected BMP signal and all of wing venations. These observations suggest that ubiquitously expressed Dpp is redistributed to specify distinct fore- and hindwing vein patterns in sawfly. Taken together, I found that the extracellular distribution of Dpp/BMP is tightly regulated and coordinated to achieve precise patterning and morphogenesis of the insect wing veins. Furthermore, this study raises an interesting possibility that changes in the directionality of Dpp/BMP diffusion may underlie distinct insect wing vein patterns.Evoluution aikana konservoituneet signalointireitit välittävät solujen välistä viestintää kehityksen aikana. Sen lisäksi, että lajitasolla solunulkoinen signaali on tarkasti säädelty dynaamisten morfogeenisten tapahtumien saavuttamiseksi, sen on oltava myös joustava luodakseen monipuolisia morfologioita kautta evoluution. Tästä huolimatta näistä tarkkuudesta ja joustavuudesta vastaavista mekanismeista tiedetään vähän. Hyönteissiiven laskimokuviointi tarjoaa erinomaisen mallin tämän perustavanlaatuisen kysymyksen tutkimiseksi, sillä lajikohtaiset siipien laskimokuvioitukset ovat monipuolistuneet evoluution edetessä. Tässä väitöskirjatyössä tutkin kuinka evoluutiossa konservoitunut bone morphogenetic protein (BMP) -tyypin ligandi määrittää monimuotoisia hyönteissiiven laskimokuvioita käyttäen kaksisiipisiin kuuluvaa banaanikärpästä (Drosophila melanogaster) ja lehtipistiäisiin kuuluvaa rapsipistiäistä (Athalia rosae) malleina. Drosophila-mallissa BMP-tyypin ligandi Decapentaplegic (Dpp) ilmentyy pitkittäissuonissa niiden ylläpitämiseksi ja poikittaissuonten indusoimiseksi kotelovaiheiden aikana. Dpp:n jakaantuminen on kuitenkin pysynyt pitkälti tuntemattomana. Käyttäen GFP-leimattua Dpp:tä osoitin, että kaksi BMP:tä sitovaa proteiinia, Short gastrulation (Sog) ja Crossveinless (Cv), kuljettaa Dpp:n pitkittäissuonista posterioriselle poikittaissuonialueelle (PCV). Sitä vastoin suurin osa Dpp:stä ei diffundoitunut pitkittäissuonista tyypin I BMP-reseptorin ja positiivisen takaisinkytkentämekanismin välityksellä. Täten aktiivinen kuljetus ja retentiomekanismit antavat diffundoituvan Dpp:n piirtää banaanikärpäsen monimutkaiset siipilaskimokuviot. Tutkiakseni kuinka BMP-signaali ohjeistaa siipilaskimoiden morfogeneesiä, johon liittyy appositiota ja solujen muodon muutoksia siiven kahden epiteelikerroksen välissä, keskityin RhoGAP Crossveinless-C:n (Cv-C) toimintaan PCV:n morfogeneesin aikana. Löysin, että cv-c välittää PCV:n morfogeneesiä BMP-signaalista alavirtaan inaktivoimalla erinäisiä Rho-tyypin pieniä GTPaaseja. Kiinnostavana löytönä huomasin cv-c:n myös olevan välttämätön Dpp:n kuljetukselle, kun Sog/Cv-välitetty BMP-signaali kulkee ohjatusti ektooppisissa siipilaskimoissa Rho-tyypin pienten GTPaasien inaktivoitumisen takia. Näiden havaintojen avulla tunnistin Dpp-kuljetuksen ja PCV:n morfogeneesin yhteen liittävän positiivisen takaisinkytkentämekanismin. Selvittääkseni kuinka BMP-signaali määrittää monipuolisia hyönteissiiven laskimokuvioita, otin käyttöön rapsipistiäisen uutena mallina. Rapsipistiäisessä huomasin dpp:n ilmentyvän kaikkialla, mutta BMP-signaalin kuvaavan erillisiä etu- ja takasiiven laskimokuvioita. Dpp:n kuljetusmekanismin osuuden selvittämiseksi siipilaskimoiden muodostumisessa Cv/Tsg tunnistettiin rapsipistiäisessä. dpp:n tai cv/tsg:n hiljentäminen RNA-interferenssillä vaikutti BMP-signaaliin ja kaikkiin siipilaskimoihin. Nämä havainnot antavat ymmärtää, että kaikkialla ilmentyvä Dpp jakaantuu uudelleen rapsipistiäisen etu- ja takasiipien erillisten laskimokuvioiden määrittämiseksi. Kaiken kaikkiaan löysin, että solunulkoinen Dpp/BMP:n jakaantuminen on tiukasti säädelty ja koordinoitu tarkan kuvioinnin ja morfogeneesin saavuttamiseksi hyönteissiipien laskimoissa. Lisäksi tämä tutkimus tuo esiin mielenkiintoisen mahdollisuuden siitä, että vaihtelut Dpp/BMP:n diffundoitumisen suuntautumisessa voivat olla erilaisten hyönteissiiven laskimokuvioiden takana

Directional transport and active retention of Dpp/BMP create wing vein patterns in Drosophila

