EGFR signaling controls directionality of epithelial multilayer formation upon loss of cell polarity.

Apical-basal polarity is maintained by distinct protein complexes that reside in membrane junctions, and polarity loss in monolayered epithelial cells can lead to formation of multilayers, cell extrusion, and/or malignant overgrowth. Yet, how polarity loss cooperates with intrinsic signals to control directional invasion toward neighboring epithelial cells remains elusive. Using the Drosophila ovarian follicular epithelium as a model, we found that posterior follicle cells with loss of lethal giant larvae (lgl) or Discs large (Dlg) accumulate apically toward germline cells, whereas cells with loss of Bazooka (Baz) or atypical protein kinase C (aPKC) expand toward the basal side of wildtype neighbors. Further studies revealed that these distinct multilayering patterns in the follicular epithelium were determined by epidermal growth factor receptor (EGFR) signaling and its downstream target Pointed, a zinc-finger transcription factor. Additionally, we identified Rho kinase as a Pointed target that regulates formation of distinct multilayering patterns. These findings provide insight into how cell polarity genes and receptor tyrosine kinase signaling interact to govern epithelial cell organization and directional growth that contribute to epithelial tumor formation.

Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut.

Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis and regeneration. In mammals, the progenies of intestinal stem cells (ISCs) can dedifferentiate to generate ISCs upon ablation of resident stem cells. However, whether and how mature tissue cells generate ISCs under physiological conditions remains unknown. Here, we show that infection of the Drosophila melanogaster intestine with pathogenic bacteria induces entry of enteroblasts (EBs), which are ISC progenies, into the mitotic cycle through upregulation of epidermal growth factor receptor (EGFR)-Ras signaling. We also show that ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive enteroblast mitosis cell autonomously. Furthermore, we find that the dividing enteroblasts do not gain ISC identity as a prerequisite to divide, and the regenerative ISCs are produced through EB mitosis. Taken together, our work uncovers a new role for EGFR-Ras signaling in driving EB mitosis and replenishing the ISC pool during fly intestinal regeneration, which may have important implications for tissue homeostasis and tumorigenesis in vertebrates.

Injury-stimulated and self-restrained BMP signaling dynamically regulates stem cell pool size during Drosophila midgut regeneration.

Many adult organs rely on resident stem cells to maintain homeostasis. Upon injury, stem cells increase proliferation, followed by lineage differentiation to replenish damaged cells. Whether stem cells also change division mode to transiently increase their population size as part of a regenerative program and, if so, what the underlying mechanism is have remained largely unexplored. Here we show that injury stimulates the production of two bone morphogenetic protein (BMP) ligands, Dpp and Gbb, which drive an expansion of intestinal stem cells (ISCs) by promoting their symmetric self-renewing division in Drosophila adult midgut. We find that BMP production in enterocytes is inhibited by BMP signaling itself, and that BMP autoinhibition is required for resetting ISC pool size to the homeostatic level after tissue repair. Our study suggests that dynamic BMP signaling controls ISC population size during midgut regeneration and reveals mechanisms that precisely control stem cell number in response to tissue needs.

Intestinal stem cell response to injury: lessons from Drosophila.

Many adult tissues and organs are maintained by resident stem cells that are activated in response to injury but the mechanisms that regulate stem cell activity during regeneration are still poorly understood. An emerging system to study such problem is the Drosophila adult midgut. Recent studies have identified both intrinsic factors and extrinsic niche signals that control the proliferation, self-renewal, and lineage differentiation of Drosophila adult intestinal stem cells (ISCs). These findings set up the stage to interrogate how niche signals are regulated and how they are integrated with cell-intrinsic factors to control ISC activity during normal homeostasis and regeneration. Here we review the current understanding of the mechanisms that control ISC self-renewal, proliferation, and lineage differentiation in Drosophila adult midgut with a focus on the niche signaling network that governs ISC activity in response to injury.

