BMP/Dpp signaling and epithelial morphogenesis in Drosophila development

In this thesis, I mainly investigate how BMP/Dpp signaling is involved in development of the early pupal wing of Drosophila, and the mechanisms coupling Dpp signaling with morphogenesis.Tässä työssä tutkitaan lähinnä sitä, miten BMP / Dpp-signalointi on mukana Drosophilan varhaisen pupalin siiven kehityksessä ja mekanismeja, jotka kytkeytyvät Dpp-signalointiin morfogeneesi kanssa

Scribbled Optimizes BMP Signaling through its Receptor Internalization to the Rab5 Endosome and Promote Robust Epithelial Morphogenesis

Epithelial cells are characterized by apical-basal polarity. Intrinsic factors underlying apical-basal polarity are crucial for tissue homeostasis and have often been identified to be tumor suppressors. Patterning and differentiation of epithelia are key processes of epithelial morphogenesis and are frequently regulated by highly conserved extrinsic factors. However, due to the complexity of morphogenesis, the mechanisms of precise interpretation of signal transduction as well as spatiotemporal control of extrinsic cues during dynamic morphogenesis remain poorly understood. Wing posterior crossvein (PCV) formation in Drosophila serves as a unique model to address how epithelial morphogenesis is regulated by secreted growth factors. Decapentaplegic (Dpp), a conserved bone morphogenetic protein (BMP)-type ligand, is directionally trafficked from longitudinal veins (LVs) into the PCV region for patterning and differentiation. Our data reveal that the basolateral determinant Scribbled (Scrib) is required for PCV formation through optimizing BMP signaling. Scrib regulates BMP-type I receptor Thickveins (Tkv) localization at the basolateral region of PCV cells and subsequently facilitates Tkv internalization to Rab5 endosomes, where Tkv is active. BMP signaling also up-regulates scrib transcription in the pupal wing to form a positive feedback loop. Our data reveal a unique mechanism in which intrinsic polarity genes and extrinsic cues are coupled to promote robust morphogenesis.Peer reviewe

Adaptive protein divergence of BMP ligands takes place under developmental and evolutionary constraints

The bone morphogenetic protein (BMP) signaling network, comprising evolutionary conserved BMP2/4/Decapentaplegic (Dpp) and Chordin/Short gastrulation (Sog), is widely utilized for dorsal-ventral (DV) patterning during animal development. A similar network is required for posterior crossvein (PCV) formation in the Drosophila pupal wing. Although both transcriptional and post-transcriptional regulation of co-factors in the network gives rise to tissue-specific and species-specific properties, their mechanisms are incompletely understood. In Drosophila, BMP5/6/7/8-type ligands, Screw (Scw) and Glass bottom boat (Gbb), form heterodimers with Dpp for DV patterning and PCV development, respectively. Sequence analysis indicates that the Scw ligand contains two N-glycosylation motifs: one being highly conserved between BMP2/4- and BMP5/6/7/8-type ligands, and the other being Scw ligand specific. Our data reveal that N-glycosylation of the Scw ligand boosts BMP signaling both in cell culture and in the embryo. In contrast, N-glycosylation modifications of Gbb or Scw ligands reduce the consistency of PCV development. These results suggest that tolerance for structural changes of BMP5/6/7/8-type ligands is dependent on developmental constraints. Furthermore, gain and loss of N-glycosylation motifs in conserved signaling molecules under evolutionary constraints appear to constitute flexible modules to adapt to developmental processes.Peer reviewe

Scribble and alpha-Catenin cooperatively regulate epithelial homeostasis and growth

Epithelial homeostasis is an emergent property of both physical and biochemical signals emanating from neighboring cells and across tissue. A recent study reveals that Scribble, an apico-basal polarity determinant, cooperates with alpha-Catenin, an adherens junction component, to regulate tissue homeostasis in the Drosophila wing imaginal disc. However, it remains to be addressed whether similar mechanisms are utilized in vertebrates. In this study, we first address how alpha-Catenin cooperates with Scribble to regulate epithelial homeostasis and growth in mammalian cells. Our data show that alpha-Catenin and Scribble interact physically in mammalian cells. We then found that both alpha-Catenin and Scribble are required for regulating nuclear translocation of YAP, an effector of the Hippo signaling pathway. Furthermore, ectopic Scribble suffices to suppress YAP in an alpha-Catenin-dependent manner. Then, to test our hypothesis that Scribble amounts impact epithelial growth, we use the Drosophila wing imaginal disc. We show that Scribble expression is complementary to Yorkie signal, the Drosophila ortholog of YAP. Ectopic expression of full-length Scribble or Scribble Leucine Rich Region (LRR):alpha-Catenin chimera sufficiently down-regulates Yorkie signal, leading to smaller wing size. Moreover, Scribble LRR:alpha-Catenin chimera rescues scribble mutant clones in the wing imaginal disc to maintain tissue homeostasis. Taken together, our studies suggest that the association of cell polarity component Scribble with alpha-Catenin plays a conserved role in epithelial homeostasis and growth.Peer reviewe

