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Prostate cancer (PCa) commonly metastasizes to the bone where the cells frequently undergo dormancy. The escape of disseminated tumor cells from cellular dormancy is a major cause of recurrence in marrow. Abscisic acid (ABA), a phytohormone, is known to regulate dormancy of plant seeds and to regulate other stress responses in plants. Recently, ABA was found to be synthesized by mammals cells and has been linked to human disease. Yet the role of ABA in regulating tumor dormancy or reactivation is unknown. We found that ABA is produced by human marrow cells, and exogenous ABA inhibits PCa cell proliferation while increasing the expression of p27, p21, and p16 and decreasing the expression of the proliferation marker, Ki67. Further, ABA significantly increased the percentage of PCa cells in the G0 phase of the cell cycle as well as the duration the cells were arrested in G0. We found that ABA regulates an increase of PPARγ receptor expression and suppressed phosphorylation of mTOR/p70S6K signaling and resulting in the induction of the cellular dormancy. We then confirmed that ABA regulates G0 cell cycle arrest through PPARγ receptor signaling in vitro and under co-culture conditions with osteoblasts. Finally, we demonstrate that ABA regulates PCa dormancy in vivo following intratibial injection in an animal model. Together these data suggest that the ABA and PPARγ signaling pathways contribute to the establishment of PCa cellular dormancy in the bone marrow microenvironment. These findings may suggest critical pathways for targeting metastatic disease.

The Nucleoporin 98KD (Nup98) is one of the most promiscuous translocation partners in hematological malignancies, contributing to at least 31 different truncation-fusion proteins. To date, nearly all disease models of Nup98 translocations involve ectopic expression of transgenes recapitulating the fusion protein under study, leaving the endogenous Nup98 loci unperturbed. Overlooked in these approaches is that translocation leads to the loss of one copy of normal Nup98 in addition to the loss of Nup96 – a second Nucleoporin encoded within the same mRNA and reading frame as Nup98. Nup98 and 96 are also mutated in a number of other cancer types and are located near a tumor suppressor region known to be epigenetically silenced, suggesting that their disruption is not limited to blood cancers. We found that reducing Nup98-96 function via an RNAi approach in Drosophila melanogaster (where the Nup98-96 shared mRNA and reading frame gene structure is conserved) de-regulates the cell cycle. We find evidence of over-proliferation in Nup98-96 deficient tissues, counteracted by elevated apoptosis and aberrant Wingless and JNK signaling associated with chronic wound healing. When the knockdown of Nup98-96 is combined with inhibition of apoptosis, we see synergism leading to dramatic tissue overgrowth, consistent with a tumor-suppressor function for endogenous Nup98 and 96. To understand how growth and proliferation become mis-regulated when Nup98-96 levels are reduced, we performed RNAseq and uncovered a gene expression signature consistent with defects in ribosome biogenesis. We found that reducing Nup 98 and 96 function limits nuclear export of the ribosome component RpL10A, leading to defects in protein synthesis. Defects in protein synthesis are sufficient to trigger JNK signaling that contributes to compensatory proliferation and hallmarks of tumorigenesis when apoptosis is inhibited. Based upon our data, we suggest that the partial loss of Nup98 and Nup96 function in translocations could de-regulate protein synthesis leading to stress signaling that cooperates with other mutations in cancer to promote tumorigenesis.HighlightsCompromising Nups 98 and 96 triggers cell death and compensatory proliferation via JNK signaling that becomes tumorigenic when apoptosis is blockedReducing Nup 98 and 96 function limits nuclear export of the ribosome stalk component RpL10A, leading to defects in protein synthesis which cause stress signaling via JNK.Reduced protein synthesis coupled with increased JNK signaling, paradoxically leads to more rapid proliferation with a gene expression signature that resembles a chronic wounding response.Overexpression of Nup98, which occurs in oncogenic fusions, leads to similar defects in protein synthesis and JNK activation.

Terminally differentiated cells of the nervous system have long been considered to be in a stable non-cycling state and are often considered to be permanently in G0. Exit from the cell cycle during development is often coincident with the differentiation of neurons, and is critical for neuronal function. But what happens in long lived postmitotic tissues that accumulate cell damage or suffer cell loss during aging? In other contexts, cells that are normally non-dividing or postmitotic can or re-enter the cell cycle and begin replicating their DNA to facilitate cellular growth in response to cell loss. This leads to a state called polyploidy, where cells contain multiple copies of the genome. A growing body of literature from several vertebrate and invertebrate model organisms has shown that polyploidy in the nervous system may be more common than previously appreciated and occurs under normal physiological conditions. Moreover, it has been found that neuronal polyploidization can play a protective role when cells are challenged with DNA damage or oxidative stress. By contrast, work over the last two and a half decades has discovered a link between cell-cycle reentry in neurons and several neurodegenerative conditions. In this context, neuronal cell cycle re-entry is widely considered to be aberrant and deleterious to neuronal health. In this review, we highlight historical and emerging reports of polyploidy in the nervous systems of various vertebrate and invertebrate organisms. We discuss the potential functions of polyploidization in the nervous system, particularly in the context of long-lived cells and age-associated polyploidization. Finally, we attempt to reconcile the seemingly disparate associations of neuronal polyploidy with both neurodegeneration and neuroprotection.