Efficient EGFR signaling and dorsal-ventral axis patterning requires syntaxin dependent Gurken trafficking.

Vesicle trafficking plays a crucial role in the establishment of cell polarity in various cellular contexts, including axis-pattern formation in the developing egg chamber of Drosophila. The EGFR ligand, Gurken (Grk), is first localized at the posterior of young oocytes for anterior-posterior axis formation and later in the dorsal anterior region for induction of the dorsal-ventral (DV) axis, but regulation of Grk localization by membrane trafficking in the oocyte remains poorly understood. Here, we report that Syntaxin 1A (Syx1A) is required for efficient trafficking of Grk protein for DV patterning. We show that Syx1A is associated with the Golgi membrane and is required for the transportation of Grk-containing vesicles along the microtubules to their dorsal anterior destination in the oocyte. Our studies reveal that the Syx1A dependent trafficking of Grk protein is required for efficient EGFR signaling during DV patterning.

Topoisomerase I is an Evolutionarily Conserved Key Regulator for Satellite DNA Transcription.

Repetitive satellite DNAs, divergent in nucleic-acid sequence and size across eukaryotes, provide a physical site for centromere assembly to orchestrate chromosome segregation during the cell cycle. These non-coding DNAs are transcribed by RNA polymerase (RNAP) II and the transcription has been shown to play a role in chromosome segregation, but a little is known about the regulation of centromeric transcription, especially in higher organisms with tandemly-repeated-DNA-sequence centromeres. Using RNA interference knockdown, chemical inhibition and AID/IAA degradation, we show that Topoisomerase I (TopI), not TopII, promotes the transcription of α-satellite DNAs, the main type of satellite on centromeres in human cells. Mechanistically, TopI localizes to centromeres, binds RNAP II and facilitates RNAP II elongation on centromeres. Interestingly, in response to DNA double-stranded breaks (DSBs) induced by chemotherapy drugs or CRSPR/Cas9, α-satellite transcription is dramatically stimulated in a DNA damage checkpoint-independent but TopI-dependent manner. These DSB-induced α-satellite RNAs were predominantly derived from the α-satellite high-order repeats of human centromeres and forms into strong speckles in the nucleus. Remarkably, TopI-dependent satellite transcription also exists in mouse 3T3 and Drosophila S2 cells and in Drosophila larval imaginal wing discs and tumor tissues. Altogether, our findings herein reveal an evolutionally conserved mechanism with TopI as a key player for the regulation of satellite transcription at both cellular and animal levels.

Topoisomerase I is an evolutionarily conserved key regulator for satellite DNA transcription.

RNA Polymerase (RNAP) II transcription on non-coding repetitive satellite DNAs plays an important role in chromosome segregation, but a little is known about the regulation of satellite transcription. We here show that Topoisomerase I (TopI), not TopII, promotes the transcription of α-satellite DNAs, the main type of satellite DNAs on human centromeres. Mechanistically, TopI localizes to centromeres, binds RNAP II and facilitates RNAP II elongation. Interestingly, in response to DNA double-stranded breaks (DSBs), α-satellite transcription is dramatically stimulated in a DNA damage checkpoint-independent but TopI-dependent manner, and these DSB-induced α-satellite RNAs form into strong speckles in the nucleus. Remarkably, TopI-dependent satellite transcription also exists in mouse 3T3 and Drosophila S2 cells and in Drosophila larval imaginal wing discs and tumor tissues. Altogether, our findings herein reveal an evolutionally conserved mechanism with TopI as a key player for the regulation of satellite transcription at both cellular and animal levels.

Involvement of Lgl and Mahjong/VprBP in cell competition.

