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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.

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.

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 (