Background Disseminated tumor cells (DTCs) have been reported in the bone marrow (BM) of patients with localized prostate cancer (PCa). However, the existence of these cells continues to be questioned, and few methods exist for viable DTC isolation. Therefore, we sought to develop novel approaches to identify and, if detected, analyze localized PCa patient DTCs. Methods We used fluorescence-activated cell sorting (FACS) to isolate a putative DTC population, which was negative for CD45, CD235a, alkaline phosphatase, and CD34, and strongly expressed EPCAM. We examined tumor cell content by bulk cell RNA sequencing (RNA-Seq) and whole-exome sequencing after whole genome amplification. We also enriched for BM DTCs with α-EPCAM immunomagnetic beads and performed quantitative reverse trancriptase polymerase chain reaction (qRT-PCR) for PCa markers. Results At a threshold of 4 cells per million BM cells, the putative DTC population was present in 10 of 58 patients (17%) with localized PCa, 4 of 8 patients with metastatic PCa of varying disease control, and 1 of 8 patients with no known cancer, and was positively correlated with patients' plasma PSA values. RNA-Seq analysis of the putative DTC population collected from samples above (3 patients) and below (5 patients) the threshold of 4 putative DTCs per million showed increased expression of PCa marker genes in 4 of 8 patients with localized PCa, but not the one normal donor who had the putative DTC population present. Whole-exome sequencing also showed the presence of single nucleotide polymorphisms and structural variants in the gene characteristics of PCa in 2 of 3 localized PCa patients. To examine the likely contaminating cell types, we used a myeloid colony formation assay, differential counts of cell smears, and analysis of the RNA-Seq data using the CIBERSORT algorithm, which most strongly suggested the presence of B-cell lineages as a contaminant. Finally, we used EPCAM enrichment and qRT-PCR for PCa markers to estimate DTC prevalence and found evidence of DTCs in 21 of 44 samples (47%). Conclusion These data support the presence of DTCs in the BM of a subset of patients with localized PCa and describe a novel FACS method for isolation and analysis of viable DTCs.

The discovery of anticancer therapeutics effective in eliminating dormant cells is a significant challenge in cancer biology. Here, we describe new synthetic polymer-based anticancer agents that mimic the mode of action of anticancer peptides. These anticancer polymers developed here are designed to capture the cationic, amphiphilic traits of anticancer peptides. The anticancer polymers are designed to target anionic lipids exposed on the cancer cell surfaces and act by disrupting the cancer cell membranes. Because the polymer mechanism is not dependent on cell proliferation, we hypothesized that the polymers were active against dormant cancer cells. The polymers exhibited cytotoxicity to proliferating prostate cancer. Importantly, the polymer killed dormant prostate cancer cells that were resistant to docetaxel. This study demonstrates a new approach to discover novel anticancer therapeutics.

Using a gain-of-function screen in Drosophila, we identified the Kruppel-like factor Cabut (Cbt) as a positive regulator of cell cycle gene expression and cell proliferation. Enforced cbt expression is sufficient to induce an extra cell division in the differentiating fly wing or eye, and also promotes intestinal stem cell divisions in the adult gut. Although inappropriate cell proliferation also results from forced expression of the E2f1 transcription factor or its target, Cyclin E, Cbt does not increase E2F1 or Cyclin E activity. Instead, Cbt regulates a large set of E2F1 target genes independently of E2F1, and our data suggest that Cbt acts via distinct binding sites in target gene promoters. Although Cbt was not required for cell proliferation during wing or eye development, Cbt is required for normal intestinal stem cell divisions in the midgut, which expresses E2F1 at relatively low levels. The E2F1-like functions of Cbt identify a distinct mechanism for cell cycle regulation that may be important in certain normal cell cycles, or in cells that cycle inappropriately, such as cancer cells.

Summary In this issue of Developmental Cell, Cohen et al. show that the Drosophila hindgut is a genetically tractable model for studying tissue regeneration. This tissue exhibits different regeneration strategies at different developmental times, demonstrating that the hindgut developmental clock, not tissue type, dictates the mode and capacity for regeneration.

Long-lived cells such as terminally differentiated postmitotic neurons and glia must cope with the accumulation of damage over the course of an animal’s lifespan. How long-lived cells deal with ageing-related damage is poorly understood. Here we show that polyploid cells accumulate in the ageing adult fly brain and that polyploidy protects against DNA damage-induced cell death. Multiple types of neurons and glia that are diploid at eclosion, become polyploid with age in the adultDrosophilabrain. The optic lobes exhibit the highest levels of polyploidy, associated with an elevated DNA damage response in this brain region with age. Inducing oxidative stress or exogenous DNA damage leads to an earlier onset of polyploidy, and polyploid cells in the adult brain are more resistant to DNA damage-induced cell death than diploid cells. Our results suggest polyploidy may serve a protective role for neurons and glia in ageingDrosophila melanogasterbrains.

While cellular proliferation is fundamental to the development of all multicellular organisms, the slowing or stopping of proliferation at the right places and times is equally important for proper tissue and organ development. The non-cycling state of cellular quiescence or “G0” is relatively understudied compared to proliferation, given its prevalence in nature. It may seem that actively proliferating cells undergo a series of dynamic events, while quiescent cells are in a passive, static state. However, studies over the last 10-15 years suggest that quiescence may be more dynamic than previously thought and must also be actively regulated and maintained. This review focuses on recent advances in understanding quiescence or G0 and in particular, on observations about the proliferation-quiescence decision in cell lines, in tissues and during development. We also discuss novel, advanced molecular tools that are likely to enable the field to address outstanding, unresolved questions about cellular quiescence and its regulation.

