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Certain copy number alterations (CNAs) are strongly associated with particular cancer types. However, the mechanisms that underlie selection of specific CNAs remain unknown. Here, we identified functional relationships between recurrent CNAs in colorectal cancers (CRCs) and adenomatous polyposis coli (APC) mutations. Quantitative phenotyping of mitotic spindles highlighted APC functions at centrosomes where APC positively regulated Aurora A kinase (AURKA). Upon APC inactivation, elevated β-catenin levels blocked AURKA activation, which caused chromosome instability and suppressed proliferation, resulting in the generation and selection of AURKA-activating CNAs. Arm-level amplification of chromosomes that contained AURKA and AURKA activator genes was observed in APC-mutant CRCs, early stage mouse tumours, and cells in culture, which was concomitant with an increase in growth potential. Our findings demonstrate a mechanism that restores tumour cell fitness through compensatory chromosome alterations to overcome adverse effects of prior mutations, which may affect the course of cancer type-specific CNA formation.

There is intense interest in developing methods to regulate proliferation and differentiation of stem cells into neuronal fates for the purposes of regenerative medicine. One way to do this is throughin vivopharmacological engineering using small molecules. However, a key challenge is identification of relevant signaling pathways and therein druggable targets to manipulate stem cell behavior efficientlyin vivo. Here, we use the planarian flatworm as a simple chemical-genetic screening model for nervous system regeneration to show that the isoquinoline drug praziquantel (PZQ) acts as a small molecule neurogenic to produce two-headed animals with integrated CNSs following regeneration. Characterization of the entire family of planarian voltage-operated Ca2+channel α subunits (Cavα), followed byin vivoRNAi of specific Cavsubunits, revealed that PZQ subverted regeneration by activation of a specific voltage-gated Ca2+channel isoform (Cav1A). PZQ-evoked Ca2+entry via Cav1A served to inhibit neuronally derived Hedgehog signals, as evidenced by data showing that RNAi of Cav1A prevented PZQ-evoked bipolarity, Ca2+entry, and decreases inwnt1andwnt11-5levels. Surprisingly, the action of PZQ was opposed by Ca2+influx through a closely related neuronal Cavisoform (Cav1B), establishing a novel interplay between specific Cav1 channel isoforms, Ca2+entry, and neuronal Hedgehog signaling. These data map PZQ efficacy to specific neuronal Cavcomplexesin vivoand underscore that both activators (Cav1A) and inhibitors (Cav1B) of Ca2+influx can act as small molecule neurogenicsin vivoon account of the unique coupling of Ca2+channels to neuronally derived polarity cues.

Studies in the Xenopus model system have provided considerable insight into the developmental role of intracellular Ca2+ signals produced by activation of IP3Rs (inositol 1,4,5-trisphosphate receptors). However, unlike mammalian systems where three IP3R subtypes have been well characterized, our molecular understanding of the IP3Rs that underpin Ca2+ signalling during Xenopus embryogenesis relate solely to the original characterization of the ‘Xenopus IP3R’ cloned and purified from Xenopus laevis oocytes several years ago. In the present study, we have identified Xenopus type 2 and type 3 IP3Rs and report the full-length sequence, genomic architecture and developmental expression profile of these additional IP3R subtypes. In the light of the emerging genomic resources and opportunities for genetic manipulation in the diploid frog Xenopus tropicalis, these data will facilitate manipulations to resolve the contribution of IP3R diversity in Ca2+ signalling events observed during vertebrate development.

A pharmacological screen identified the H+ and K+ ATPase transporter as obligatory for normal orientation of the left-right body axis in Xenopus. Maternal H+/K+-ATPase mRNA is symmetrically expressed in the 1-cell Xenopus embryo but becomes localized during the first two cell divisions, demonstrating that asymmetry is generated within two hours postfertilization. Although H+/K+-ATPase subunit mRNAs are symmetrically localized in chick embryos, an endogenous H+/K+-ATPase-dependent difference in membrane voltage potential exists between the left and right sides of the primitive streak. In both species, pharmacologic or genetic perturbation of endogenous H+/K+-ATPase randomized the sided pattern of asymmetrically expressed genes and induced organ heterotaxia. Thus, LR asymmetry determination depends on a very early differential ion flux created by H+/K+-ATPase activity.

