Doctor of Philosophy / Division of Biology / Susan J. Brown / A segmented body plan is conserved in vertebrates and arthropods. Comparative studies implicate a conserved mode of A-P axis patterning and segmentation among vertebrates: Wnt signaling is involved in fate map patterning along the A-P axis and in segmentation in the posterior region of the embryo. On the other hand, comparative studies in arthropods have revealed distinct modes of development between long and short germ insects, which differ both morphologically and genetically. In the short germ insect Tribolium, a Wnt activity gradient contributes to A-P axis patterning and generates a posterior Tc-cad expression gradient that regulates segmentation through a pair-rule gene clock, forming segments sequentially as in vertebrates. In contrast, instead of Wnt activity, a hierarchy of regulatory genes regionalizes the blastoderm and defines segments simultaneously in the long germ insect Drosophila. In Tribolium, Tc-zen-1, Tc-otd-1 and Tc-cad play key roles in patterning serosa, head and trunk regions, respectively, of the blastoderm fate map, which are impacted by changes in Wnt activity levels. However, interactions between these genetic factors have not been described. My work revealed that cross talk between the Wnt and Dpp signaling pathways regulates the expression of Tc-zen-1 to determine the serosa. Furthermore, mutually repression between Tcotd-1 and Tc-cad defines the head and trunk regions while mutual repression between Tc-zen-1 and cad determines the posterior extent of the dorsal serosa. Analysis of target genes of the posterior Tc-cad gradient indicates that the Tc-cad gradient regulates the serial expression of gap genes, which are predominately regulators of Hox genes. Thus the posterior Tc-cad gradient determines segment formation through regulation of pair-rule genes in the insect segmentation clock, and defines segmental identity through regulation of gap genes. Given its effect on Tc-zen-1 and Tc-cad, the Wnt activity gradient is a key organizer of the Tribolium blastoderm fate map. Since homologs of these genes as well as the Wnt signaling pathway have also been identified in several other short germ band insects, and affect cell fates along the A-P body axis, this work provides a new paradigm for the short germ mode of development to guide studies in other arthropods.

The zebrafish has emerged as an important genetic model system for the study of vertebrate development. However, while genetics is a powerful tool for the study of early gene functions, the approach is more limited when it comes to understanding later functions of genes that have essential roles in early embryogenesis. There is thus a need to manipulate gene expression at different times, and ideally only in some regions of the developing embryo. Methods for conditional gene regulation have been established in Drosophila, C.elegans and the mouse, utilizing conditional gene activation systems such as the Gal4-UAS system (fly) and the cre/lox recombination system (mouse). While these tools are also being developed in zebrafish, the accessibility of the zebrafish embryo makes other approaches both possible and desirable. We have taken advantage of a heat-shock inducible system that uses the hsp70 promoter that is activated by cellular stress, such as heat. Having established that this global heat shock method allows temporal control of gene expression, we aimed to spatially control gene expression by applying controlled thermal heat to only a small region of the embryo. This would allow us to determine cell- and tissue-autonomous roles for developmentally important genes in an embryo with otherwise normal gene function. We have now developed a device that uses a laser to heat a defined region of the embryo, and thus activate the hsp70 promoter only in restricted regions of the embryo. The output of a 75 mW red laser pointer was focused into the 50 µm diameter core of an optical fiber, whose cleaved and coated end was used to heat, and thus induce, gene expression in a defined area. We have established conditions that allow controlled heating and trans-gene activation in small regions of the embryo without inducing cell death. This new tool will allow us to study the cell-autonomous roles of embryonic signaling molecules in cell differentiation, proliferation, and survival in a variety of tissues and at different times.