During development, cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. Here we examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. We find that a cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa wing. We show that in the wing, ecdysone signaling at the larva-to-puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva-to-puparium pulse during early metamorphosis, Broad expression plummets, allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing., Summary: Pulsed ecdysone signaling remodels cell cycle dynamics, causing distinct primary and secondary cell cycle arrests in Drosophila cells, analogous to those observed in the wing during metamorphosis.

The Drosophila melanogaster male accessory gland is a functional analog of the mammalian prostate containing two secretory epithelial cell types, termed main and secondary cells. This tissue is responsible for making and secreting seminal fluid proteins and other molecules that contribute to successful reproduction. Here we show that similar to the mammalian prostate, this tissue grows with age and can undergo functional regeneration post-damage. We describe a post-eclosion endocycle required for gland maturation, that is dependent on juvenile hormone signaling. In addition, the differentiated binucleated main cells of the adult accessory gland continue to undergo rare endocycles throughout physiological aging to increase DNA content and cell size. Accessory gland cells remain poised to endocycle and upregulation of signals that promote endocycling and tissue growth are sufficient to trigger dramatic increases in cell ploidy. We provide evidence that the main cells of this tissue remain poised to enter the endocycle to assist in tissue regrowth after damage, providing a novel model for postmitotic regeneration in a binucleate tissue.

During terminal differentiation most cells will exit the cell cycle and enter into a prolonged or permanent G0. Cell cycle exit is usually initiated through the repression of cell cycle gene expression by formation of a transcriptional repressor complex called DREAM. However when DREAM repressive function is compromised during terminal differentiation, additional unknown mechanisms act to stably repress cycling and ensure robust cell cycle exit. Here we provide evidence that developmentally programmed, temporal changes in chromatin accessibility at a subset of critical cell cycle genes acts to enforce cell cycle exit during terminal differentiation in the Drosophila melanogaster wing. We show that during terminal differentiation, chromatin closes at a set of pupal wing enhancers for the key rate-limiting cell cycle regulators cycE, e2f1 and stg. This closing coincides with wing cells entering a robust postmitotic state that is strongly refractory to cell cycle re-activation. When cell cycle exit is genetically disrupted, chromatin accessibility at cell cycle genes remains largely unaffected and the closing of enhancers at cycE, e2f1 and stg proceeds independent of the cell cycling status. Instead, disruption of cell cycle exit leads to changes in accessibility and expression of a subset of hormone-induced transcription factors involved in the progression of terminal differentiation. Our results uncover a mechanism that acts as a cell cycle-independent timer to limit aberrant cycling in terminally differentiating tissues. In addition, we provide a new molecular description of the cross-talk between cell cycle exit and terminal differentiation during metamorphosis.

Many barriers discourage underrepresented students from pursuing science careers. To access graduate education, undergraduate students must first gain exposure to a particular subject and subsequently accumulate related coursework and research experience. Many underrepresented students lack exposure to developmental biology due to limited undergraduate course offerings and finite resources at smaller institutions. To address this disparity, a group of University of Michigan graduate students and postdoctoral fellows created a portable short course focusing on developmental biology, titled “Developing Future Biologists” (DFB). This weeklong educational initiative provides hands-on laboratory sessions, interactive lectures, and professional development workshops to teach students about developmental biology and increase awareness of scientific career options. To evaluate course effectiveness, we developed a pre-post assessment, incorporating main ideas from the BioCore Guide. Student understanding of basic concepts and perceived experience in developmental biology increased in DFB participants, despite the abbreviated nature of the course. Here, we provide all course materials and an in-depth analysis of the assessment we created. The DFB portable short course model is an easily adaptable tool that connects undergraduate students with opportunities for advanced study and lowers barriers for underrepresented students in science, technology, engineering, and mathematics.

Prostate cancer (PCa) is known to develop resistance to chemotherapy. Growth arrest-specific 6 (GAS6), plays a role in tumor progression by regulating growth in many cancers. Here, we explored how GAS6 regulates the cell cycle and apoptosis of PCa cells in response to chemotherapy. We found that GAS6 is sufficient to significantly increase the fraction of cells in G1 and the duration of phase in PCa cells. Importantly, the effect of GAS6 on G1 is potentiated during docetaxel chemotherapy. GAS6 altered the levels of several key cell cycle regulators, including the downregulation of Cyclin B1 (G2 /M phase), CDC25A, Cyclin E1, and CDK2 (S phase entry), while the upregulation of cell cycle inhibitors p27 and p21, Cyclin D1, and CDK4. Importantly, these changes became further accentuated during docetaxel treatment in the presence of GAS6. Moreover, GAS6 alters the apoptotic response of PCa cells during docetaxel chemotherapy. Docetaxel induced PCa cell apoptosis is efficiently suppressed in PCa cell culture in the presence of GAS6 or GAS6 secreted from co-cultured osteoblasts. Similarly, the GAS6-expressing bone environment protects PCa cells from apoptosis within primary tumors in vivo studies. Docetaxel induced significant levels of Caspase-3 and PARP cleavage in PCa cells, while GAS6 protected PCa cells from docetaxel-induced apoptotic signaling. Together, these data suggest that GAS6, expressed by osteoblasts in the bone marrow, plays a significant role in the regulation of PCa cell survival during chemotherapy, which will have important implications for targeting metastatic disease. J. Cell. Biochem. 117: 2815-2824, 2016. © 2016 Wiley Periodicals, Inc.