Coupling between dynamic 3D tissue architecture and BMP morphogen signaling during Drosophila wing morphogenesis

At the level of organ formation, tissue morphogenesis drives developmental processes in animals, often involving the rearrangement of two-dimensional (2D) structures into more complex three-dimensional (3D) tissues. These processes can be directed by growth factor signaling pathways. However, little is known about how such morphological changes affect the spatiotemporal distribution of growth factor signaling. Here, using the Drosophila pupal wing, we address how decapentaplegic (Dpp)/bone morphogenetic protein (BMP) signaling and 3D wing morphogenesis are coordinated. Dpp, expressed in the longitudinal veins (LVs) of the pupal wing, initially diffuses laterally within both dorsal and ventral wing epithelia during the inflation stage to regulate cell proliferation. Dpp localization is then refined to the LVs within each epithelial plane, but with active interplanar signaling for vein patterning/differentiation, as the two epithelia appose. Our data further suggest that the 3D architecture of the wing epithelia and the spatial distribution of BMP signaling are tightly coupled, revealing that 3D morphogenesis is an emergent property of the interactions between extracellular signaling and tissue shape changes.Peer reviewe

Amyloid-beta/Fyn–Induced Synaptic, Network, and Cognitive Impairments Depend on Tau Levels in Multiple Mouse Models of Alzheimer’s Disease

Alzheimer\u27s disease (AD), the most common neurodegenerative disorder, is a growing public health problem and still lacks effective treatments. Recent evidence suggests that microtubule-associated protein tau may mediate amyloid-β peptide (Aβ) toxicity by modulating the tyrosine kinase Fyn.Weshowed previously that tau reduction prevents, and Fyn overexpression exacerbates, cognitive deficits in human amyloid precursor protein (hAPP) transgenic mice overexpressing Aβ. However, the mechanisms by which Aβ, tau, and Fyn cooperate in AD-related pathogenesis remain to be fully elucidated. Here we examined the synaptic and network effects of this pathogenic triad. Tau reduction prevented cognitive decline induced by synergistic effects of Aβ and Fyn. Tau reduction also prevented synaptic transmission and plasticity deficits in hAPP mice. Using electroencephalography to examine network effects, we found that tau reduction prevented spontaneous epileptiform activity in multiple lines of hAPP mice. Tau reduction also reduced the severity of spontaneous and chemically induced seizures in mice overexpressing both Aβ and Fyn. To better understand these protective effects, we recorded wholecell currents in acute hippocampal slices from hAPP mice with and without tau. hAPP mice with tau had increased spontaneous and evoked excitatory currents, reduced inhibitory currents, and NMDA receptor dysfunction. Tau reduction increased inhibitory currents and normalized excitation/inhibition balance and NMDA receptor-mediated currents in hAPP mice. Our results indicate that Aβ, tau, and Fyn jointly impair synaptic and network function and suggest that disrupting the copathogenic relationship between these factors could be of therapeutic benefit

Two distinct waves of transcriptome and translatome changes drive Drosophila germline stem cell differentiation.

Funder: Herchel Smith FundThe tight control of fate transitions during stem cell differentiation is essential for proper tissue development and maintenance. However, the challenges in studying sparsely distributed adult stem cells in a systematic manner have hindered efforts to identify how the multilayered regulation of gene expression programs orchestrates stem cell differentiation in vivo. Here, we synchronised Drosophila female germline stem cell (GSC) differentiation in vivo to perform in-depth transcriptome and translatome analyses at high temporal resolution. This characterisation revealed widespread and dynamic changes in mRNA level, promoter usage, exon inclusion, and translation efficiency. Transient expression of the master regulator, Bam, drives a first wave of expression changes, primarily modifying the cell cycle program. Surprisingly, as Bam levels recede, differentiating cells return to a remarkably stem cell-like transcription and translation program, with a few crucial changes feeding into a second phase driving terminal differentiation to form the oocyte. Altogether, these findings reveal that rather than a unidirectional accumulation of changes, the in vivo differentiation of stem cells relies on distinctly regulated and developmentally sequential waves

Two distinct waves of transcriptome and translatome changes drive Drosophila germline stem cell differentiation.

