Notch signalling defines critical boundary during budding in Hydra.

Boundary formation is an important mechanism of development and has been studied in a number of bilaterian model organisms where it is often controlled by Notch, FGF and Wnt signalling. Tissue boundaries are also formed in simple pre-bilaterian animals. The boundary between parent and bud during asexual reproduction in the fresh water polyp Hydra vulgaris is an example. The Hydra homolog of the FGF-receptor FGFR (kringelchen) and some components of the Wnt signalling pathway are expressed at this boundary, but their precise functions are unknown. In this work we have discovered an important role for Notch signalling at this boundary. Notch signalling is needed to sharpen the kringelchen expression zone during the final budding stages from an initially broad band into a clear line demarcating the boundary between bud and parent. Expression of the Notch target gene HyHes and the putative matrix metalloprotease MMP-A3 was observed at the boundary shortly before the bud began to constrict and differentiate foot cells. When Notch signalling was inhibited with the presenilin inhibitor DAPT the expression pattern for kringelchen changed dramatically into a diffused pattern. The expression of both HyHes and MMP-A3 was abolished. Moreover, morphogenesis of the bud was not completed and buds did not constrict, failed to form a foot and never detached from the parent. This resulted in the formation of two-headed animals. We suggest that the function of Notch signalling during budding in Hydra is in promoting the formation of two stripes of differing gene expression, which are needed to differentiate the foot of the bud and a progressing narrowing of the mesoglea on the side of the parent.

Tube Is an IRAK-4 homolog in a Toll pathway adapted for development and immunity.

Acting through the Pelle and IRAK family of protein kinases, Toll receptors mediate innate immune responses in animals ranging from insects to humans. In flies, the Toll pathway also functions in patterning of the syncytial embryo and requires Tube, a Drosophila -specific adaptor protein lacking a catalytic domain. Here we provide evidence that the Tube, Pelle, and IRAK proteins originated from a common ancestral gene. Following gene duplication, IRAK-4, Tube-like kinases, and Tube diverged from IRAK-1, Pelle, and related kinases. Remarkably, the function of Tube and Pelle in Drosophila embryos can be reconstituted in a chimera modeled on the predicted progenitor gene. In addition, a divergent property of downstream transcription factors was correlated with developmental function. Together, these studies reveal previously unrecognized parallels in Toll signaling in fly and human innate immunity and shed light on the evolution of pathway organization and function.

A kappaB sequence code for pathway-specific innate immune responses.

The Toll and Imd pathways induce humoral innate immune responses in Drosophila by activating NF-kappaB proteins that bind kappaB target sites. Here, we delineate a kappaB site sequence code that directs pathway-specific expression of innate immune loci. Using bioinformatic analysis of expression and sequence data, we identify shared properties of Imd- and Toll-specific response elements. Employing synthetic kappaB sites in luciferase reporter and in vitro binding assays, we demonstrate that the length of the (G)(n) element in the 5' half-site and of the central (A,T)-rich region combine to specify responsiveness to one or both pathways. We also show that multiple sites function to enhance the response to either or both pathways. Together, these studies elucidate the mechanism by which kappaB motifs direct binding by particular Drosophila NF-kappaB family members and thereby induce specialized innate immune repertoires.

The Notch signaling pathway in the cnidarian Hydra.

Many of the major pathways that govern early development in higher animals have been identified in cnidarians, including the Wnt, TGFbeta and tyrosine kinase signaling pathways. We show here that Notch signaling is also conserved in these early metazoans. We describe the Hydra Notch receptor (HvNotch) and provide evidence for the conservation of the Notch signaling mode via regulated intramembrane proteolysis. We observed that nuclear translocation of the Notch intracellular domain (NID) was inhibited by the synthetic gamma-secretase inhibitor DAPT. Moreover, DAPT treatment of hydra polyps caused distinct differentiation defects in their interstitial stem cell lineage. Nerve cell differentiation proceeded normally but post-mitotic nematocyte differentiation was dramatically reduced. Early female germ cell differentiation was inhibited before exit from mitosis. From these results we conclude that gamma-secretase activity and presumably Notch signaling are required to control differentiation events in the interstitial cell lineage of Hydra.

Regulated assembly of the Toll signaling complex drives Drosophila dorsoventral patterning.

In Drosophila, the Toll pathway establishes the embryonic dorsoventral axis and triggers innate immune responses to infection. The transmembrane receptor Toll acts through three death domain-containing proteins, the kinase Pelle and the adapters Tube and MyD88, in signaling to downstream NF-kappaB-like transcription factors. Here, we delineate the critical events in the earliest stages of Toll signaling. Mutational studies based on structural modeling reveal that the direct interaction of the bivalent Tube death domain with MyD88 is critical for signaling in vivo. The complex of MyD88 and Tube forms prior to signaling and is localized to the embryonic plasma membrane by MyD88. Upon Toll homodimerization, this complex is rapidly recruited to Toll. Binding of Pelle to the MyD88-Tube complex promotes Pelle activation, leading to degradation of the IkappaB-like inhibitor, Cactus. Together, these experiments convert a linear picture of gene function into a dynamic mechanistic and structural understanding of signaling complex assembly and function.