controls Wnt-mediated cytoskeletal reorganization during vertebrategastrulation.

doi:10.1016/j.ydbio.2008.05.164

Program/Abstract # 153δ-Catenin regulates Xenopus developmental morphogenesisDongmin Gu a, Amy K. Sater c, Hong Ji a, Melissa Clark d, Sabrina A.Stratton a, Michelle C. Barton a, Qun Lu, Pierre D. McCrea a

a Department of Biochemistry and Molecular Biology, UT MD AndersonCancer Center, Houston, TX, USAb Program in Genes and Development, UT GSBS, Houston, TX, USAc Department of Biology and Biochemistry, University of Houston,Houston, TX, USAd Department of Anatomy and Cell Biology, East Carolina University,Greenville, NC, USA

Catenins of the p120 sub-class display an array of intracellularlocalizations and functions. While the genetic knock-out of mouse δ-catenin resulted in mild cognitive dysfunctions and aberrant neuronaldendritic forms, we report severe effects upon its depletion in Xeno-pus. We find that Xenopus δ-catenin is transcribed as a full-lengthmRNA, or as three (or more) alternatively spliced isoforms. Furtherstructural and functional complexity is suggested by three predictedand alternative translation initiation sites. Unlike the primarily neuralexpression of δ-catenin reported in mammals, Xenopus δ-catenin isdetectable in most adult Xenopus tissues, although enriched in neuralstructures. To characterize δ-catenin's functions in amphibian devel-opment, we employed anti-sensemorpholinos targeted to inhibit pre-RNA splicing events. δ-catenin knock-down leads to developmentaldefects in gastrulation and neural crest migration, phenotypes thatwere specific based upon self-rescue experiments. Biochemical assaysindicated that δ-catenin depletion results in reduced C-cadherin levelsas well as activation of RhoA. Indeed, titrated doses of C-cadherin ordominant-negative RhoA significantly rescued δ-catenin depletion.Collectively, our experiments indicate that δ-catenin plays an essentialrole in amphibian development, with contributing functional links tocadherins and Rho family GTPases.

doi:10.1016/j.ydbio.2008.05.165

Program/Abstract # 154Heterotaxin: A novel pyridine compound that perturbs left–rightasymmetric organ morphogenesisMeredith Parr a, Doug Young b, Michael Dush a, Alex Dieters b,Nanette Nascone-Yoder a

a Molecular Biomedical Sciences, College of Veterinary Medicine, NorthCarolina State University, Raleigh, NC, USAb Department Chemistry, North Carolina State University, Raleigh, NC,USA

Proper orientation of internal organ situs is dependent on correctinterpretation of left–right asymmetric cues by developing primordia.To investigate the molecular mechanisms of asymmetric organmorphogenesis, we employed a phenotype-based chemical geneticscreen in embryos of the frog Xenopus laevis, which develop organasymmetries analogous to higher vertebrates. In a pilot screen of 44natural product-like compounds, synthesized and screened asmixtures of regioisomers, one compound mixture specificallyreversed or isomerized the asymmetry of the heart and gut without

affecting other aspects of development. Purification and rescreeningof the individual components of this mixture revealed a single activepyridine compound, which we termed “Heterotaxin”. The effect ofHeterotaxin on organ situs is both dose- and stage-dependent, andoccurs with high penetrance. Heterotaxin-treated embryos haveeither unilateral left, unilateral right, bilateral or absent Pitx2expression in the lateral plate mesoderm, suggesting that globalleft–right asymmetry is randomized by Heterotaxin. In contrast tocontrol embryos, which have a well-defined, polarized intestinalepithelium, Heterotaxin-treated embryos have cohesive masses ofdisorganized, rounded cells protruding into the gut lumen, suggestingthat epithelial morphogenesis is involved in the generation ofdigestive organ asymmetry. The discovery of Heterotaxin thusprovides a novel tool to uncover the etiology of heterotaxia, andunderscores the utility of a chemical genetic approach to organmorphogenesis.

