The major questions we focus on are: 1) What are the upstream regulatory components that specify the animal pole domain (APD) of the sea urchin embryo, which contains nerves and cells bearing long, immotile cilia? 2) What are the signals transmitted during early cleavage and blastula stages that are required for endomesoderm specification and timely gastrulation? 3) What is the molecular basis for the embryos ability to change its morphology to adapt to changing food concentrations? In each case, we assay the expression of all predicted genes in the genome in order to gain a complete understanding of the gene regulatory networks that underlie these processes.[unreadable] [unreadable] Six3 is necessary and sufficient for APD specification (45%) (Zheng Wei, Junko Yaguchi, Shunsuke Yaguchi, Lynne Angerer)[unreadable] [unreadable] During the last several years, using bioinformatics approaches and molecular screens, we identified many genes encoding regulatory proteins expressed specifically in the primary neurogenic domain of the sea urchin embryo. Two of the earliest, FoxQ2 and Six3, control early decisions in ectodermal patterning. Experiments conducted during 2008 showed that Six3 is necessary and sufficient for all known features of APD development, including all neurons formed during embryogenesis and for the large majority of APD-specific regulatory proteins previously identified, including FoxQ2. We identified the full regulatory repertoire of genes that depends on Six3, and discovered that Six3 can also suppress canonical Wnt and TGF-beta signals that pattern the rest of the embryo. Our work revealed that Six3 function in the sea urchin embryo APD and the vertebrate forebrain share some features. Because our studies have identified many additional regulatory genes that are Six3-dependent and expressed in the APD, they are likely to facilitate understanding of vertebrate forebrain development. A manuscript reporting this work (Wei et al.) is in review. [unreadable] [unreadable] FoxQ2 is a checkpoint coordinating cell fate specification along the primary and secondary developmental axes (5%)(Shunsuke and Junko Yaguchi) [unreadable] [unreadable] FoxQ2 negatively regulates TGF-beta signaling, which is required for patterning along the secondary axis of all regions of ectoderm except that at the animal pole. In order for TGf-beta signaling to occur, FoxQ2 must be eliminated from the lateral ectoderm, an event that depends on canonical Wnt, the primary axis signaling system. FoxQ2 provides a checkpoint coordinating patterning along the primary and secondary axes (Yaguchi et al., Developmental Cell 14, 97-107, 2008). It also functions in the differentiation of different cell types in the APD and may mediate Six3s role. Whole-genome screens show that is required for the expression of nearly 500 genes. The achievements this year provide strong impetus for determining the structure and cell type specificity of the FoxQ2-dependent GRN, as well as the Six3-dependent GRN, in the neurogenic domain at the animal pole.[unreadable] [unreadable] ActivinB/ALK4/5/7 and Delta/Notch are early signals that induce specification of endomesoderm and endoderm (25%)(Adi Sethi, Radhika Wikramanayake) [unreadable] [unreadable] Previous experiments showed that both ectopic and normal endomesoderm induction requires ActivinB and that this signaling factor executes all of the endomesoderm inducing functions of the previously unknown, but long-sought, early micromere signal. His work is the first to connect ActivinB signaling to specific components of an endomesoderm gene regulatory (GRN) network, the largest and best developed in any embryo, and it has led to new insights into its operation. During this year additional experiments carried out in response to reviews have confirmed previous work and an improved, revised manuscript is in review. Previous work blocking Notch function reveal new functions for this pathway in separating two primary germ layers, the endoderm and mesoderm, within the endomesodermal field. During 2008, we found that micromere Delta signals not only aactivate gcm, the cardinal pigment cell GRN regulator, but also to repress several critical genes required for early endoderm specification. Delta signaling to endoderm is earlier than previously reported and necessary for expression of early endoderm specification genes. Because macromere Delta depends on prior micromere Delta signals, new methods to knock down it down in specific blastomeres have been developed. These include injection of a morpholino blocking Delta translation in single blastomeres or inducing loss of Delta transcription in specific blastomeres using antisense intron RNAs. Preliminary experiments suggest that both methods will be useful.[unreadable] [unreadable] Dopaminergic neurons regulate the embryos response to food density (25%) (Diane Adams)[unreadable] [unreadable] A new line of investigation aims to understand the molecular pathways by which larvae sense and respond to the concentration of food by adjusting arm structure to maximize food intake. It is a high-risk project that bridges larval developmental biology, larval physiology and the ecology of larval dispersal. Diane has found that changes in the post-oral skeletal rod length in response to food density is regulated by dopamine and that dopaminergic neurons are positioned correctly at the base of the post-oral arms to regulate the sensory response. To determine the components of the food sensing pathway, she is assaying the whole geneme reponse to food concentration and to dopamine antagonists and agonists using microarrays. She has also established a collaboration with Dr. Andrew Cameron at Caltech to search for G-coupled protein receptors that might function in larval sensing of food during larval stages.