Consistent visceral asymmetry in vertebrates raises fascinating questions about the developmental mechanisms and evolutionary origin of fixed chirality of the left-right axis. One persistent controversy is whether consistently biased asymmetry is a later innovation imposed on a bilaterally symmetrical primitive body-plan, or whether asymmetry is a fundamental property predating the bilateria. The morphology of planaria suggests proximity to the origin of the bilateral body-plan, and they are commonly thought to be left-right symmetrical, as no consistent anatomical asymmetries have been described despite over a century of study of regeneration. Here, we show that D. japonica possess a consistent functional asymmetry in eye patterning defects caused by inhibition of H+/K+-ATPase activity (an ion flux mechanism recently shown to be an important early step in the asymmetry of several vertebrate embryos). Moreover, an endogenous transcript of the non-gastric H+/K+-ATPase subunit alpha is expressed in the head blastema shortly after amputation. Taken together, these data suggest that (1) left-right asymmetry is at least as old as planaria, (2) subtle functional asymmetries should be sought in other more primitive model systems that are believed to be symmetrical, and (3) symmetrical paired structures may in fact contain information about their position on the L or R side.

Planaria possess remarkable powers of regeneration. After bisection, one blastema regenerates a head, while the other forms a tail. The ability of previously-adjacent cells to adopt radically different fates could be due to long-range signaling allowing determination of position relative to, and the identity of, remaining tissue. However, this process is not understood at the molecular level. Following the hypothesis that gap-junctional communication (GJC) may underlie this signaling, we cloned and characterized the expression of the Innexin gene family during planarian regeneration. Planarian innexins fall into 3 groups according to both sequence and expression. The concordance between expression-based and phylogenetic grouping suggests diversification of 3 ancestral innexin genes into the large family of planarian innexins. Innexin expression was detected throughout the animal, as well as specifically in regeneration blastemas, consistent with a role in long-range signaling relevant to specification of blastema positional identity. Exposure to a GJC-blocking reagent which does not distinguish among gap junctions composed of different Innexin proteins (is not subject to compensation or redundancy) often resulted in bipolar (2-headed) animals. Taken together, the expression data and the respecification of the posterior blastema to an anteriorized fate by GJC loss-of-function suggest that innexin-based GJC mediates instructive signaling during regeneration.

Hox genes are pivotal molecules in the control of morphogenesis along the anterior-posterior (AP) axis in various bilaterians. Planarians are key animals for understanding the evolution of the bilaterian body plan. Furthermore, they are also known for their strong regeneration ability and are thought to use the Hox genes in the process of reconstruction of the AP axis. In the present paper, the identification and analysis of expression of two posterior (Abdominal-B-like) genes, DjAbd-Ba and DjAbd-Bb, is reported in the planarian Dugesia japonica. DjAbd-Ba is expressed in the entire tail region and its anterior boundary is the posterior pharyngeal region. In contrast, DjAbd-Bb is expressed in several types of cells throughout the body. During regeneration, the expression of DjAbd-Ba rapidly recovers a pattern similar to that in the normal worm. These findings suggest the possibility that DjAbd-Ba is involved in the specification of the tail region. The anterior boundary of the expression domain of the posterior gene DjAbd-Ba is anterior to the domains of the central genes Plox4-Dj and Plox5-Dj. These expression patterns of planarian Hox genes seem out of the rule of spatial colinearity and may reflect an ancestral feature of bilaterian Hox genes.