doi:10.1016/j.ydbio.2008.05.166

Program/Abstract # 155Basolumenal endoderm intercalation: A geometrically uniqueexecution of convergent extension during gut tube elongationNanette Nascone-Yoder, Rachel Reed, Mandy Womble, Michael Dush,Stephanie Bloom, Read Tull, Allison MorckelMolecular Biomedical Sciences, College of Veterinary Medicine, NorthCarolina State University, Raleigh, USA

The vertebrate gut tube undergoes dramatic elongation androtation, but the morphogenetic mechanisms underlying thesetopological transformations are poorly understood. We found thatendoderm cells in the late stage Xenopus embryo adopt a fusiformbipolar morphology andmediolateral orientation, reminiscent of axialmesoderm cells undergoing convergent extension in the gastrula.However, the endoderm cells rearrange in the unique three-dimen-sional context of the gut tube cylinder and appear to be “captured” asmonopolar types at both the basement membrane and expandingcentral lumen, gradually reorienting themselves to radially-arrangedbasolumenal “spokes”. The novel geometry of these endoderm cellrearrangements accomplishes gut elongation, provides a morphoge-netic mechanism for generating curvature and rotation, and ulti-mately facilitates the development of themature digestive epithelium.Moreover, gut-specific expression of a dominant negative mutantversion of RhoA, or exposure of embryos to small-molecule inhibitorsof Rho kinase and myosin II, perturbs the cell shape and adhesionpatterns necessary for endoderm intercalation, and concomitantlyinduces severe defects in gut tube elongation, intestinal rotation andepithelial morphogenesis. These results provide insight into theetiology of human digestive deformities, and suggest that themorphogenetic events driving gut elongation via endoderm inter-calation are surprisingly analogous to the mechanisms directing axialelongation in gastrulating mesoderm.

doi:10.1016/j.ydbio.2008.05.167

Program/Abstract # 156Stiffening of the vertebrate embryo during axis elongationdepends on actomyosin contractilityLance Davidson, Hye Young Kim, Jian ZhouDepartment of Bioengineering, University of Pittsburgh, Pittsburgh, PA,USA

513ABSTRACTS / Developmental Biology 319 (2008) 502–520

Physical forces drive the movement of tissues within the earlyembryo. Classical and modern approaches have been used to inferand in rare cases measure mechanical properties and the locationand magnitude of forces within embryos. Elongation of the dorsalaxis is a critical event in early vertebrate development yet themechanics of dorsal tissues in driving embryonic elongation thatlater support neural tube closure and formation of the centralnervous system are not known. Among vertebrates, amphibianembryos allow complex physical manipulation of embryonic tissuesthat are required to measure the mechanical properties of tissues.We measure the stiffness of dorsal isolate explants of frog (Xenopuslaevis) from gastrulation to neurulation. We find that tissues stiffenfrom less than 20 Pa to over 80 Pa in less than 8 h. Furthermore, byisolating specific axial and paraxial tissues we find that paraxialsomitic mesoderm is nearly twice as stiff as either the notochord orneural plate and at least 20-fold stiffer than the endoderm. Dorsalisolates treated with acute acting cytoskeletal inhibitors or preparedfrom fibronectin knock-down embryos reveal that stiffness is theproduct of a contractile actomyosin cortex rather than a fibrillarfibronectin extracellular matrix. Increasing dorsal stiffness andspatial variation between germ layers during neurulation suggestsa common vertebrate dorsal tissue architecture that may insurerobust neural tube closure and provide positional cell identity priorto organogenesis.

doi:10.1016/j.ydbio.2008.05.168

Program/Abstract # 157Regulation of cytoarchitecture in development: The roles ofIQGAP2, C-cadherin and Cdc42 in the early Xenopus embryosQinghua Tao, Sumeda Nandadasa, Stephanie Lang, Janet Heasman,Chris WylieDivision of Developmental Biology, Children's Hospital Medical Center,USA