AbstractThe bone morphogenetic protein (BMP) family ligand decapentaplegic (Dpp) plays critical roles in wing vein development during pupal stages in Drosophila. However, how the diffusible Dpp specifies elaborate wing vein patterns remains unknown. Here, we visualized Dpp distribution in the pupal wing and found that it tightly reflects the wing vein patterns. We show that Dpp is directionally transported from the longitudinal veins (LVs) into the posterior crossvein (PCV) primordial region by the extracellular BMP-binding proteins, short gastrulation (Sog) and crossveinless (Cv). Another BMP-type ligand, glass bottom boat (Gbb), also moves into the PCV region and is required for Dpp distribution, presumably as a Dpp–Gbb heterodimer. In contrast, we found that most of the Dpp is actively retained in the LVs by the BMP type I receptor thickveins (Tkv) and a positive feedback mechanism. We provide evidence that the directionality of Dpp transport is manifested by sog transcription that prepatterns the PCV position in a Dpp signal-independent manner. Taken together, our data suggest that spatial distribution of Dpp is tightly regulated at the extracellular level by combination of long-range facilitated transport toward the PCV and short-range signaling by active retention in the LVs, thereby allowing diffusible ligands to form elaborate wing vein patterns

Rhodopsin-mediated light-off-induced protein kinase A activation in mouse rod photoreceptor cells

網膜の知られざる光応答を顕微鏡観察で発見 --光センサー細胞が暗黒に反応した--. 京都大学プレスリリース. 2020-10-14.Detect with PKAchu. 京都大学プレスリリース. 2020-10-30.Light-induced extrasynaptic dopamine release in the retina reduces adenosine 3′, 5′-cyclic monophosphate (cAMP) in rod photoreceptor cells, which is thought to mediate light-dependent desensitization. However, the fine time course of the cAMP dynamics in rods remains elusive due to technical difficulty. Here, we visualized the spatiotemporal regulation of cAMP-dependent protein kinase (PKA) in mouse rods by two-photon live imaging of retinal explants of PKAchu mice, which express a fluorescent biosensor for PKA. Unexpectedly, in addition to the light-on-induced suppression, we observed prominent light-off-induced PKA activation. This activation required photopic light intensity and was confined to the illuminated rods. The estimated maximum spectral sensitivity of 489 nm and loss of the light-off-induced PKA activation in rod-transducin-knockout retinas strongly suggest the involvement of rhodopsin. In support of this notion, rhodopsin-deficient retinal explants showed only the light-on-induced PKA suppression. Taken together, these results suggest that, upon photopic light stimulation, rhodopsin and dopamine signals are integrated to shape the light-off-induced cAMP production and following PKA activation. This may support the dark adaptation of rods

Dally is not essential for Dpp spreading or internalization but for Dpp stability by antagonizing Tkv-mediated Dpp internalization

Dpp/BMP acts as a morphogen to provide positional information in the Drosophila wing disc. Key cell-surface molecules to control Dpp morphogen gradient and signaling are heparan sulfate proteoglycans (HSPGs). In the wing discs, two HSPGs, the glypicans Division abnormally delayed (Dally) and Dally-like (Dlp) have been suggested to act redundantly to control these processes through direct interaction of their heparan sulfate (HS) chains with Dpp. Based on this assumption, a number of models on how glypicans control Dpp gradient formation and signaling have been proposed, including facilitating or hindering Dpp spreading, stabilizing Dpp on the cell surface, or recycling Dpp. However, how distinct HSPGs act remains largely unknown. Here we generate genome-engineering platforms for the two HSPGs and find that only Dally is critical for Dpp gradient formation and signaling through interaction of its core protein with Dpp. However, we find that this interaction is not sufficient and the HS chains of Dally are essential for Dpp gradient formation and signaling, but surprisingly, largely without interacting with Dpp. We provide evidence that the HS chains of Dally are not essential for spreading or recycling of Dpp but for stabilizing Dpp on the cell surface by antagonizing Dpp internalization through Tkv. These results provide new insights into how distinct HSPGs control morphogen gradient formation and signaling during development

Shaping and interpretation of Dpp morphogen gradient by endocytic trafficking

Dpp/BMP is a morphogen that controls patterning and growth in the Drosophila wing disc. Contrast with the extracellular and nuclear regulation, how Dpp morphogen gradient is shaped and interpreted by endocytic trafficking remains unclear. To address this, here we generate novel fluorescent protein tagged dpp alleles that allow to visualize both extracellular and intracellular Dpp distribution. Using these alleles, we found that, while blocking endocytosis expanded the extracellular Dpp gradient and impaired Dpp signaling, blocking early endosome expanded not only the extracellular Dpp gradient but also Dpp signaling range due to impaired downregulation of activated receptors. We show that blocking multivesicular body (MVB) formation, but not late endosome, expanded Dpp signaling and caused accumulation of the intracellular Dpp without affecting the extracellular Dpp gradient. These results indicate that, while the early endocytosis acts as a sink for Dpp and initiates Dpp signaling, termination of Dpp signaling at MVB is required for interpretation of the extracellular Dpp gradient. Taken together, our results reveal that extracellular Dpp morphogen gradient is shaped and interpreted by distinct endocytic trafficking pathways

Significance of ethnic values in the process of the youth self-definition in Northern Caucasus

В статье выявляются факторы, воздействующие на этническую социализацию в поликультурном пространстве Северного Кавказа. Рассматривается специфичность этнической идентичности юности.In this paper factors, affecting the increasing significance of ethnic socialization in policultural space of North Caucasus are considered. The specificity of the youth ethnic identity forming is shown and the necessity of other identity kinds development, allowing to develop constructive forms of intergroup reciprocity is demonstrated