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo. However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed Drosophila, Rbf, E2F and Myb/Multi-vulva class B (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression.

// Younghun Jung 1 , Ann M. Decker 1 , Jingcheng Wang 1 , Eunsohl Lee 1 , Lulia A. Kana 1 , Kenji Yumoto 1 , Frank C. Cackowski 1, 2 , James Rhee 1 , Peter Carmeliet 3, 4 , Laura Buttitta 5 , Todd M. Morgan 6 , Russell S. Taichman 1 1 Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI, USA 2 Department of Internal Medicine, Division of Hematology and Oncology, University of Michigan School of Medicine, Ann Arbor, MI, USA 3 Laboratory of Angiogenesis and Vascular Metabolism, Vesalius Research Center (VRC), VIB, K.U. Leuven, Belgium 4 Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, K.U. Leuven, Belgium 5 Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA 6 Department of Urology, University of Michigan School of Medicine, Ann Arbor, MI, USA Correspondence to: Russell S. Taichman, e-mail: rtaich@umich.edu Keywords: endogenous GAS6, Mer, prostate cancer, cancer stem cells, bone marrow Received: December 01, 2015 Accepted: March 07, 2016 Published: March 25, 2016 ABSTRACT GAS6 and its receptors (Tryo 3, Axl, Mer or “TAM”) are known to play a role in regulating tumor progression in a number of settings. Previously we have demonstrated that GAS6 signaling regulates invasion, proliferation, chemotherapy-induced apoptosis of prostate cancer (PCa) cells. We have also demonstrated that GAS6 secreted from osteoblasts in the bone marrow environment plays a critical role in establishing prostate tumor cell dormancy. Here we investigated the role that endogenous GAS6 and Mer receptor signaling plays in establishing prostate cancer stem cells in the bone marrow microenvironment. We first observed that high levels of endogenous GAS6 are expressed by disseminated tumor cells (DTCs) in the bone marrow, whereas relatively low levels of endogenous GAS6 are expressed in PCa tumors grown in a s.c. setting. Interestingly, elevated levels of endogenous GAS6 were identified in putative cancer stem cells (CSCs, CD133+/CD44+) compared to non-CSCs (CD133–/CD44–) isolated from PCa/osteoblast cocultures in vitro and in DTCs isolated from the bone marrow 24 hours after intracardiac injection. Moreover, we found that endogenous GAS6 expression is associated with Mer receptor expression in growth arrested (G 1 ) PCa cells, which correlates with the increase of the CSC populations. Importantly, we found that overexpression of GAS6 activates phosphorylation of Mer receptor signaling and subsequent induction of the CSC phenotype in vitro and in vivo . Together these data suggest that endogenous GAS6 and Mer receptor signaling contribute to the establishment of PCa CSCs in the bone marrow microenvironment, which may have important implications for targeting metastatic disease.

While cellular proliferation is fundamental to the development of all multicellular organisms, the slowing or stopping of proliferation at the right places and times is equally important for proper tissue and organ development. The non-cycling state of cellular quiescence or "G

Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. Here, we show that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the Drosophila wing, we demonstrate that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. We also uncover a temporal cascade of transcription factors following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, we show that the ecdysone-induced transcription factor E93 controls temporal identity by directly regulating chromatin accessibility across the genome. Notably, we found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility.

Cancer stem cells likely survive chemotherapy or radiotherapy by acquiring mutations that inactivate the endogenous apoptotic machinery or by cycling slowly. Thus, knowledge about the mechanisms linking the activation of an alternative cell death modality and the cell cycle machinery could have a transformative impact on the development of new cancer therapies, but the mechanisms remain completely unknown. We investigated the regulation of alternative cell death in Drosophila larval brain neural stem cells (neuroblasts) in which apoptosis is normally repressed. From a screen, we identified two novel loss-of-function alleles of the Cdc20/fizzy (fzy) gene that lead to premature brain neuroblast loss without perturbing cell proliferation in other diploid cell types. Fzy is an evolutionarily conserved regulator of anaphase promoting complex/cyclosome (APC/C). Neuroblasts carrying the novel fzy allele or exhibiting reduced APC/C function display hallmarks of necrosis. By contrast, neuroblasts overexpressing the non-degradable form of canonical APC/C substrates required for cell cycle progression undergo mitotic catastrophe. These data strongly suggest that Fzy can elicit a novel pro-survival function of APC/C by suppressing necrosis. Neuroblasts experiencing catastrophic cellular stress, or overexpressing p53, lose Fzy expression and undergo necrosis. Co-expression of fzy suppresses the death of these neuroblasts. Consequently, attenuation of the Fzy-dependent survival mechanism functions downstream of catastrophic cellular stress and p53 to eliminate neuroblasts by necrosis. Strategies that target the Fzy-dependent survival mechanism might lead to the discovery of new treatments or complement the pre-existing therapies to eliminate apoptosis-resistant cancer stem cells by necrosis.