The control of cytoarchitecture is fundamentally important inmulticellular organisms, both for tissue assembly during embryonicdevelopment and for adult tissue remodeling. The Xenopus blastulahas proven to be a useful model system for studying the mechanismsof cytoarchitecture control during early development. Becausecortical actin assembly at late blastula stage is controlled by mRNAsmaternally transcribed and stored in the egg, depletion of individualstored mRNAs allows rapid loss of function, as well as epistaticexperiments. We have previously shown that the cortical actinnetwork is essential for rigidity and shape in the early embryos, andthat the level of expression of C-cadherin on the cell surface is ratelimiting for cortical actin assembly at the blastula stage. We have alsoidentified two G protein-coupled receptors (GPCRs) that controlcortical actin assembly by controlling the expression level of C-cadherin on the cell surface in the late Xenopus blastulae. However, itis unclear how the activity of C-cadherin is controlled at the cellsurface to nucleate cortical actin assembly. Here, we provide evidencethat cdc42, a member of small Rho GTPases, is essential to mediate C-cadherin controlled cortical actin assembly. IQGAP2, a multi-domainscaffold protein is identified as a major effector protein of Cdc42 thatis necessary for cortical actin assembly controlled by C-cadherin atthe cell surface. We also show that actin nucleation factor Arp2/3complex is required for C-cadherin to assemble cortical actin at thecell surface.

doi:10.1016/j.ydbio.2008.05.169

Program/Abstract # 158A tale of two tails: Multiple pathways regulate cell adhesion andmorphogenesis in the zebrafish tailChris Thorpe, Yi YangDivision of Biology, Kansas State University, Manhattan, KS, USA

Multiple signaling pathways regulate development of the posteriorvertebrate body, which is derived from a population of progenitor cellscalled the tailbud. Fate specification and differentiation are closelylinked with cell migration to ensure that, as some cells exit the tailbudand differentiate, other cells are retained in the tailbud as undiffer-entiated precursors to support later tail outgrowth.

doi:10.1016/j.ydbio.2008.05.170

Program/Abstract # 159Neuregulin-mediated ErbB3 signaling is required for DRG neuronformationYasuko Honjo, Judith S. EisenInstitute of Neuroscience, University of Oregon, Eugene 97403-1254, USA

ErbB3 is a receptor-type tyrosine kinase that has been shown tohave important roles in neural and glial development. We found thaterbb3 mutant zebrafish have a defect in dorsal root ganglion (DRG)formation, as revealed early in development by absence of neuro-genin1-positive nascent DRG neurons and later by absence of HuC/D-positive DRG neurons. To learn whether neural crest cell migration isaffected in erbb3 mutants, we followed neural crest migration in livetransgenic embryos inwhich GFP expression is driven by the zebrafishsox10 promoter. In erbb3mutants, overall motility of neural crest cellsappeared unaffected. However, migrating neural crest cells failed topause near the positionwhere DRGs normally form. This is in contrasttowild types, inwhich a subset of migrating neural crest cells pause inthis location for many hours. Transient blockade of ErbB receptorsrevealed that ErbB receptor signaling is required for DRG neuronformation around the time migrating neural crest cells pause.Together these results suggest that ErbB3 signaling is required forDRG progenitors to recognize their target position during migration.We isolated genes encoding the ErbB3 ligands Neuregulin 1 (Nrg1)and Neuregulin 2 (Nrg2) and found that zebrafish has one nrg1 andtwo nrg2 (nrg2a and nrg2b) genes. To learn whether any of the Nrgsare involved in DRG neuron formation, we knock them downindividually and in combination using morpholinos and found thatNrg1 and Nrg2a act together during neural crest migration and DRGformation.

doi:10.1016/j.ydbio.2008.05.171

Program/Abstract # 160A role for zic genes during neural tube morphogenesis in zebrafishYevgenya Grinblat a, Molly Nyholm a, Aaron Taylor a, Shawn Burgess b

a Zoology and Anatomy, University of Wisconsin, Madison, WI, USAb NIH, Bethesda, MD, USA

Our studies are aimed at understanding the conserved roles of Zictranscription factors (TFs) during vertebrate brain development. Zicsare required for correct neurulation inmammals via amechanism thatis poorly understood. We have uncovered an essential role for zic2aand zic5 in hinge point (HP) formation during cranial neurulation inzebrafish. HP formation is an essential step in primary neurulation,

514 ABSTRACTS / Developmental Biology 319 (2008) 502–520