syndecan-4 in non-canonical wnt signaling and gastrulation movements in xenopus embryos

We have studied the embryos of two foam nesting frogs,Engystomops randi and E. coloradorum (Leiuperidae) fromfertilization to tadpole hatching and prepared a normal table ofdevelopment, which was divided into 23 stages. The foam nests,built on standing water, each contain about 200 white eggs (1.1and 1.3 mm in diameter, respectively) that resemble X. laevisalbino eggs and develop at almost the same rate. Embryomorphology was evaluated in whole mount preparations andvibratome sections. Somites were analyzed morphologicallyand by immunostaining with an anti-myosin antibody. Althoughboth species approach the egg size and developmental rates ofX. laevis, their development differs in significant ways, anaspect that may relate to the different breeding strategies ofthese frogs. The morphology of cleavage and gastrulationresembles the X. laevis developmental pattern. Similarly,elongation of the archenteron and notochord begins beforeblastopore closure. At later stages, however, embryos of En-gystomops differ greatly from X. laevis not only in the generalshape of the embryo at the tailbud stage, but also in the patternof somite differentiation. The first somites were detected in themid-neurula instead of in the late gastrula. The pattern ofmyotome differentiation includes the intercalation of numerouscells, similar to that which occurs in Gastrotheca riobambaeand Bombina variegata. This pattern greatly differs frommyotome differentiation in X. laevis. This is the first descriptionof early development in frogs of the genus Engystomops.

doi:10.1016/j.ydbio.2007.03.590

Program/Abstract # 290Gastrulation in four species of dendrobatid frogsPaola C. Montenegro-Larrea, Eugenia M. Del PinoPont. Cath. Univ. Ecuador, Sch. of Biol. C., Quito, Ecuador

We have studied gastrulation of four closely related speciesof dendrobatid frogs, Epipedobates anthonyi, Epipedobatestricolor, Epipedobates ingeri and Dendronbates auratus.Developmental patterns have been compared with Xenopuslaevis and a marsupial frog, Gastrotheca riobambae. Gastrula-tion morphology was analyzed in whole mount and in cross-sections stained for cell nuclei. We observed great variability inegg size amongst the dendrobatids (egg diameters, 1.6 to3.5 mm). Eggs of some dendrobatids are larger than those of G.riobambae (3 mm in diameter). G. riobambae has one of themost divergent patterns of frog gastrulation, with the formationof an embryonic disk. Gastrulae of most dendrobatid frogs sharewith G. riobambae a delayed elongation of the archenteron andthe formation of a large circumblastoporal collar. We thereforeasked whether the formation of an embryonic disk is associatedwith egg size. The results show that despite the large egg size,there are similar features of gastrulation among dendrobatids,for example gastrulation time, time of archenteron expansion,thickness of the roof of both the blastocoele and archenteronand the degree of cell accumulation in the blastopore lip.Nevertheless, an embryonic disk was not formed. Egg size

variation and similarity of gastrulation processes suggest thatthe gastrulation pattern is highly conserved among dendroba-tids, and that egg size is not related to the formation of anembryonic disk. Gastrulation in dendrobatids is a useful subjectfor further study, as it greatly differs from X. laevis gastrulation.

doi:10.1016/j.ydbio.2007.03.591

Program/Abstract # 291SAGE analysis of dorsal and ventral transcriptome ofXenopus tropicalis gastrulaFernando Faunes 1, Javier Castellanos 2, Rodrigo Malig 2,Francisco Melo 2, Juan Larrain 1

1 Dep. of Cell and Molecular Biology2 Dep. of Molecular Genetics and Microbiology3 Catholic University of Chile

Xenopus has been a favourable model for studying genefunction during early embryonic development. Substantialprogress has been made in the identification of genes andmolecular mechanisms involved in dorsoventral patterning ofXenopus embryos. However, all these pre-genomic approacheswere generally biased to the detection of a limited group oftranscripts. In addition, global approaches in several specieshave demonstrated that transcriptomes are more complex thanexpected. At present, genomic approaches can be performed inXenopus tropicalis because its diploid genome sequence isavailable. In this work, we used the global approach SAGE toidentify novel genes involved in dorsoventral patterning ofXenopus embryos. SAGE permits a qualitative and quantitativeanalysis of the transcriptomes and the detection of noveltranscripts. SAGE libraries were prepared from dorsal andventral explants of Xenopus tropicalis gastrula and 30,000 tags/library have been obtained. Here, we present the comparativeanalysis of these libraries. We performed tag-mapping by usingboth genome sequence and known transcripts because ∼35% ofthe experimental tags have no match in known transcriptsdatabases. We modified a novel bioinformatics method, namedHierarchical Gene Assignment, for proper tag-mapping in thisgenome. We have begun to experimentally verify tag assign-ments by RT-PCR and novel transcripts identified will be usedfor functional studies. This is the first SAGE experiment inXenopus tropicalis and we expect to find novel genes involvedin dorsoventral patterning.

doi:10.1016/j.ydbio.2007.03.592

Program/Abstract # 292Syndecan-4 in non-canonical Wnt signaling andgastrulation movements in Xenopus embryosRosana Muñoz, Loreto Carvallo, Alejandro Burga,Juan LarraínFONDAP Biomedicine. Fac. Cs. Biol, PUC, Chile

