Program/Abstract # 397Heterotaxin: A novel TGF-beta signaling inhibitor identified in amulti-phenotype profiling screen in Xenopus embryosNanette M. Nascone-Yodera, Michael Dusha, Andrew McIverc,Meredith Parra, Douglas Youngc, Julie Fisherb,Marlene Hauckb, Alexander DeiterscaDept. of Molecular Biomedical Sciences, College of Veterinary Medicine,USAbDept. of Clinical Science, College of Veterinary Medicine, USAcDept. of Chemistry, North Carolina State University, Raleigh, NC 27606,USA

Disruptions of anatomical left–right asymmetry result in life-threaten-ing heterotaxic birth defects in vital organs. We performed a smallmolecule screen for left–right asymmetryphenotypes inXenopusembryosand discovered a novel pyridine analog, heterotaxin, which disrupts bothcardiovascular and digestive organ laterality and inhibits TGF-β-depen-dent left–right asymmetric gene expression. Heterotaxin analogs alsoperturb vascular development, melanogenesis, cell migration and adhe-sion, and inhibit the phosphorylation of Smad2, an intracellular mediatorof TGF-β receptor activation. This combined phenotypic profile identifiesthese compounds as a novel class of TGF-β signaling inhibitors. Notably,heterotaxin analogs also inhibit angiogenesis in human cell culture,revealing their broad applicability. As TGF-β inhibitors are excellentcandidates for anti-fibrotic or anti-metastatic drugs, our discovery of anew class of inhibitors with demonstrated in vivo efficacy may lead toimportant new therapeutics. Our results also illustrate that embryonicphenotypic profiling, in which multiple organ, tissue, cellular andmolecular parameters are assessed in a whole organism context, is avaluable strategy for identifying the mechanism of action of novelcompounds. Finally, this study reveals the utility of frog embryos as anemerging model for chemical genomics and in vivo drug discovery.

doi:10.1016/j.ydbio.2010.05.483

Program/Abstract # 398Transmembrane voltage gradient in GlyR-expressing nichecells controls behavior of neural crest derivatives in vivoDouglas Blackiston, Dany Adams, Joan Lemire, Michael LevinDept. of Biol., Tufts University, Medford, MA, USA

Understanding the mechanisms that guide stem cell behavior duringcomplex morphogenesis is a high priority for developmental biology,regenerative medicine, and oncology. Like chemical cues, endogenousbioelectric signals are important regulators of morphogenesis. Weexploited the native glycine receptor chloride channel (GlyR) toinvestigate the role of niche cells' transmembrane potential in controllingembryonic stem cell function in Xenopus laevis. The neural crest gives riseto a number of tissue types includingmelanocytes, the pigmented cells ofthe epidermis. Molecular-genetic or pharmacological depolarization of asparse, widely-distributed set of GlyR-expressing cells confers a neoplas-tic-like phenotype on distant melanocytes: they overproliferate, acquirean arborized cell shape, and migrate inappropriately, invading numeroustissues in a metalloprotease-dependent fashion. A similar effect isobserved in human melanocytes in culture. The pathway linking depo-larization of GlyR-expressing cells to metastatic behavior in melanocytesinvolves increase of extracellular serotonin levels by SERT and the up-regulation of Slug and Sox10. These data identify GlyR as a unique markerof cells with the ability to instruct neural crest, reveal a novel non-cell-autonomous aspect of the stemcell-cancer cell transitionbased onvoltagegradients, identify a new role for non-neural serotonin, and suggest newstrategies for manipulating embryonic stem cell behavior.

doi:10.1016/j.ydbio.2010.05.484

Program/Abstract # 399Effects of methyl mercury (MeHg) on neural development in X.laevis and in regenerating planariaMaitreyi D. Nagarkar, Margaret S. SahaDept. of Biology, College of William and Mary, VA, USA

