Planar Cell Polarity Signaling in Collective Cell Movements During Morphogenesis and Disease

Collective and directed cell movements are crucial for diverse developmental processes in the animal kingdom, but they are also involved in wound repair and disease. During these processes groups of cells are oriented within the tissue plane, which is referred to as planar cell polarity (PCP). This requires a tight regulation that is in part conducted by the PCP pathway. Although this pathway was initially characterized in flies, subsequent studies in vertebrates revealed a set of conserved core factors but also effector molecules and signal modulators, which build the fundamental PCP machinery. The PCP pathway in Drosophila regulates several developmental processes involving collective cell movements such as border cell migration during oogenesis, ommatidial rotation during eye development, and embryonic dorsal closure. During vertebrate embryogenesis, PCP signaling also controls collective and directed cell movements including convergent extension during gastrulation, neural tube closure, neural crest cell migration, or heart morphogenesis. Similarly, PCP signaling is linked to processes such as wound repair, and cancer invasion and metastasis in adults. As a consequence, disruption of PCP signaling leads to pathological conditions. In this review, we will summarize recent findings about the role of PCP signaling in collective cell movements in flies and vertebrates. In addition, we will focus on how studies in Drosophila have been relevant to our understanding of the PCP molecular machinery and will describe several developmental defects and human disorders in which PCP signaling is compromised. Therefore, new discoveries about the contribution of this pathway to collective cell movements could provide new potential diagnostic and therapeutic targets for these disorders

A DPP-mediated feed-forward loop canalizes morphogenesis during Drosophila dorsal closure

Development is robust because nature has selected various mechanisms to buffer the deleterious effects of environmental and genetic variations to deliver phenotypic stability. Robustness relies on smart network motifs such as feed-forward loops (FFLs) that ensure the reliable interpretation of developmental signals. In this paper, we show that Decapentaplegic (DPP) and JNK form a coherent FFL that controls the specification and differentiation of leading edge cells during Drosophila melanogaster dorsal closure (DC). We provide molecular evidence that through repression by Brinker (Brk), the DPP branch of the FFL filters unwanted JNK activity. High-throughput live imaging revealed that this DPP/Brk branch is dispensable for DC under normal conditions but is required when embryos are subjected to thermal stress. Our results indicate that the wiring of DPP signaling buffers against environmental challenges and canalizes cell identity. We propose that the main function of DPP pathway during Drosophila DC is to ensure robust morphogenesis, a distinct function from its well-established ability to spread spatial information

Coordinated waves of actomyosin flow and apical cell constriction immediately after wounding

© 2013 Antunes et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).Epithelial wound healing relies on tissue movements and cell shape changes. Our work shows that, immediately after wounding, there was a dramatic cytoskeleton remodeling consisting of a pulse of actomyosin filaments that assembled in cells around the wound edge and flowed from cell to cell toward the margin of the wound. We show that this actomyosin flow was regulated by Diaphanous and ROCK and that it elicited a wave of apical cell constriction that culminated in the formation of the leading edge actomyosin cable, a structure that is essential for wound closure. Calcium signaling played an important role in this process, as its intracellular concentration increased dramatically immediately after wounding, and down-regulation of transient receptor potential channel M, a stress-activated calcium channel, also impaired the actomyosin flow. Lowering the activity of Gelsolin, a known calcium-activated actin filament-severing protein, also impaired the wound response, indicating that cleaving the existing actin filament network is an important part of the cytoskeleton remodeling process.info:eu-repo/semantics/publishedVersio

Transcriptional Activity and Nuclear Localization of Cabut, the Drosophila Ortholog of Vertebrate TGF-β-Inducible Early-Response Gene (TIEG) Proteins

BackgroundCabut (Cbt) is a C2H2-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein.Methodology/Principal FindingsTo determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using β-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2.Conclusions/SignificanceOur results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of similarities and differences in both aspects of Cbt function between the insect and the vertebrate TIEG proteins

The Cabut zinc finger region is not essential for nuclear localization in CHO-K1 cells.