During the initial stages of carcinogenesis, transformation events occur in a single cell within an epithelial monolayer. However, it remains unknown what happens at the interface between normal and transformed epithelial cells during this process. In Drosophila, it has been recently shown that normal and transformed cells compete with each other for survival in an epithelial tissue; however the molecular mechanisms whereby "loser cells" undergo apoptosis are not clearly understood. Lgl (lethal giant larvae) is a tumor suppressor protein and plays a crucial role in oncogenesis in flies and mammals. Here we have examined the involvement of Lgl in cell competition and shown that a novel Lgl-binding protein is involved in Lgl-mediated cell competition. Using biochemical immunoprecipitation methods, we first identified Mahjong as a novel binding partner of Lgl in both flies and mammals. In Drosophila, Mahjong is an essential gene, but zygotic mahjong mutants (mahj(-/-)) do not have obvious patterning defects during embryonic or larval development. However, mahj(-/-) cells undergo apoptosis when surrounded by wild-type cells in the wing disc epithelium. Importantly, comparable phenomena also occur in Mahjong-knockdown mammalian cells; Mahjong-knockdown Madin-Darby canine kidney epithelial cells undergo apoptosis, only when surrounded by non-transformed cells. Similarly, apoptosis of lgl(-/-) cells is induced when they are surrounded by wild-type cells in Drosophila wing discs. Phosphorylation of the c-Jun N-terminal kinase (JNK) is increased in mahj(-/-) or lgl(-/-) mutant cells, and expression of Puckered (Puc), an inhibitor of the JNK pathway, suppresses apoptosis of these mutant cells surrounded by wild-type cells, suggesting that the JNK pathway is involved in mahj- or lgl-mediated cell competition. Finally, we have shown that overexpression of Mahj in lgl(-/-) cells strongly suppresses JNK activation and blocks apoptosis of lgl(-/-) cells in the wild-type wing disc epithelium. These data indicate that Mahjong interacts with Lgl biochemically and genetically and that Mahjong and Lgl function in the same pathway to regulate cellular competitiveness. As far as we are aware, this is the first report that cell competition can occur in a mammalian cell culture system.

Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants.

NAC transcription factors play important roles in plant growth, development and stress responses. Previously, we identified multiple NAC genes in soybean (Glycine max). Here, we identify the roles of two genes, GmNAC11 and GmNAC20, in stress responses and other processes. The two genes were differentially induced by multiple abiotic stresses and plant hormones, and their transcripts were abundant in roots and cotyledons. Both genes encoded proteins that localized to the nucleus and bound to the core DNA sequence CGT[G/A]. In the protoplast assay system, GmNAC11 acts as a transcriptional activator, whereas GmNAC20 functions as a mild repressor; however, the C-terminal end of GmANC20 has transcriptional activation activity. Over-expression of GmNAC20 enhances salt and freezing tolerance in transgenic Arabidopsis plants; however, GmNAC11 over-expression only improves salt tolerance. Over-expression of GmNAC20 also promotes lateral root formation. GmNAC20 may regulate stress tolerance through activation of the DREB/CBF-COR pathway, and may control lateral root development by altering auxin signaling-related genes. GmNAC11 probably regulates DREB1A and other stress-related genes. The roles of the two GmNAC genes in stress tolerance were further analyzed in soybean transgenic hairy roots. These results provide a basis for genetic manipulation to improve the agronomic traits of important crops.

Wheat WRKY genes TaWRKY2 and TaWRKY19 regulate abiotic stress tolerance in transgenic Arabidopsis plants.

WRKY-type transcription factors are involved in multiple aspects of plant growth, development and stress response. WRKY genes have been found to be responsive to abiotic stresses; however, their roles in abiotic stress tolerance are largely unknown especially in crops. Here, we identified stress-responsive WRKY genes from wheat (Triticum aestivum L.) and studied their functions in stress tolerance. Forty-three putative TaWRKY genes were identified and two multiple stress-induced genes, TaWRKY2 and TaWRKY19, were further characterized. TaWRKY2 and TaWRKY19 are nuclear proteins, and displayed specific binding to typical cis-element W box. Transgenic Arabidopsis plants overexpressing TaWRKY2 exhibited salt and drought tolerance compared with controls. Overexpression of TaWRKY19 conferred tolerance to salt, drought and freezing stresses in transgenic plants. TaWRKY2 enhanced expressions of STZ and RD29B, and bound to their promoters. TaWRKY19 activated expressions of DREB2A, RD29A, RD29B and Cor6.6, and bound to DREB2A and Cor6.6 promoters. The two TaWRKY proteins may regulate the downstream genes through direct binding to the gene promoter or via indirect mechanism. Manipulation of TaWRKY2 and TaWRKY19 in wheat or other crops should improve their performance under various abiotic stress conditions.

