Lack of apoptosis leads to cellular senescence and tumorigenesis in Drosophila epithelial cells.

Programmed cell death (apoptosis) is a homeostasis program of animal tissues designed to remove cells that are unwanted or are damaged by physiological insults. To assess the functional role of apoptosis, we have studied the consequences of subjecting Drosophila epithelial cells defective in apoptosis to stress or genetic perturbations that normally cause massive cell death. We find that many of those cells acquire persistent activity of the JNK pathway, which drives them into senescent status, characterized by arrest of cell division, cell hypertrophy, Senescent Associated ß-gal activity (SA-ß-gal), reactive oxygen species (ROS) production, Senescent Associated Secretory Phenotype (SASP) and migratory behaviour. We have identified two classes of senescent cells in the wing disc: 1) those that localize to the appendage part of the disc, express the upd, wg and dpp signalling genes and generate tumour overgrowths, and 2) those located in the thoracic region do not express wg and dpp nor they induce tumour overgrowths. Whether to become tumorigenic or non-tumorigenic depends on the original identity of the cell prior to the transformation. We also find that the p53 gene contributes to senescence by enhancing the activity of JNK.

Regulation and coordination of the different DNA damage responses in Drosophila.

Cells have evolved mechanisms that allow them to respond to DNA damage to preserve genomic integrity and maintain tissue homeostasis. These responses include the activation of the cell cycle checkpoints and the repair mechanisms or the induction of apoptosis that eventually will eliminate damaged cells. These "life" vs. "death" decisions differ depending on the cell type, stages of development, and the proliferation status of the cell. The apoptotic response after DNA damage is of special interest as defects in its induction could contribute to tumorigenesis or the resistance of cancer cells to therapeutic agents such as radiotherapy. Multiples studies have elucidated the molecular mechanisms that mediate the activation of the DNA damage response pathway (DDR) and specifically the role of p53. However, much less is known about how the different cellular responses such as cell proliferation control and apoptosis are coordinated to maintain tissue homeostasis. Another interesting question is how the differential apoptotic response to DNA damage is regulated in distinct cell types. The use of Drosophila melanogaster as a model organism has been fundamental to understand the molecular and cellular mechanisms triggered by genotoxic stress. Here, we review the current knowledge regarding the cellular responses to ionizing radiation as the cause of DNA damage with special attention to apoptosis in Drosophila: how these responses are regulated and coordinated in different cellular contexts and in different tissues. The existence of intrinsic mechanisms that might attenuate the apoptotic pathway in response to this sort of DNA damage may well be informative for the differences in the clinical responsiveness of tumor cells after radiation therapy.

Coordination between cell proliferation and apoptosis after DNA damage in Drosophila.

Exposure to genotoxic stress promotes cell cycle arrest and DNA repair or apoptosis. These "life" or "death" cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, the precise regulation of p53 is essential to maintain tissue homeostasis and to prevent cancer development. However, how cell cycle progression has an impact on p53 cell fate decision-making is mostly unknown. In this work, we demonstrate that Drosophila p53 proapoptotic activity can be impacted by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation-induced apoptosis. We show that p53 binding to the regulatory elements of the proapoptotic genes and its ability to activate their expression is compromised in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 proapoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 proapoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.

The transcription factor Dysfusion promotes fold and joint morphogenesis through regulation of Rho1.

The mechanisms that control tissue patterning and cell behavior are extensively studied separately, but much less is known about how these two processes are coordinated. Here we show that the Drosophila transcription factor Dysfusion (Dysf) directs leg epithelial folding and joint formation through the regulation of Rho1 activity. We found that Dysf-induced Rho1 activity promotes apical constriction specifically in folding epithelial cells. Here we show that downregulation of Rho1 or its downstream effectors cause defects in fold and joint formation. In addition, Rho1 and its effectors are sufficient to induce the formation of epithelial folds when misexpressed in a flat epithelium. Furthermore, as apoptotic cells can actively control tissue remodeling, we analyzed the role of cell death in the formation of tarsal folds and its relation to Rho1 activity. Surprisingly, we found no defects in this process when apoptosis is inhibited. Our results highlight the coordination between a patterning transcription factor and the cellular processes that cause the cell shape changes necessary to sculpt a flat epithelium into a three dimensional structure.

Annexin A2 is critical for embryo adhesiveness to the human endometrium by RhoA activation through F-actin regulation.

Annexin A2 (ANXA2) is present in vivo in the mid- and late-secretory endometria and is mainly localized in the luminal epithelium. Our aim was to evaluate its function in regulating the human implantation process. With an in vitro adhesion model, constructed to evaluate how the mouse embryo and JEG-3 spheroids attach to human endometrial epithelial cells, we demonstrated that ANXA2 inhibition significantly diminishes embryo adhesiveness. ANXA2 is also implicated in endometrial epithelial cell migration and trophoblast outgrowth. ANXA2 was seen to be linked to the RhoA/ROCK pathway and to regulate cell adhesion. We noted that ANXA2 inhibition significantly reduces active RhoA, although RhoA inactivation does not alter the ANXA2 levels. RhoA inactivation and ROCK inhibition also moderate embryo adhesiveness to endometrial epithelial cells. We corroborated that the induction of constitutively active RhoA partially reverses the effects of ANXA2 inhibition on endometrial adhesiveness. These molecules colocalize on the plasma membrane of endometrial epithelial cells, and a large proportion of ANXA2 and RhoA are colocalized in the F-actin networks. The functional effects of ANXA2 inhibition and RhoA/ROCK inactivation are associated with significant alterations in F-actin organization and its depolymerization. ANXA2 may act upstream of the RhoA/ROCK pathway by regulating F-actin remodeling and is a key factor in human endometrial adhesiveness.