Background: The Decapentaplegic (Dpp) signaling pathway is used in many developmental and homeostatic contexts, each time resulting in cellular responses particular to that biological niche. The flexibility of Dpp signaling is clearly evident in epithelial cells of the Drosophila wing imaginal disc. During larval stages of development, Dpp functions as a morphogen, patterning the wing developmental field and stimulating tissue growth. A short time later, however, as wing-epithelial cells exit the cell cycle and begin to differentiate, Dpp is a critical determinant of vein-cell fate. It is likely that the Dpp signaling pathway regulates different sets of target genes at these two developmental time points. Results: To identify mechanisms that temporally control the transcriptional output of Dpp signaling in this system, we have taken a gene expression profiling approach. We identified genes affected by Dpp signaling at late larval or early pupal developmental time points, thereby identifying patterning- and differentiation-specific downstream targets, respectively. Conclusions: Analysis of target genes and transcription factor binding sites associated with these groups of genes revealed potential mechanisms by which target-gene specificity of the Dpp signaling pathway is temporally regulated. In addition, this approach revealed novel mechanisms by which Dpp affects the cellular differentiation of wing-veins. Developmental Dynamics 243:818–832, 2014. © 2014 Wiley Periodicals, Inc.

During terminal differentiation, most cells exit the cell cycle and enter into a prolonged or permanent G0 in which they are refractory to mitogenic signals. Entry into G0 is usually initiated through the repression of cell cycle gene expression by formation of a transcriptional repressor complex called dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM). However, when DREAM repressive function is compromised during terminal differentiation, additional unknown mechanisms act to stably repress cycling and ensure robust cell cycle exit. Here, we provide evidence that developmentally programmed, temporal changes in chromatin accessibility at a small subset of critical cell cycle genes act to enforce cell cycle exit during terminal differentiation in the Drosophila melanogaster wing. We show that during terminal differentiation, chromatin closes at a set of pupal wing enhancers for the key rate-limiting cell cycle regulators Cyclin E (cycE), E2F transcription factor 1 (e2f1), and string (stg). This closing coincides with wing cells entering a robust postmitotic state that is strongly refractory to cell cycle reactivation, and the regions that close contain known binding sites for effectors of mitogenic signaling pathways such as Yorkie and Notch. When cell cycle exit is genetically disrupted, chromatin accessibility at cell cycle genes remains unaffected, and the closing of distal enhancers at cycE, e2f1, and stg proceeds independent of the cell cycling status. Instead, disruption of cell cycle exit leads to changes in accessibility and expression of a subset of hormone-induced transcription factors involved in the progression of terminal differentiation. Our results uncover a mechanism that acts as a cell cycle–independent timer to limit the response to mitogenic signaling and aberrant cycling in terminally differentiating tissues. In addition, we provide a new molecular description of the cross talk between cell cycle exit and terminal differentiation during metamorphosis., The longer a cell remains in G0, the more refractory it becomes to re-entering the cell cycle. This study shows that in terminally differentiated cells in vivo, regulatory elements at genes encoding just three key cell cycle regulators (cycE, e2f1 and stg) become inaccessible, limiting their aberrant activation and maintaining a prolonged, robust G0.

Prostate cancer (PCa) is known to develop resistance to chemotherapy. Growth arrest-specific 6 (GAS6), plays a role in tumor progression by regulating growth in many cancers. Here, we explored how GAS6 regulates the cell cycle and apoptosis of PCa cells in response to chemotherapy. We found that GAS6 is sufficient to significantly increase the number and duration of G1 phase in PCa cells. Importantly, GAS6 further increased the number of G1 arrested cells during docetaxel chemotherapy. GAS6 altered the signals of key cell cycle regulators: Cyclin B1 (G2/M phase), CDC25A, Cyclin E1, and CDK2 (S phase entry) were all downregulated, while p27, p21, Cyclin D1, and CDK4 (G0/G1 phase) were upregulated. Importantly, these signaling events were further accentuated during docetaxel treatment in the presence of GAS6. Moreover, the apoptotic response of PCa cells to GAS6 was examined during docetaxel chemotherapy. Docetaxel induced PCa cell apoptosis. However, this apoptotic response was abrogated in PCa cell cultures in the presence of GAS6 or GAS6 secreted from co-cultured osteoblasts. Similarly, the GAS6-expressing bone environment protects PCa cells from apoptosis within primary tumors in vivo studies. In addition, docetaxel induced significant levels of Caspase-3 and PARP cleavages in PCa cells, while GAS6 protected PCa cells from docetaxel-induced apoptotic signaling. Together, these data suggest that GAS6, expressed by osteoblasts in the bone marrow, plays a significant role in the regulation of PCa cell survival during chemotherapy, which may have important implications for targeting metastatic disease.