Tumor models in various Drosophila tissues.

The development of cancer is a complex multistage process. Over the past few decades, the model organism Drosophila melanogaster has been crucial in identifying cancer-related genes and pathways and elucidating mechanisms underlying growth regulation in development. Investigations using Drosophila has yielded new insights into the molecular mechanisms involved in tumor initiation and progression. In this review, we describe various tumor models that have been developed in recent years using different Drosophila tissues, such as the imaginal tissue, the neural tissue, the gut, the ovary, and hematopoietic cells. We discuss underlying genetic alterations, cancer-like characteristics, as well as similarities and key differences among these models. We also discuss how disruptions in stem cell division and differentiation result in tumor formation in diverse tissues, and highlight new concepts developed using the fly model to understand context-dependent tumorigenesis. We further discuss the progress made in Drosophila to explore tumor-host interactions that involve the innate immune response to tumor growth and the cachexia wasting phenotype. This article is categorized under: Cancer > Genetics/Genomics/Epigenetics Cancer > Stem Cells and Development Cancer > Molecular and Cellular Physiology.

Par-1 and Tau regulate the anterior-posterior gradient of microtubules in Drosophila oocytes.

The formation of an anterior-posterior (AP) gradient of microtubules in Drosophila oocytes is essential for specification of the AP axis. Proper microtubule organization in the oocyte requires the function of serine/threonine kinase Par-1. The N1S isoform of Par-1 is enriched at the posterior cortex of the oocyte from stage 7 of oogenesis. Here we report that posterior restriction of Par-1 (N1S) kinase activity is critical for microtubule AP gradient formation. Egg chambers with excessive and ectopic Par-1 (N1S) kinase activity in the germline cells display phenotypes similar to those of egg chambers treated with the microtubule-depolymerizing drug colcemid: depolymerization of microtubules in the oocyte and disruption of oocyte nucleus localization. A phosphorylation target of Par-1, the microtubule-associated protein Tau, is also involved in oocyte polarity formation, and overexpression of Tau alleviates the phenotypes caused by ectopic Par-1 (N1S) kinase activity, suggesting that Par-1 regulates oocyte polarity at least partly through Tau. Our findings reveal that maintaining proper levels of Par-1 at correct position in the oocyte is key to oocyte polarity formation and that the conserved role of Par-1 and Tau is crucial for the establishment of an AP gradient of microtubules and for AP axis specification.

Gilgamesh (Gish)/CK1γ regulates tissue homeostasis and aging in adult Drosophila midgut.

Adult tissues and organs rely on resident stem cells to generate new cells that replenish damaged cells. To maintain homeostasis, stem cell activity needs to be tightly controlled throughout the adult life. Here, we show that the membrane-associated kinase Gilgamesh (Gish)/CK1γ maintains Drosophila adult midgut homeostasis by restricting JNK pathway activity and that Gish is essential for intestinal stem cell (ISC) maintenance under stress conditions. Inactivation of Gish resulted in aberrant JNK pathway activation and excessive production of multiple cytokines and growth factors that drive ISC overproliferation. Mechanistically, Gish restricts JNK activation by phosphorylating and destabilizing a small GTPase, Rho1. Interestingly, we find that Gish expression is down-regulated in aging guts and that increasing Gish activity in aging guts can restore tissue homeostasis. Hence, our study identifies Gish/CK1γ as a novel regulator of Rho1 and gatekeeper of tissue homeostasis whose activity is compromised in aging guts.

Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants.

WRKY-type transcription factors have multiple roles in the plant defence response and developmental processes. Their roles in the abiotic stress response remain obscure. In this study, 64 GmWRKY genes from soybean were identified, and were found to be differentially expressed under abiotic stresses. Nine GmWRKY proteins were tested for their transcription activation in the yeast assay system, and five showed such ability. In a DNA-binding assay, three proteins (GmWRKY13, GmWRKY27 and GmWRKY54) with a conserved WRKYGQK sequence in their DNA-binding domain could bind to the W-box (TTGAC). However, GmWRKY6 and GmWRKY21, with an altered sequence WRKYGKK, lost the ability to bind to the W-box. The function of three stress-induced genes, GmWRKY13, GmWRKY21 and GmWRKY54, was further investigated using a transgenic approach. GmWRKY21-transgenic Arabidopsis plants were tolerant to cold stress, whereas GmWRKY54 conferred salt and drought tolerance, possibly through the regulation of DREB2A and STZ/Zat10. Transgenic plants over-expressing GmWRKY13 showed increased sensitivity to salt and mannitol stress, but decreased sensitivity to abscisic acid, when compared with wild-type plants. In addition, GmWRKY13-transgenic plants showed an increase in lateral roots. These results indicate that the three GmWRKY genes play differential roles in abiotic stress tolerance, and that GmWRKY13 may function in both lateral root development and the abiotic stress response.

Dual role of BMP signaling in the regulation of Drosophila intestinal stem cell self-renewal.

Many adult organs including Drosophila adult midguts rely on resident stem cells to replenish damaged cells during tissue homeostasis and regeneration. Previous studies have shown that, upon injury, intestinal stem cells (ISCs) in the midguts can increase proliferation and lineage differentiation to meet the demand for tissue repair. Our recent study has demonstrated that, in response to certain injury, midguts can expand ISC population size as an additional regenerative mechanism. We found that injury elicited by bleomycin feeding or bacterial infection increased the production of two BMP ligands (Dpp and Gbb) in enterocytes (ECs), leading to elevated BMP signaling in progenitor cells that drove an expansion of ISCs by promoting their symmetric self-renewing division. Interestingly, we also found that BMP signaling in ECs inhibits the production of Dpp and Gbb, and that this negative feedback mechanism is required to reset ISC pool size to the homeostatic state. Our findings suggest that BMP signaling exerts two opposing influences on stem cell activity depending on where it acts: BMP signaling in progenitor cells promotes ISC self-renewal while BMP signaling in ECs restricts ISC self-renewal by preventing excessive production of BMP ligands. Our results further suggest that transient expansion of ISC population in conjunction with increasing ISC proliferation provides a more effective strategy for tissue regeneration.

Injury-stimulated Hedgehog signaling promotes regenerative proliferation of Drosophila intestinal stem cells.

Many adult tissues are maintained by resident stem cells that elevate their proliferation in response to injury. The regulatory mechanisms underlying regenerative proliferation are still poorly understood. Here we show that injury induces Hedgehog (Hh) signaling in enteroblasts (EBs) to promote intestinal stem cell (ISC) proliferation in Drosophila melanogaster adult midgut. Elevated Hh signaling by patched (ptc) mutations drove ISC proliferation noncell autonomously. Inhibition of Hh signaling in the ISC lineage compromised injury-induced ISC proliferation but had little if any effect on homeostatic proliferation. Hh signaling acted in EBs to regulate the production of Upd2, which activated the JAK-STAT pathway to promote ISC proliferation. Furthermore, we show that Hh signaling is stimulated by DSS through the JNK pathway and that inhibition of Hh signaling in EBs prevented DSS-stimulated ISC proliferation. Hence, our study uncovers a JNK-Hh-JAK-STAT signaling axis in the regulation of regenerative stem cell proliferation.