Inhibition of histone deacetylase activity in human endometrial stromal cells promotes extracellular matrix remodelling and limits embryo invasion.

Invasion of the trophoblast into the maternal decidua is regulated by both the trophoectoderm and the endometrial stroma, and entails the action of tissue remodeling enzymes. Trophoblast invasion requires the action of metalloproteinases (MMPs) to degrade extracellular matrix (ECM) proteins and in turn, decidual cells express tissue inhibitors of MMPs (TIMPs). The balance between these promoting and restraining factors is a key event for the successful outcome of pregnancy. Gene expression is post-transcriptionally regulated by histone deacetylases (HDACs) that unpacks condensed chromatin activating gene expression. In this study we analyze the effect of histone acetylation on the expression of tissue remodeling enzymes and activity of human endometrial stromal cells (hESCs) related to trophoblast invasion control. Treatment of hESCs with the HDAC inhibitor trichostatin A (TSA) increased the expression of TIMP-1 and TIMP-3 while decreased MMP-2, MMP-9 and uPA and have an inhibitory effect on trophoblast invasion. Moreover, histone acetylation is detected at the promoters of TIMP-1 and TIMP-3 genes in TSA-treated. In addition, in an in vitro decidualized hESCs model, the increase of TIMP-1 and TIMP-3 expression is associated with histone acetylation at the promoters of these genes. Our results demonstrate that histone acetylation disrupt the balance of ECM modulators provoking a restrain of trophoblast invasion. These findings are important as an epigenetic mechanism that can be used to control trophoblast invasion.

Molecular integration of wingless, decapentaplegic, and autoregulatory inputs into Distalless during Drosophila leg development.

The development of the Drosophila leg requires both Decapentaplegic (Dpp) and Wingless (Wg), two signals that establish the proximo-distal (PD) axis by activating target genes such as Distalless (Dll). Dll expression in the leg depends on a Dpp- and Wg-dependent phase and a maintenance phase that is independent of these signals. Here, we show that accurate Dll expression in the leg results from the synergistic interaction between two cis-regulatory elements. The Leg Trigger (LT) element directly integrates Wg and Dpp inputs and is only active in cells receiving high levels of both signals. The Maintenance (M) element is able to maintain Wg- and Dpp-independent expression, but only when in cis to LT. M, which includes the native Dll promoter, functions as an autoregulatory element by directly binding Dll. The "trigger-maintenance" model describes a mechanism by which secreted morphogens act combinatorially to induce the stable expression of target genes.

Logic of Wg and Dpp induction of distal and medial fates in the Drosophila leg.

Drosophila leg development requires the cooperation of two secreted signals, Decapentaplegic (Dpp) and Wingless (Wg), to form the proximodistal (PD) axis. Wg and Dpp are also required to pattern the dorsoventral (DV) axis of the leg. Here, we show that Distalless (Dll) and dachshund (dac), genes expressed at different positions along the PD axis, are activated by Wg signaling and repressed by Brinker (Brk), a transcriptional repressor in the Dpp pathway. The levels of both Brk and Wg determine which of these PD genes is activated. Surprisingly, Brk does not play a role in DV axis specification in the leg, suggesting that Dpp uses two distinct mechanisms for generating the PD and DV axes. Based on these results, we present a model for how Dpp and Wg, which are present as dorsal and ventral gradients, respectively, induce nearly circular domains of gene expression along the PD axis.

The role of buttonhead and Sp1 in the development of the ventral imaginal discs of Drosophila.

The related genes buttonhead (btd) and Drosophila Sp1 (the Drosophila homologue of the human SP1 gene) encode zinc-finger transcription factors known to play a developmental role in the formation of the Drosophila head segments and the mechanosensory larval organs. We report a novel function of btd and Sp1: they induce the formation and are required for the growth of the ventral imaginal discs. They act as activators of the headcase (hdc) and Distal-less (Dll) genes, which allocate the cells of the disc primordia. The requirement for btd and Sp1 persists during the development of ventral discs: inactivation by RNA interference results in a strong reduction of the size of legs and antennae. Ectopic expression of btd in the dorsal imaginal discs (eyes, wings and halteres) results in the formation of the corresponding ventral structures (antennae and legs). However, these structures are not patterned by the morphogenetic signals present in the dorsal discs; the cells expressing btd generate their own signalling system, including the establishment of a sharp boundary of engrailed expression, and the local activation of the wingless and decapentaplegic genes. Thus, the Btd product has the capacity to induce the activity of the entire genetic network necessary for ventral imaginal discs development. We propose that this property is a reflection of the initial function of the btd/Sp1 genes that consists of establishing the fate of the ventral disc primordia and determining their pattern and growth.