Environmental mercury is known to have severe neurologicaleffects during development. In humans, fetal exposure can occurthrough the mother's consumption of seafood, and very smallconcentrations of methyl mercury are sufficient to cause subtledevelopmental changes. However, the effects of chronic low levelsof mercury exposure on neural development have not beenadequately characterized. Here we use Xenopus laevis as well asplanarian species to analyze in more detail the effects of MeHg ondevelopment and regeneration, in particular of neurotransmitterpathways. Work from other laboratories has demonstrated thatcertain pathways essential to X. laevis neural development,including Notch signaling, as well as dopaminergic and GABAergicpathways, are functionally disrupted following MeHg exposure.Preliminary results indicate that X. laevis embryos that have beenexposed to increasing concentrations of MeHg-containing mediadevelop almost (morphologically) normally below 0.1 ppm, butexperience an almost complete arrest of development at MeHgconcentrations even slightly higher than this. Exposure at levelsintermediate between 0.1 ppm and 0.01 ppm in conjunction withanalysis of neural markers will demonstrate which neurotransmit-ter phenotypes and developmental pathways are being affected byMeHg exposure. Additionally, the use of regeneration experimentsin planarian species, and subsequent neural expression analysis,will offer an important comparative perspective.

doi:10.1016/j.ydbio.2010.05.485

Program/Abstract # 400Using zebrafish to understand the neurodevelopment role ofsusceptibility genes for autism spectrum disorderBrian KeySchool of Biomedical Sciences, University of Queensland, Australia

Several studies in the last three years have revealed that membersof a synaptic cell adhesion network are candidate susceptibility genesfor autism spectrum disorder (ASD). ASD is increasingly attributed toa disorder of brain function rather than brain anatomy. We havebegun to address the role of gene–gene interactions within thesynaptic cell adhesion pathway involved in neural circuits associatedwith simple behaviours using the zebrafish animal model. We arefocusing on interactions between identified susceptibility genesNLGN-1, NLGN-4, NRXN-1α, Shank3 and CNTNAP2 as well as oninteractions of these genes with other known synaptic cell adhesionpathway genes (LRRTM2, PSD-95 and CASK) in order to begin tounderstand the function of gene networks underlying the emergenceof early behaviours. Knock down of either NRXN-1aα, NRXN1bβ orCNTAP2 significantly reduced the touch response at 30 hpf to asimilar extent. The high penetrance of these phenotypes (71–84%)suggests that these genes are playing a major role in the developmentof the underlying neural circuitry responsible for this behaviour. Incontrast, at 45 hpf knock down of NRXN-1aα had no effect on theescape response, knock down of NRXN1bβ either extinguished orreduced the response, while knock down of CNTNAP2 produced anabnormal response. These very different phenotypes suggest verydifferent roles of these synaptic adhesion network genes in theunderlying neural circuitry. Our analyses are beginning to reveal the

526 Abstracts

most critical genes involved in development of neural circuitsunderlying a simple behaviour.

doi:10.1016/j.ydbio.2010.05.486

Program/Abstract # 401Withdrawn

doi:10.1016/j.ydbio.2010.05.487

Program/Abstract # 402Regulatory elements controlling the expression of short-stature(Shox) genes during developmentJohn A. Cobb, Jessica RosinDepartment of Biological Sciences, University of Calgary, Alberta, Canada

The murine Shox2 (mShox2) gene is required for development ofthe proximal limbs, palate, jaws and heart. Deficiencies of the closelyrelated human SHOX (hSHOX) gene cause the limb abnormalitiesassociated with Turner, Léri–Weill and Langer syndromes. Some Léri–

Weill and Langer patients have an intact hSHOX coding region, butdeletions far downstream of the gene, suggesting that long-rangeenhancer elements are required for hSHOX expression in limbs. Tobegin to understand the regulation of Shox genes, we used transgenicmouse embryos to analyze the regulation of mShox2 and hSHOX. Firstwe analyzed the regulatory potential of sequences near each gene byinserting a LacZ reporter at the start codon of each coding sequenceon an appropriate bacterial artificial chromosome (BAC). Transgenicembryos produced from the mShox2 construct revealed the presenceof regulatory elements driving expression in proximal limbs, sensoryneurons and the hindbrain. Further analysis revealed that sensoryneuron expression is associated with sequences close to the Shox2gene, whereas limb and hindbrain expression requires an evolutio-narily conserved region approximately 35 kb downstream. In contrasttransgenic embryos containing the hSHOX-LacZ BAC showed expres-sion only in the first pharyngeal arch, a known expression domain ofhSHOX. Therefore we are now searching for hSHOX limb enhancers onBACs mapping further downstream of the gene. One prominentcandidate is a conserved sequence with homology to the mShox2limb/hindbrain enhancer region.

doi:10.1016/j.ydbio.2010.05.488

527Abstracts