<p>(A) Schematic representation of the eGFPCbt fusion constructs used to transfect CHO-K1 cells. The locations of the SR region (black rectangle) and the zinc fingers (1, 2, 3, grey rectangles) are indicated. (B–G) Localization of eGFP fusion proteins in CHO-K1 cells transiently transfected with the constructs shown in (A). Cells were immunostained with anti-eGFP (green, first panel) and DAPI (blue; second panel). Differential interference contrast (DIC) was used to visualize cell boundaries (third panel). The overlay panel shows DIC and anti-eGFP staining. The localization of the fusion proteins is shown in the fourth panel (C, cytoplasmic; N, nuclear). Wild-type eGFP was located in the cytoplasm and the nucleus (B), but the eGFPCbt fusion protein translocates to the nucleus (C). With the exception of the peGFPCbt_262–428 fusion protein, which lacks the N-terminal region of the Cbt protein (G), all Cbt deletions affecting the zinc finger region showed cytoplasmic localization (D–F). Scale bar: 10 µm. (H) Western blots of protein extracts from CHO-K1 cells transfected with the constructs shown in (A) and stained with anti-GFP. (1) Non-transfected cells, (2) empty peGFP vector (∼27 kDa), (3) peGFPCbt_1–428 (∼70 kDa), (4) peGFPCbt_1–322 (∼60 kDa), (5) peGFPCbt_1–292 (∼60 kDa) and (6) peGFPCbt_1–262 (∼50 kDa).</p

The ₇₁PNKKPRL₇₇ sequence is the NLS of the Cabut protein.

<p>(A) Schematic representation of the β-GalCbt fusion constructs transfected into S2 cells to determine whether the ₁₆₂KMNRKRAAEVALPPVQTPETPVAKLVTPP₁₉₀ and/or ₇₁PNKKPRL₇₇ sequences are functional NLSs. The Serine-rich (SR) domain is shown in black and the PNKKPRL sequence in fuchsia. (B–J) Localization of β-Gal fusion proteins in S2 cells transiently transfected with the constructs shown in (A). Cells were stained with anti-β-Gal (red; first panel) and anti-Lam (green; second panel) to mark nuclear membranes. The overlay panel depicts double staining of cells with both antibodies, and the localization of the fusion proteins is shown in the fourth panel (C, cytoplasmic; N, nuclear). Note that fusion proteins lacking or containing a mutated ₇₁PNKKPRL₇₇ sequence (E and J) were cytoplasmic. The ₇₁PNKKPRL₇₇ sequence was able to translocate β-Gal to the nucleus when fused to either the N- or the C-terminus of the protein (H and I). Scale bar: 10 µm. (K) Western blots of protein extracts from S2 cells transfected with the constructs shown in (A) and stained with anti-β-Gal. (1) pIE-β-GalCbt_1–141 (∼140 kDa), (2) pIE-β-GalCbt_1–108 (∼140 kDa), (3) and (7) pIE-β-GalCbt_1–77 (∼135 kDa), (4) pIE-β-GalCbt_1–70 (∼135 kDa), (5) pIE-_PNKKPRL-β-Gal (∼120 kDa), (6) pIE-β-Gal-_PNKKPRL (∼120 kDa) and (8) pIE-β-Gal-Cbt_K73N–K74N (∼135 kDa).</p

Cabut functions as a transcriptional repressor in S2 cells and regulates its own transcription.

<p>(A) Expression of Gal4Cbt_1–428 led to repression of luciferase activity levels relative to a control Gal4 protein. Luciferase activity was measured 48 h after transfection. pRENILLA was used to normalize for cell number, transfection efficiency, and general effects on transcription (luciferase activity = firefly luciferase/renilla luciferase). (B) S2 cells transfected with <i>Prom1-2</i>-GFP as a control or co-transfected with <i>Prom1-2</i>-GFP and MT-<i>cbt</i>. The MT promoter was induced by exposing the cells to medium containing copper (upper picture, no copper; lower picture, plus copper). Fluorescence levels were reduced following transcriptional induction of Cbt by copper in cells co-transfected with <i>Prom1-2</i>-GPF and MT-<i>cbt</i> (left panels). Fluorescence was measured 48 h after induction (right panel). pCHERRY was used for normalization (Fluorescence levels = GFP/CHERRY). In A and B, data are presented as the mean ± SD of three replicates.</p