The complexity and plasticity of cell fate determination has intrigued cell and developmental biologists for decades. Cellular differentiation is the acquisition of specialized characteristics; which is intimately associated with changes in gene expression, alterations of chromatin, and changes in nuclear architecture. Differentiating tissues exhibit a progressive restriction of cellular plasticity. However, the regenerative ability of some organisms has revealed an amazing capacity for dramatic switches in cell fate, through trans-differentiation and de-differentiation (Sanchez Alvarado and Tsonis, 2006). Furthermore, the groundbreaking work on somatic cell nuclear reprogramming and induced pluripotency has revealed that commitment to cell fate can be far more flexible than previously thought (Lensch and Mummery, 2013). In this research topic on cell fate we aimed to highlight new developments and outstanding questions in our understanding of how chromatin dynamics impact cell fate and cellular reprogramming. We include articles discussing cell fate decisions in a wide variety of contexts and model organisms. The contributions to this topic include review articles, mini-reviews, original research, and perspectives. The work described here encompasses organisms ranging from C. elegans to humans and deals with global cell fate issues of sex determination (Lau and Csankovszki), lineage choice (Chin), preventing premature differentiation (Foret et al.) cell fate and cell cycle regulation (Oyama et al.; Julian and Blais; Ma et al.; Parker), nuclear architecture (Talamas and Capelson) and how dynamic transcriptional repressors promote cell fate choices (Kok and Arnosti). We thank the authors, reviewers and editors for contributing to the stimulating discussion of the open questions in this rapidly changing field.

The miR-200 microRNA family plays important tumor suppressive roles. The sole Drosophila miR-200 ortholog, miR-8 plays conserved roles in Wingless, Notch and Insulin signaling - pathways linked to tumorigenesis, yet homozygous null animals are viable and often appear morphologically normal. We observed that wing tissues mosaic for miR-8 levels by genetic loss or gain of function exhibited patterns of cell death consistent with a role for miR-8 in modulating cell survival in vivo. Here we show that miR-8 levels impact several actin cytoskeletal regulators that can affect cell survival and epithelial organization. We show that loss of miR-8 can confer resistance to apoptosis independent of an epithelial to mesenchymal transition while the persistence of cells expressing high levels of miR-8 in the wing epithelium leads to increased JNK signaling, aberrant expression of extracellular matrix remodeling proteins and disruption of proper wing epithelial organization. Altogether our results suggest that very low as well as very high levels of miR-8 can contribute to hallmarks associated with cancer, suggesting approaches to increase miR-200 microRNAs in cancer treatment should be moderate.

Overexpression of both CycE and E2F is necessary to trigger cell cycle reentry and overproliferation of terminally differentiated wing cells., Terminally differentiated cells in Drosophila melanogaster wings and eyes are largely resistant to proliferation upon deregulation of either E2F or cyclin E (CycE), but exogenous expression of both factors together can bypass cell cycle exit. In this study, we show this is the result of cooperation of cell cycle control mechanisms that limit E2F-CycE positive feedback and prevent cycling after terminal differentiation. Aberrant CycE activity after differentiation leads to the degradation of E2F activator complexes, which increases the proportion of CycE-resistant E2F repressor complexes, resulting in stable E2F target gene repression. If E2F-dependent repression is lost after differentiation, high anaphase-promoting complex/cyclosome (APC/C) activity degrades key E2F targets to limit cell cycle reentry. Providing both CycE and E2F activities bypasses exit by simultaneously inhibiting the APC/C and inducing a group of E2F target genes essential for cell cycle reentry after differentiation. These mechanisms are essential for proper development, as evading them leads to tissue outgrowths composed of dividing but terminally differentiated cells.

Since the earliest observations of cells undergoing mitosis, it has been clear that there is an intimate relationship between the cell cycle and nuclear chromatin architecture. The nuclear envelope and chromatin undergo robust assembly and disassembly during the cell cycle, and transcriptional and post-transcriptional regulation of histone biogenesis and chromatin modification is controlled in a cell cycle-dependent manner. Chromatin binding proteins and chromatin modifications in turn influence the expression of critical cell cycle regulators, the accessibility of origins for DNA replication, DNA repair, and cell fate. In this review we aim to provide an integrated discussion of how the cell cycle machinery impacts nuclear architecture and vice-versa. We highlight recent advances in understanding cell cycle-dependent histone biogenesis and histone modification deposition, how cell cycle regulators control histone modifier activities, the contribution of chromatin modifications to origin firing for DNA replication, and newly identified roles for nucleoporins in regulating cell cycle gene expression, gene expression memory and differentiation. We close with a discussion of how cell cycle status may impact chromatin to influence cell fate decisions, under normal contexts of differentiation as well as in instances of cell fate re-programming.

Protein phosphatase type 2A complex (PP2A) has been known as a tumor suppressor for over two decades, but it remains unclear exactly how it suppresses tumor growth. Here we provide data indicating a novel role for PP2A in promoting the transition to quiescence upon terminal differentiation in vivo. Using Drosophila eyes and wings as a model, we find that compromising PP2A activity during the final cell cycle prior to a developmentally controlled cell cycle exit leads to extra cell divisions and delayed entry into quiescence. By systematically testing the regulatory subunits of Drosophila PP2A, we find that the B56 family member widerborst (wdb) is required for the role of PP2A in promoting the transition to quiescence. Cells in differentiating tissues with compromised PP2A retain high Cdk2 activity when they should be quiescent, and genetic epistasis tests demonstrate that ectopic CyclinE/Cdk2 activity is responsible for the extra cell cycles caused by PP2A inhibition. The loss of wdb/PP2A function cooperates with aberrantly high Cyclin E protein, allowing cells to bypass a robust G0 late in development. This provides an example of how loss of PP2A can cooperate with oncogenic mutations in cancer. We propose that the wdb/PP2A complex plays a novel role in differentiating tissues to promote developmentally controlled quiescence through the regulation of CyclinE/Cdk2 activity.