397ABSTRACTS / Developmental Biology 306 (2007) 396–410

During gastrulation, morphogenetic cell movements arecritical for the organization of the three germ layers. In ourrecent paper published in Nat. Cell Biology (Muñoz, R., et al.8, 492–500. 2006) we demonstrated that a cell surfaceheparan sulphate proteoglycan (HSPG), Syndecan-4 (xSyn4),is critical during Xenopus laevis gastrulation, regulatingspecific activation of the non-canonical Wnt signaling.During convergence and extension movement (CE) mesoder-mal cells are polarized and aligned mediolaterally, thenintercalated. To test whether xSyn4 is required in this process,the CE in dorsal marginal zone (DMZ) explants wereobserved using confocal microscopy. xSyn4 morpholino(xSyn4MO) and mRFP were co-injected into one of thetwo dorsal blastomeres at four-cell stage. In turns, controlmorpholino (MoCo) and mGFP were co-injected into theother dorsal blastomere. At gastrula stage, DMZ explantswere obtained and cultured on a cover glass. DMZ adhered tothe surface, and subsequently CE movements occurred,rendering an elongated mesoderm. The xSyn4MO-injectedcells (red cells) were morphologically round-shaped, notpolarized, and its intercalation was perturbed. In contrast, thenon-red cells (lacking xSyn4MO) were polarized and showedcorrect CE, which clearly demonstrated the specificity of theexperiment. In accordance with its morphologically appear-ance, xSyn4MO injected cells showed no protrusive activityand complete absence of filopodia. However, these cellsshowed lamellipodia-like membrane extensions. These resultssuggest that xSyn4 is important for cell polarization andintercalation during CE.

doi:10.1016/j.ydbio.2007.03.593

Program/Abstract # 293Xenopus laevis ATF1, a novel target gene of Notchsignaling, functions during gastrulation of XenopusembryosTamaru Tatsuya, Sakamoto Chiharu, Takakura KotaDept. of Bioscience, Kwansei Gakuin Univ., Japan

The Notch signaling pathway is widely conserved fromvertebrate to invertebrate and mediates the specification ofnumerous cell fates during developmental processes. In Xe-nopus laevis, the role of Notch signaling in neurogenesis hasbeen studied in detail, whereas Notch signaling in gastrulaembryo remains unknown. In Xenopus gastrula embryo,Xdelta1, one of the Notch ligands, is expressed in theprospective mesoderm prior to Xbrachyury expression.However XESR1, one of the Notch target genes, is notdetected during gastrulation. In order to understand the role ofXdelta1 at gastrula stage, novel Notch target genes werescreened by using microarray analysis. The screeningidentified 11 candidate genes that showed Notch-dependentexpression. Within these candidates, 4 genes showed Nodal-dependent genes expression. In gain of function experiment,only one among 4 genes caused the defective gastrulation.

Basing on DNA sequencing and homology search, wedesignated this gene Xenopus ATF1, XATF1. XATF1 is amember of cAMP response element-binding/activation tran-scription factor 1 (CREB/ATF1) family. To examine the roleof XATF1 during gastrulation, we constructed the dominantnegative form (DN) of XATF1. Micro-injection of DN-XATF1 mRNA caused the downregulation of Xbrachyury andthe severe gastrulation defect. Knockdown of XSu(H)2 usingmorpholino oligo DNA caused the similar defective embryo,which was rescued by co-injection of XATF1. These resultssuggest that XATF1 is involved in the gastrulation of Xeno-pus embryo in Notch-dependent manner.

doi:10.1016/j.ydbio.2007.03.594

Program/Abstract # 294Molecular and morphogenetic analysis of gastrulation inannual fishLuisa Pereiro 1, Felix Loosli 2, Jochen Wittbrodt 2,Miguel Concha 1

1 ICBM, Facultad de Medicina, Universidad de Chile2 EMBL, Heidelberg, Germany

Vertebrate gastrulation is a morphogenetic process thatinvolves a dramatic redistribution of cells that gives rise tothe germ layers. Among vertebrates there are differences inthe spatial pattern of gastrulation thought to respond, at leastpartially, to differences in the geometry/shape of the gastrula.Gastrulation in most teleosts occurs in a cup shaped gastrulaand is characterized by the internalization of cells at themargin of the embryo, a process that is accompanied byepiboly and by convergent extension cellular movements. Incontrast to this pattern, we observe that in annual fish of thegenus Cynolebias, epiboly is temporally separated fromgerm layer formation and convergent extension. Duringepiboly, blastoderm cells become dispersed over the yolk.After this is complete, gastrulation starts through theaggregation of cells in a discrete region of the embryo toform a discoidal structure. We are currently addressingwhether the pattern of gastrulation in annual fish resemblesthat seen in other discoidal embryos such as the chickblastodisc. We have cloned and analyzed the expressionpattern of organizer marker genes (e.g. gsc) and genes thatare expressed early during the formation of the embryonicaxis (e.g. bra), and we are currently performing amorphogenetic analysis of gastrulation using time-lapsemicroscopy and histological techniques. Our preliminaryresults suggest that gastrulation in annual fish occurs througha combination of morphogenetic mechanisms resemblingboth discoidal (chick) and other teleost (zebrafish) embryos.Funding: HHMI, PBCT ACT47, ICM P04-068-F, DAAD,ICBM.

doi:10.1016/j.ydbio.2007.03.595

398 ABSTRACTS / Developmental Biology 306 (2007) 396–410