The tight control of fate transitions during stem cell differentiation is essential for proper tissue development and maintenance. However, the challenges in studying sparsely distributed adult stem cells in a systematic manner have hindered efforts to identify how the multilayered regulation of gene expression programs orchestrates stem cell differentiation in vivo. Here, we synchronised Drosophila female germline stem cell (GSC) differentiation in vivo to perform in-depth transcriptome and translatome analyses at high temporal resolution. This characterisation revealed widespread and dynamic changes in mRNA level, promoter usage, exon inclusion, and translation efficiency. Transient expression of the master regulator, Bam, drives a first wave of expression changes, primarily modifying the cell cycle program. Surprisingly, as Bam levels recede, differentiating cells return to a remarkably stem cell-like transcription and translation program, with a few crucial changes feeding into a second phase driving terminal differentiation to form the oocyte. Altogether, these findings reveal that rather than a unidirectional accumulation of changes, the in vivo differentiation of stem cells relies on distinctly regulated and developmentally sequential waves

LRR domain of Scrib interacts with Tkv.

<p>(A) Diagram of different fragments of Scrib. Intensities of interaction between Scrib fragments and Tkv are shown at the right. (B, C) Co-IP of Scrib (full length or fragments) and Tkv. Scrib-MYC and Tkv-GFP were expressed in S2 cells, and cell lysates were immunopreciated by anti-GFP. Cell lysates (input) and immunoprecipitated proteins (IP: GFP) were analysed by Western blots probed with anti-GFP and anti-MYC antibodies. (D) Co-IP of LRR, Tkv and Mad. LRR-RFP, caTkv-GFP and Mad-FLAG were expressed in S2 cells, and cell lysates were immunopreciated by anti-RFP. Cell lysates (input) and immunoprecipitated proteins (IP: RFP) were analysed by Western blots probed with anti-GFP, anti-FLAG and anti-pMad antibodies. (E) Expression of Scrib (full length), LRR domain or Scrib without LRR domain in <i>scrib</i> mutant clones labeled by GFP. pMad staining in PCV region at 24 h AP. Dashed lines delineate the clone boundaries. (F) Quantification of pMad-positive cells in E. (G) Effects of <i>scrib</i>^<i>5</i> mutant clones on pMad (red) at 24 h AP in the PCV region. Mutant GFP-labeled cells (green) were generated using MARCM. Results shown are representative of one of three independent experiments (B-D).</p

Scrib regulates spatial distribution of Tkv in the PCV region.

<p>(A) A schematic of different planes (1 and 2) of PCV cells along the apicobasal axis in B. (B) Loss of Scrib affects Tkv distribution. <i>scrib</i> mutant clones are marked by mRFP and absence of Scrib staining. Tkv-YFP staining in the PCV region at 24 h AP. (C) Optical cross sections of B. Note that Tkv is more enriched basally in control but restricted more apically in <i>scrib</i> mutant cells. (D, E) Wild-type pupal wing. Tkv-YFP, Scrib and Rab5 staining in the PCV region at 24 h AP (D). Tkv-YFP, pMad and Scrib staining in the PCV region at 24 h AP (E). Arrows indicate that Tkv-YFP puncta co-localize with Scrib and Rab5 (D), or pMad and Scrib (E). (F) Tkv-YFP and Rab5 staining at the basolateral plane in control (<i>nub</i>^<i>ts</i>) and <i>scrib</i> RNAi (<i>nub</i>^<i>ts</i> <i>> scrib RNAi</i>) in the PCV region at 24 h AP. Arrows indicate that Tkv-YFP puncta co-localize with Rab5. (G) Quantification of co-localization of Tkv-YFP with Rab5. n = 20 cells for each sample. (H) Quantification of Tkv-YFP or Rab5 puncta. n = 20 cells for each sample. Error bar, SEM. **<i>P</i><0.01 in f, two-paired <i>t</i>-test. Images are the composite of sections with 2 μm thickness in D-F. Images have been taken by a 25X objective for B, C, and 63X objective for D-F.</p