To elucidate the mechanisms underlying pharynx regeneration in planarians, we transplanted pieces excised from various regions of the body into the prepharyngeal or postpharyngeal region, since it has been shown that such transplantation experiments can induce ectopic pharynx formation. We confirmed the ectopic formation of pharynxes by expression of the myosin heavy chain gene specific to pharynx muscles (DjMHC-A). To investigate the cellular events after grafting, we also stained such transplanted worms by in situ hybridization using neuronal cell- and mucous producing cell-type-specific marker genes which can detect formation of brain and prepharyngeal region, respectively. When the head piece was transplanted into the tail region, ectopic formation of the head, prepharyngeal and pharynx region was observed in the postpharyngeal region anterior to the graft, while these organs were formed in the reversed polarity along the anterior-posterior (A-P) axis. Furthermore, in the tail region posterior to the graft, ectopic formation of the prepharyngeal and pharynx region was observed. In the reverse combination, when a tail piece was transplanted into the prepharyngeal region, ectopic formation of prepharyngeal and pharynx region was observed in the region between the head and the graft, and an additional ectopic pharynx was also formed in reverse polarity in the region between the graft and host pharynx. These results clearly indicated that ectopic pharynxes were formed as a consequence of the regional reorganization induced by interaction between the host and graft. Furthermore, chimeric analyses demonstrated that the cells participating in ectopic pharynx formation were not exclusively derived from the host or donor cells in the worm, suggesting that the stem cells of the host and donor may change their differentiation pattern due to altered regionality. To further investigate if regional reorganization is induced after grafting, expression of a Hox gene was analyzed in the transplanted worms by whole-mount in situ hybridization. The expression of the Hox gene along the A-P axis was apparently rearranged after grafting of the head piece into the tail region. These results suggest that grafting of the head piece may rearrange the regionality of the host tail, and that stem cells in the region newly defined as pharynx-forming may start to regenerate a pharynx.

Recently, the EB1 and XMAP215/TOG families of microtubule binding proteins have been demonstrated to bind autonomously to the growing plus ends of microtubules and regulate their behaviour in in vitro systems. However, their functional redundancy or difference in cells remains obscure. Here, we compared the nanoscale distributions of EB1 and ch-TOG along microtubules using high-resolution microscopy techniques, and also their roles in microtubule organisation in interphase HeLa cells. The ch-TOG accumulation sites protruded ∼100 nm from the EB1 comets. Overexpression experiments showed that ch-TOG and EB1 did not interfere with each other's localisation, confirming that they recognise distinct regions at the ends of microtubules. While both EB1 and ch-TOG showed similar effects on microtubule plus end dynamics and additively increased microtubule dynamicity, only EB1 exhibited microtubule-cell cortex attachment activity. These observations indicate that EB1 and ch-TOG regulate microtubule organisation differently via distinct regions in the plus ends of microtubules.

Concurrent session 3: Mechanisms of developmental patterning Program/Abstract # 18 Dynamic interpretation of the Hedgehog gradient drives wing disc patterning in Drosophila Marcos Nahmad, Angela Stathopoulos Department of CDS, Caltech, Pasadena, CA, USA Div. of Biol., Caltech, Pasadena, CA, USA Morphogens are classically defined as molecules that control patterning by acting at a distance to regulate gene expression in a concentration-dependent manner. In the Drosophila wing imaginal disc, secreted Hedgehog (Hh) forms an extracellular gradient that organizes patterning along the anterior–posterior axis by defining at least three different domains of gene expression. However, a direct spatial correspondence between the boundaries of these patterns and Hh-concentration thresholds has not been demonstrated. We have acquired evidence that the interpretation of Hh signaling depends on the history of exposure to Hh and that a single concentration threshold is sufficient to support multiple outputs. Mathematical modeling and computer simulations suggest that a spatial ‘overshoot’ of the Hh gradient occurs, that is, a transient gradient in which the Hh-concentration profile is expanded compared to the steady-state gradient. This overshoot results from the gene network architecture, in particular, it depends on Hh-dependent up-regulation of the Hh receptor, Patched (Ptc). Through a temporal examination of Hh-target gene expression in vivo, we observe that particular borders of expression shift posteriorly, and subsequently refine. Furthermore, when the network structure is altered such that Ptc transcription is no longer activated in response to Hh signaling, we find that the patterns of gene expression, which have distinct borders in wild-type discs, now in fact overlap. Taken together, our results demonstrate that the overshoot of the Hh gradient is necessary to establish different patterns of gene expression. doi:10.1016/j.ydbio.2009.05.025 Program/Abstract # 19 Planarian anterior–posterior axis is established by interaction of voltage-dependent Ca influx and β-catenin signaling at the early stage of regeneration Taisaku Nogi, Dan Zhang, John D. Chan, Jonathan S. Marchant Department of Pharmacology and The Stem Cell Institute, University of Minnesota Medical School, MN, USA Since the classic experiments of Marsh and Beams in the 1950s, it has been suggested that endogenous voltage gradients play an important role in planarian regeneration. Here, we show voltagedependent Ca influx is a key regulator of anterior–posterior (AP) axis specification during planarian regeneration. Using a pharmacological screen of Ca signaling modulators in the planarian D. japonica, we identified that praziquantel (PZQ) – a voltage-operated Ca channel (VOCC) activator – had a robust effect to yield a bipolar (two-headed) phenotype from regenerating trunk fragments. PZQtreated fragments displayed a mirror-imaged pharynx and posterior, rather than ectopic, head. This effect was Ca-dependent and increased by a variety of depolarizing agents known to couple to VOCCs. Time-window experiments and in situ hybridization of marker gene expression demonstrated PZQ treatment impacted the early stages of regeneration (