Sonic hedgehog (Shh) signaling is essential for sclerotome development in the mouse. Gli2 and Gli3 are thought to be the primary transcriptional mediators of Shh signaling; however, their roles in Shh induction of sclerotomal genes have not been investigated. Using a combination of mutant analysis and in vitro explant assays, we demonstrate that Gli2 and Gli3 are required for Shh-dependent sclerotome induction. Gli2 –/– Gli3 –/– embryos exhibit a severe loss of sclerotomal gene expression, and somitic mesoderm from these embryos cannot activate sclerotomal genes in response to exogenous Shh. We find that one copy of either Gli2 or Gli3 is required to mediate Shh induction of sclerotomal markers Pax1 and Pax9 in vivo and in vitro. Although Gli2 is generally considered an activator and Gli3 a repressor, our results also reveal a repressor function for Gli2 and an activator function for Gli3 in the developing somite. To further dissect the function of each Gli, we used adenovirus to overexpress Gli1, Gli2 and Gli3 in presomitic mesoderm explants. We find that each Gli preferentially activates a distinct set of Shh target genes, suggesting that the functions of Shh in patterning, growth and negative feedback are divided preferentially between different Gli proteins in the somite.

We have developed a new computational approach to track and analyze data from cells engineered with fluorescent cell cycle gene reporters. Gene reporters are a rapidly growing tool to gather information captured with time-lapse imaging, and can give insights to real-time metabolic conditions, cell cycle state, cancer stem cell state, and many other applications. Gene reporters may reflect cellular conditions that are cyclical rather than monotonic. Currently, the primary mechanism for interpretation of time lapse gene reporter data is reporting the mean fluorescence of images over time. This simplistic analysis discards information such as the cycle length and variation between of gene reporters between cells, and can be biased due to the overall fluorescence shift from population growth. More nuanced data acquisition and analysis approaches have been limited due to requirement of laborious hand-on data analysis. We have implemented a high-throughput computational pipeline for interpretation of cell cycle information using a combination of commercial and newly developed software to partition the activities of individual cells. The fluorescence information for each cell is smoothed and the cycle states for each cell are assigned adaptively. Our approach captures and quantifies the total time each individual cell spends in each presumptive state, and then analyses state information from the cohort of observed cells using censored survival time data methods such as the Cox Proportional Hazards Model to account for cell death or movement out of the observed area. As a proof of concept for our pipeline, we engineered 3T3 fibroblast cells with a cell cycle reporter construct including mCherry-CDT1 and mVenous-p27K- reporters which can discriminate cells in G0 phase from G1 phase. We calculated the sample size required for statistical power of 80% to detect a hazard ratio (HR) of 0.8 between groups at α = 0.05 at 386 tracked cells per group. As our treatment model, we used the Protein Phosphatase 2A inhibitor Okadaic Acid (OA) an agent known to promote departure from G0. We captured a distinct total of 601 vehicle cells and 479 treatment cells in a 24 hour monitoring period with 20 minute capture intervals. We were able to discern subtle differences between control and treatment G0 time, including a subtle, statistically significant decrease in G0 time after treatment with OA (p=0.0166, HR = 0.84). Here we have developed a computational pipeline with the capability for high-throughput image-stack data processing and nuanced quantification. Our approach assigns cycling cell states adaptively and the time until each cell departs its current state is tallied and interpreted statistically with survival time statistical analysis. The cell-level analysis affords a high degree of statistical power to discriminate group differences. Our pipeline is highly adaptable and has a multitude of applications in cancer biology research. Citation Format: Alexander T. Pearson, Dan Sun, Zhengda Li, Jacques Nor, Qiong Yang, Laura Buttitta. A computational and statistical approach for interpreting real-time in-vitro gene reporter data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 880. doi:10.1158/1538-7445.AM2017-880

Dorsoventral polarity of the somitic mesoderm is established by competitive signals originating from adjacent tissues. The ventrally located notochord provides the ventralizing signals to specify the sclerotome, while the dorsally located surface ectoderm and dorsal neural tube provide the dorsalizing signals to specify the dermomyotome. Noggin and SHH-N have been implicated as the ventralizing signals produced by the notochord. Members of the WNT family of proteins, on the other hand, have been implicated as the dorsalizing signals derived from the ectoderm and dorsal neural tube. When presomitic explants are confronted with cells secreting SHH-N and WNT1 simultaneously, competition to specify the sclerotome and dermomyotome domains within the naive mesoderm can be observed. Here, using these explant cultures, we provide evidence that SHH-N competes with WNT1, not only by upregulating its own receptor Ptc1, but also by upregulating Sfrp2 (Secreted frizzled-related protein 2), which encodes a potential WNT antagonist. Among the four known Sfrps, Sfrp2 is the only member expressed in the sclerotome and upregulated by SHH-N recombinant protein. We further show that SFRP2-expressing cells can reduce the dermomyotome-inducing activity of WNT1 and WNT4, but not that of WNT3a. Together, our results support the model that SHH-N at least in part employs SFRP2 to reduce WNT1/4 activity in the somitic mesoderm.