Intestinal epithelium-derived BMP controls stem cell self-renewal in Drosophila adult midgut.

Stem cells are maintained in a specialized microenvironment called niche but the nature of stem cell niche remains poorly defined in many systems. Here we demonstrate that intestinal epithelium-derived BMP serves as a niche signal for intestinal stem cell (ISC) self-renewal in Drosophila adult midgut. We find that BMP signaling is asymmetric between ISC and its differentiated daughter cell. Two BMP ligands, Dpp and Gbb, are produced by enterocytes and act in conjunction to promote ISC self-renewal by antagonizing Notch signaling. Furthermore, the basement membrane-associated type IV collagens regulate ISC self-renewal by confining higher BMP signaling to ISCs. The employment of gut epithelia as a niche for stem cell self-renewal may provide a mechanism for direct communication between the niche and the environment, allowing niche signal production and stem cell number to be fine-tuned in response to various physiological and pathological stimuli. DOI: http://dx.doi.org/10.7554/eLife.01857.001.

The cell adhesion molecule echinoid functions as a tumor suppressor and upstream regulator of the Hippo signaling pathway.

The Hippo (Hpo) signaling pathway controls tissue growth and organ size in species ranging from Drosophila to mammals and is deregulated in a wide range of human cancers. The core pathway consists of the Hpo/Warts (Wts) kinase cassette that phosphorylates and inactivates the transcriptional coactivator Yorkie (Yki). Here, we report that Echinoid (Ed), an immunoglobulin domain-containing cell adhesion molecule, acts as an upstream regulator of the Hpo pathway. Loss of Ed compromises Yki phosphorylation, resulting in elevated Yki activity that increases Hpo target gene expression and drives tissue overgrowth. Ed physically interacts with and stabilizes the Hpo-binding partner Salvador (Sav) at adherens junctions. Ed/Sav interaction is promoted by cell-cell contact and requires dimerization of Ed cytoplasmic domain. Overexpression of Sav or dimerized Ed cytoplasmic domain suppressed loss-of-Ed phenotypes. We propose that Ed may link cell-cell contact to Hpo signaling through binding and stabilizing Sav, thus modulating the Hpo kinase activity.

Soybean Trihelix transcription factors GmGT-2A and GmGT-2B improve plant tolerance to abiotic stresses in transgenic Arabidopsis.

BACKGROUND: Trihelix transcription factors play important roles in light-regulated responses and other developmental processes. However, their functions in abiotic stress response are largely unclear. In this study, we identified two trihelix transcription factor genes GmGT-2A and GmGT-2B from soybean and further characterized their roles in abiotic stress tolerance. FINDINGS: Both genes can be induced by various abiotic stresses, and the encoded proteins were localized in nuclear region. In yeast assay, GmGT-2B but not GmGT-2A exhibits ability of transcriptional activation and dimerization. The N-terminal peptide of 153 residues in GmGT-2B was the minimal activation domain and the middle region between the two trihelices mediated the dimerization of the GmGT-2B. Transactivation activity of the GmGT-2B was also confirmed in plant cells. DNA binding analysis using yeast one-hybrid assay revealed that GmGT-2A could bind to GT-1bx, GT-2bx, mGT-2bx-2 and D1 whereas GmGT-2B could bind to the latter three elements. Overexpression of the GmGT-2A and GmGT-2B improved plant tolerance to salt, freezing and drought stress in transgenic Arabidopsis plants. Moreover, GmGT-2B-transgenic plants had more green seedlings compared to Col-0 under ABA treatment. Many stress-responsive genes were altered in GmGT-2A- and GmGT-2B-transgenic plants. CONCLUSION: These results indicate that GmGT-2A and GmGT-2B confer stress tolerance through regulation of a common set of genes and specific sets of genes. GmGT-2B also affects ABA sensitivity.