A GATA/homeodomain transcriptional code regulates axon guidance through the Unc-5 receptor.

Transcription factor codes play an essential role in neuronal specification and axonal guidance in both vertebrate and invertebrate organisms. However, how transcription codes regulate axon pathfinding remains poorly understood. One such code defined by the homeodomain transcription factor Even-skipped (Eve) and by the GATA 2/3 homologue Grain (Grn) is specifically required for motor axon projection towards dorsal muscles in Drosophila. Using different mutant combinations, we present genetic evidence that both Grn and Eve are in the same pathway as Unc-5 in dorsal motoneurons (dMNs). In grn mutants, in which dMNs fail to reach their muscle targets, dMNs show significantly reduced levels of unc-5 mRNA expression and this phenotype can be partially rescued by the reintroduction of unc-5. We also show that both eve and grn are required independently to induce expression of unc-5 in dMNs. Reconstitution of the eve-grn transcriptional code of a dMN in dMP2 neurons, which do not project to lateral muscles in Drosophila, is able to reprogramme those cells accordingly; they robustly express unc-5 and project towards the muscle field as dMNs. Each transcription factor can independently induce unc-5 expression but unc-5 expression is more robust when both factors are expressed together. Furthermore, dMP2 exit is dependent on the level of unc-5 induced by eve and grn. Taken together, our data strongly suggests that the eve-grn transcriptional code controls axon guidance, in part, by regulating the level of unc-5 expression.

Smad3 protein levels are modulated by Ras activity and during the cell cycle to dictate transforming growth factor-beta responses.

Transforming growth factor beta (TGF-beta) regulates many biological processes, and aberrant TGF-beta signaling is implicated in tumor development. Smad3 is a central component of the TGF-beta signaling pathway, and once activated, Smad3 forms complexes with Smad4 or other receptor-regulated Smads, which accumulate in the nucleus to transcriptionally regulate TGF-beta target genes. Because Smad3 plays a significant role in mediating the activities of TGF-beta, we examined its regulation during tumor development using a well characterized tumor model. We demonstrate that Smad3 levels are dramatically reduced in the tumorigenic cell line transformed with activated H-Ras compared with the normal parental epithelial cells. Interestingly, we also observe a cell cycle-dependent regulation of Smad3 in both cell types, with high Smad3 levels in quiescent cells and a significant drop in Smad3 protein levels in proliferating cells. Smad3 is regulated at the mRNA level and at the level of protein stability. In addition, functional analysis indicates that down-regulation of Smad3 levels is required for the tumor cells to proliferate in the presence of TGF-beta, because ectopic expression of Smad3 in the tumorigenic cell line restores the growth inhibitory response to TGF-beta. In contrast, expression of high levels of Smad3 did not interfere with the ability of these cells to undergo epithelial to mesenchymal transition upon TGF-beta stimulation. Altogether, our results suggest that the level of Smad3 protein is an important determinant of the progression of tumorigenesis. High levels of Smad3 are required for the tumor suppressor activities of TGF-beta, whereas lower levels are sufficient for the tumor promoting functions.

The Unc-5 Receptor Is Directly Regulated by Tinman in the Developing Drosophila Dorsal Vessel.

During early heart morphogenesis cardiac cells migrate in two bilateral opposing rows, meet at the dorsal midline and fuse to form a hollow tube known as the primary heart field in vertebrates or dorsal vessel (DV) in Drosophila. Guidance receptors are thought to mediate this evolutionarily conserved process. A core of transcription factors from the NK2, GATA and T-box families are also believed to orchestrate this process in both vertebrates and invertebrates. Nevertheless, whether they accomplish their function, at least in part, through direct or indirect transcriptional regulation of guidance receptors is currently unknown. In our work, we demonstrate how Tinman (Tin), the Drosophila homolog of the Nkx-2.5 transcription factor, regulates the Unc-5 receptor during DV tube morphogenesis. We use genetics, expression analysis with single cell mRNA resolution and enhancer-reporter assays in vitro or in vivo to demonstrate that Tin is required for Unc-5 receptor expression specifically in cardioblasts. We show that Tin can bind to evolutionary conserved sites within an Unc-5 DV enhancer and that these sites are required for Tin-dependent transactivation both in vitro and in vivo.

Transforming growth factor beta-induced Smad1/5 phosphorylation in epithelial cells is mediated by novel receptor complexes and is essential for anchorage-independent growth.

Transforming growth factor beta (TGF-beta) signals predominantly through a receptor complex comprising ALK5 and TbetaRII to activate receptor-regulated Smads (R-Smads) Smad2 and Smad3. In endothelial cells, however, TGF-beta can additionally activate Smad1 and Smad5. Here, we report that TGF-beta also strongly induces phosphorylation of Smad1/5 in many different normal epithelial cells, epithelium-derived tumor cells, and fibroblasts. We demonstrate that TbetaRII and ALK5, as well as ALK2 and/or ALK3, are required for TGF-beta-induced Smad1/5 phosphorylation. We show that the simultaneous activation of the R-Smads Smad2/3 and Smad1/5 by TGF-beta results in the formation of mixed R-Smad complexes, containing, for example, phosphorylated Smad1 and Smad2. The prevalence of these mixed R-Smad complexes explains why TGF-beta-induced Smad1/5 phosphorylation does not result in transcriptional activation via bone morphogenetic protein (BMP)-responsive elements, which bind activated Smad1/5-Smad4 complexes that are induced by BMP stimulation. Thus, TGF-beta induces two parallel pathways: one signaling via Smad2-Smad4 or Smad3-Smad4 complexes and the other signaling via mixed R-Smad complexes. Finally, we assess the function of the novel arm of TGF-beta signaling and show that TGF-beta-induced Smad1/5 activation is not required for the growth-inhibitory effects of TGF-beta but is specifically required for TGF-beta-induced anchorage-independent growth.