Background Approximately 200 million people worldwide harbour parasitic flatworm infections that cause schistosomiasis. A single drug—praziquantel (PZQ)—has served as the mainstay pharmacotherapy for schistosome infections since the 1980s. However, the relevant in vivo target(s) of praziquantel remain undefined. Methods and Findings Here, we provide fresh perspective on the molecular basis of praziquantel efficacy in vivo consequent to the discovery of a remarkable action of PZQ on regeneration in a species of free-living flatworm (Dugesia japonica). Specifically, PZQ caused a robust (100% penetrance) and complete duplication of the entire anterior-posterior axis during flatworm regeneration to yield two-headed organisms with duplicated, integrated central nervous and organ systems. Exploiting this phenotype as a readout for proteins impacting praziquantel efficacy, we demonstrate that PZQ-evoked bipolarity was selectively ablated by in vivo RNAi of voltage-operated calcium channel (VOCC) β subunits, but not by knockdown of a VOCC α subunit. At higher doses of PZQ, knockdown of VOCC β subunits also conferred resistance to PZQ in lethality assays. Conclusions This study identifies a new biological activity of the antischistosomal drug praziquantel on regenerative polarity in a species of free-living flatworm. Ablation of the bipolar regenerative phenotype evoked by PZQ via in vivo RNAi of VOCC β subunits provides the first genetic evidence implicating a molecular target crucial for in vivo PZQ activity and supports the ‘VOCC hypothesis’ of PZQ efficacy. Further, in terms of regenerative biology and Ca2+ signaling, these data highlight a novel role for voltage-operated Ca2+ entry in regulating in vivo stem cell differentiation and regenerative patterning., Author Summary Praziquantel is the major drug used to treat people infected with parasitic worms that cause the neglected tropical disease schistosomiasis. Despite being in widespread clinical use, it is surprising that scientists have not identified how praziquantel works to kill pathogenic schistosomes. This lack of pathobiological insight is a major roadblock to the directed design of new drugs to treat schistosomiasis, as the relevant in vivo target molecule/pathway of praziquantel remains undefined. In this report, we have discovered a new biological activity of praziquantel that enables us to bring a unique chemical genetic perspective to the problem of identifying molecules needed for in vivo praziquantel efficacy. Specifically, we show that praziquantel miscues regenerative patterning in a species of free-living flatworm to yield bipolar (two-headed) organisms. By using this phenotype to screen for molecules underpinning this activity, we provide in vivo support for the ‘Ca2+ channel hypothesis’ of PZQ efficacy, and show that manipulation of specific subunits of voltage-gated Ca2+ channels prevent this effect, and lessen praziquantel-mediated toxicity. These data provide further impetus to studying the role of these proteins in schistosome pharmacotherapy.