Coenzyme A derivatives by acyl-CoA synthetases. We have identified eight long-chain acyl-CoA synthetase (acsl) genes in the zebrafish. One of these, acsl4a, is an ortholog of the mammalian enzyme that prefers long-chain polyunsaturated fatty acids (PUFA). PUFAs and their metabolites can act as potent signaling molecules to influence a number of cellular processes. To elucidate a role for PUFAs during embryonic development, we designed targeted anti-sense oligonucleotides (mor- pholinos) to knock down acsl4a expression in developing zebrafish. Remarkably, loss of Acsl4a results in embryos with dorsoventral patterning defects, with dorsal tissues expanded at the expense of ventral tissues. Cell fates along the dorsoventral axis are patterned via a gradient of bone morphogenetic protein (BMP), and we have demon- strated that this gradient is perturbed in acsl4a morphant embryos. These data reveal a novel role for fatty acid metabolism in embryonic morphogenesis. Ongoing studies using biochemical techniques and microarray analysis are directed at elucidating the link between PUFAs and the BMP gradient. doi:10.1016/j.ydbio.2010.05.223 Program/Abstract # 183 Complex control of Wnt signaling determines the size of the initial neurogenic territory at the animal pole of the sea urchin embryo Ryan Range, Robert Angerer, Lynne Angerer National Institutes of Health, Bethesda, MD, USA The mature animal pole domain (APD) of the sea urchin embryo is the earliest neurogenic region and it acts as a patterning center that can oppose Wnt signals. During its initial specification in the early cleavage stages, the APD extends throughout the animal hemisphere and is progressively restricted to the animal pole by a process that depends on canonical Wnt signaling. To gain insights into the mechanisms that restrict the APD and protect it from Wnt inhibitory signals, we tested the functions of several potential Wnt antagonists expressed in the APD as well as two Frizzled receptors expressed in the animal hemi- sphere during blastula stages, when restriction occurs. Misexpression of either Dkk1 or sFrp1/5 shows that either can prevent restriction of the APD to the animal pole. Furthermore, dominant negative interference with the activity of Frizzled 5/8 and Frizzled 1/2/7 resulted in a similar phenotype. These data suggest that signaling by an unknown Wnt through one or both of these receptors prevents APD specification in the lateral ectoderm. Interestingly, morpholino knock- downs showed that regulatory interactions among the putative Wnt antagonists, Dkk1, sFrp1/5 and Dkk3, control the position of the boundary between the anterior neuroectoderm and lateral ectoderm. Our data support a model in which initial neuroectoderm patterning requires Wnt-dependent suppression of APD regulatory genes in the lateral ectoderm. Subsequently, Wnt antagonists at the animal pole protect these factors from Wnt signals, solidifying the boundary between the neuroectoderm and lateral ectoderm. doi:10.1016/j.ydbio.2010.05.224 Program/Abstract # 184 Genomic programs for endoderm specification in sea urchin embryos Isabelle Peter, Eric H. Davidson Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA Regulatory interactions directly encoded in the genome determine the spatial domains of gene expression and also the changes in gene expression which occur as cells undergo specification. The gene regulatory network underlying endoderm specification in pre-gastrula stage sea urchin embryos has been analyzed by perturbation approaches which involved all regulatory genes known to be expressed in endoderm precursor cells. These data were combined with selected cis-regulatory analyses and spatial gene expression data which identify the regulatory territories at different stages of development. The emerging model shows that there are two regulatory networks used to specify endoderm, which consist of both unique and shared regulatory genes. Differences in the topology of regulatory interactions account for the different dynamics of specifica- tion in the two endodermal cell lineages. The separation of anterior and posterior endodermal cell fates is thus established even before the formation of the archenteron. Furthermore, endoderm specification is initiated in cells which will also give rise to mesodermal cell types. Our results indicate that endomesoderm precursor cells are not specified by a unique regulatory network, but rather by the independent activation of endoderm- and mesoderm-specific networks. Indeed, regulatory genes underlying mesoderm specification are controlled by Delta/Notch signaling, whereas many regulatory genes in the endoderm network directly respond to wnt signaling. Both signaling pathways are activated in endomesoderm precursor cells and later in exclusive cells which drives the separation of these two cell fates. doi:10.1016/j.ydbio.2010.05.225 Program/Abstract # 185 IrxA expression and function in establishing the sea urchin endoderm–ectoderm boundary Daniel C. McIntyre, Winn Seay, David R. McClay Biology Dept., Duke University, Durham NC, USA The sea urchin embryo is patterned by a series of waves of signaling emanating from the vegetal pole of the embryo and moving progressively towards the animal pole. These signals activate sub- networks of the sea urchin gene regulatory network and establish the basic tissues in the early embryo. While the mechanisms delineating the mesoderm and endoderm in the embryo have been well studied, the regulatory events leading up to the establishment of the endoderm–ectoderm boundary remain unknown. We show that the gene IrxA, along with several other transcription factors is expressed in the ectoderm at this boundary, and that their expression is dependent on signaling from the endoderm. Additionally, while the expression of IrxA is initially limited to the aboral ectoderm boundary, it is later expressed throughout the aboral ectorderm and in the mid-gut, indicating multiple regulatory inputs. Therefore, we hypothesize that IrxA is required to reinforce the formation of the endoderm–ectoderm boundary. doi:10.1016/j.ydbio.2010.05.226 Program/Abstract # 186 Bazooka/PAR-3 and PKC protein localization correlate with spindle position during asymmetric cleavage in the early Ilyanassa embryo Ayaki Nakamoto a , James R. Cooley a,b , Jessica E. Wandelt c , Lisa M. Nagy a a Dep. of MCB, Univ. of Arizona, Tucson, AZ, USA b Dep. of CBA., Univ. of Arizona, Tucson, AZ, USA c School of Biol. Sciences, Univ. of Texas at Austin, Austin, TX, USA During asymmetric cleavages, mother cells are polarized along a specified axis and the mitotic spindle aligns with this polarity, and 469 Abstracts