Developmental patterning relies on morphogen gradients, which generally involve feedback loops to buffer against perturbations caused by fluctuations in gene dosage and expression. Although many gene components involved in such feedback loops have been identified, how they work together to generate a robust pattern remains unclear. Here we study the network of extracellular proteins that patterns the dorsal region of the Drosophila embryo by establishing a graded activation of the bone morphogenic protein (BMP) pathway. We find that the BMP activation gradient itself is robust to changes in gene dosage. Computational search for networks that support robustness shows that transport of the BMP class ligands (Scw and Dpp) into the dorsal midline by the BMP inhibitor Sog is the key event in this patterning process. The mechanism underlying robustness relies on the ability to store an excess of signalling molecules in a restricted spatial domain where Sog is largely absent. It requires extensive diffusion of the BMP-Sog complexes, coupled with restricted diffusion of the free ligands. We show experimentally that Dpp is widely diffusible in the presence of Sog but tightly localized in its absence, thus validating a central prediction of our theoretical study.

DEVELOPMENT 385Morphogens act as graded positional cues that control cell fate specification in many developing tissues. This concept, in which a signalling gradient regulates differential gene expression in a concentration-dependent manner, provides a basis for understanding many patterning processes. It also raises several mechanistic issues, such as how responding cells perceive and interpret the concentration-dependent information provided by a morphogen to generate precise patterns of gene expression and cell differentiation in developing tissues. Here, we review recent work on the molecular features of morphogen signalling that facilitate the interpretation of graded signals and attempt to identify some emerging common principles. IntroductionThe transformation of the spatial distribution of naïve cells in a developing tissue into an organised arrangement of cell differentiation is fundamental to the development of multicellular organisms. More than a century ago, evidence began to accumulate that cells receive 'positional information' that instructs them to develop in specific ways, depending on their location within a tissue (Wolpert, 1996). Over the intervening decades, the potential for signalling gradients to provide this positional information has become a much-investigated and -debated subject, and the term 'morphogen' has been coined to describe such signals. Today the morphogen concept continues to form the basis of many models of pattern formation (Lewis et al., 1977;Green and Smith, 1991;Gurdon and Bourillot, 2001;Tabata and Takei, 2004). Typically, in current models it is proposed that a signal produced from a defined localised source forms a concentration gradient as it spreads through surrounding tissue (Fig. 1A). The graded signal then acts directly on cells, in a concentration-dependent manner, to specify gene expression changes and cell fate selection. Thus, the concentration of ligand provides cells with a measure of their position relative to the source of the signal and organises the pattern of cell differentiation. Experimental evidence from tissues in both vertebrates and invertebrates indicates that several molecules appear to function as graded signals. The roles of these signals range from the establishment of the initial polarities of embryos to specification of cell identity in specific tissues, notably limb appendages and the nervous system in both vertebrates and Drosophila. The examples we focus on in this review are introduced in Fig. 1. Evidence in support of these signals acting as graded morphogens has been summarised in recent reviews (Gurdon and Bourillot, 2001;Tabata and Takei, 2004).Although the morphogen concept has provided an enduring and valid framework for understanding pattern formation, it raises many mechanistic issues. Much attention has focused on how the distribution of a morphogen through a tissue establishes and maintains a gradient of activity (Vincent and Dubois, 2002;Tabata and Takei, 2004); however, how the signal is perceived and interpreted in a grad...

Dorsal-ventral patterning in vertebrate and invertebrate embryos is mediated by a conserved system of secreted proteins that establishes a bone morphogenetic protein (BMP) gradient. Although the Drosophila embryonic Decapentaplegic (Dpp) gradient has served as a model to understand how morphogen gradients are established, no role for the extracellular matrix has been previously described. Here we show that type IV collagen extracellular matrix proteins bind Dpp and regulate its signalling in both the Drosophila embryo and ovary. We provide evidence that the interaction between Dpp and type IV collagen augments Dpp signalling in the embryo by promoting gradient formation, yet it restricts the signalling range in the ovary through sequestration of the Dpp ligand. Together, these results identify a critical function of type IV collagens in modulating Dpp in the extracellular space during Drosophila development. On the basis of our findings that human type IV collagen binds BMP4, we predict that this role of type IV collagens will be conserved.

We have identified novel nuclear transcripts in the human ␤-globin locus using nuclear run-on analysis in erythroid cell lines and in situ hybridization analysis of erythroid tissue. These transcripts extend across the LCR and intergenic regions but are undetectable in nonerythroid cells. Surprisingly, transient transfection of a ␤-globin gene (⑀, ␥, or ␤) induces transcription of the LCR and intergenic regions from the chromosomal ␤-globin locus in nonerythroid cell lines. The ␤-globin genes themselves, however, remain transcriptionally silent. Induction is dependent on transcription of the globin gene in the transfected plasmid but does not require protein expression. Using in situ hybridization analysis, we show that the plasmid colocalizes with the endogenous ␤-globin locus providing insight into the mechanism of transinduction.

SummaryDrosophila ovarian germline stem cells (GSCs) are maintained by Dpp signaling and the Pumilio (Pum) and Nanos (Nos) translational repressors. Upon division, Dpp signaling is extinguished, and Nos is downregulated in one daughter cell, causing it to switch to a differentiating cystoblast (CB). However, downstream effectors of Pum-Nos remain unknown, and how CBs lose their responsiveness to Dpp is unclear. Here, we identify Brain Tumor (Brat) as a potent differentiation factor and target of Pum-Nos regulation. Brat is excluded from GSCs by Pum-Nos but functions with Pum in CBs to translationally repress distinct targets, including the Mad and dMyc mRNAs. Regulation of both targets simultaneously lowers cellular responsiveness to Dpp signaling, forcing the cell to become refractory to the self-renewal signal. Mathematical modeling elucidates bistability of cell fate in the Brat-mediated system, revealing how autoregulation of GSC number can arise from Brat coupling extracellular Dpp regulation to intracellular interpretation.