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

Background The transformation of a developing epithelium into an adult structure is a complex process, which often involves coordinated changes in cell proliferation, metabolism, adhesion, and shape. To identify genetic mechanisms that control epithelial differentiation, we analyzed the temporal patterns of gene expression during metamorphosis of the Drosophila wing. Results We found that a striking number of genes, approximately 50% of the Drosophila transcriptome, exhibited changes in expression during a time course of wing development. While cis-acting enhancer sequences clearly correlated with these changes, a stronger correlation was discovered between core-promoter types and the dynamic patterns of gene expression within this differentiating tissue. In support of the hypothesis that core-promoter type influences the dynamics of expression, expression levels of several TATA-box binding protein associated factors (TAFs) and other core promoter-associated components changed during this developmental time course, and a testes-specific TAF (tTAF) played a critical role in timing cellular differentiation within the wing. Conclusions Our results suggest that the combinatorial control of gene expression via cis-acting enhancer sequences and core-promoter types, determine the complex changes in gene expression that drive morphogenesis and terminal differentiation of the Drosophila wing epithelium.

Coordinating terminal differentiation with permanent exit from the cell cycle is critical for proper organogenesis, yet how the cell cycle is blocked in differentiated tissues remains unclear. Important roles for Retinoblastoma family proteins and Cyclin-dependent kinase inhibitors have been delineated, but in many cases it remains unclear what triggers cell cycle exit. This review focuses on describing recent advances in deciphering how terminal differentiation and exit from the cell cycle are coordinated.

Terminal differentiation is often coupled with permanent exit from the cell cycle, yet it is unclear how cell proliferation is blocked in differentiated tissues. We examined the process of cell cycle exit in Drosophila wings and eyes and discovered that cell cycle exit can be prevented or even reversed in terminally differentiating cells by the simultaneous activation of E2F1 and either Cyclin E/Cdk2 or Cyclin D/Cdk4. Enforcing both E2F and Cyclin/Cdk activities is required to bypass exit because feedback between E2F and Cyclin E/Cdk2 is inhibited after cells differentiate, ensuring that cell cycle exit is robust. In some differentiating cell types (e.g., neurons), known inhibitors including the retinoblastoma homolog Rbf and the p27 homolog Dacapo contribute to parallel repression of E2F and Cyclin E/Cdk2. In other cell types, however (e.g., wing epithelial cells), unknown mechanisms inhibit E2F and Cyclin/Cdk activity in parallel to enforce permanent cell cycle exit upon terminal differentiation.

WNT signaling plays a major role in patterning the dermomyotome of the somitic mesoderm. However, knowledge of downstream target genes and their regulation is limited. To identify new genes involved in the development and early patterning of the somite, we performed a comparison of gene expression by microarray between the presomitic mesoderm and the 5 most recently formed somites of the mouse at embryonic day 9.5. We identified 207 genes upregulated and 120 genes downregulated in somite formation. Expression analysis and functional categorization of these genes demonstrate this to be a diverse pool that provides a valuable resource for studying somite development. Thus far, we have found three genes expressed in the dermomyotome of the early somite. Consistent with their expression patterns, these genes are transcriptional targets of WNT signals, but display differential activation by different WNTs. We further demonstrate that 1 of these genes, Troy, is a direct target of canonical WNT signaling, while the other 2 genes, Selp and Arl4, are not. Thus, our microarray study using microdissected tissues not only provides global information on gene expression during somite development, it also provides novel targets to study the inductive signaling pathways that direct somite patterning.

Heterogeneity in genetic networks across different signaling molecular contexts can suggest molecular regulatory mechanisms. Here we describe a comparative chi-square analysis (CPχ2) method, considerably more flexible and effective than other alternatives, to screen large gene expression data sets for conserved and differential interactions. CPχ2 decomposes interactions across conditions to assess homogeneity and heterogeneity. Theoretically, we prove an asymptotic chi-square null distribution for the interaction heterogeneity statistic. Empirically, on synthetic yeast cell cycle data, CPχ2 achieved much higher statistical power in detecting differential networks than alternative approaches. We applied CPχ2 to Drosophila melanogaster wing gene expression arrays collected under normal conditions, and conditions with overexpressed E2F and Cabut, two transcription factor complexes that promote ectopic cell cycling. The resulting differential networks suggest a mechanism by which E2F and Cabut regulate distinct gene interactions, while still sharing a small core network. Thus, CPχ2 is sensitive in detecting network rewiring, useful in comparing related biological systems.

The dorsal–ventral polarity of the somite is controlled by antagonistic signals from the dorsal neural tube/surface ectoderm, mediated by WNTs, and from the ventral notochord, mediated by sonic hedgehog (SHH). Each factor can act over a distance greater than a somite diameter in vitro , suggesting they must limit each other's actions within their own patterning domains in vivo . We show here that the growth-arrest specific gene 1 ( Gas1 ), which is expressed in the dorsal somite, is induced by WNTs and encodes a protein that can bind to SHH. Furthermore, ectopic expression of Gas1 in presomitic cells attenuates the response of these cells to SHH in vitro . Taken together, these data suggest that GAS1 functions to reduce the availability of active SHH within the dorsal somite.

Emerging evidence strongly suggests that stem cells and their differentiated progeny display distinct metabolic profiles, but how metabolic changes are coupled with organogenesis has remained unclear. Homem et al. (2014) now reveal a hormone-dependent pathway that couples metabolic changes with stem cell differentiation, thereby terminating neurogenesis in the Drosophila brain.