Hilary L. Ashe

Robustness of the BMP morphogen gradient in Drosophila embryonic patterning.

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.

The interpretation of morphogen gradients

Morphogens 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.

Type IV collagens regulate BMP signalling in Drosophila

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.

Local inhibition and long-range enhancement of Dpp signal transduction by Sog

Extracellular gradients of signalling molecules can specify different thresholds of gene activity in development. A gradient of Decapentaplegic (Dpp) activity subdivides the dorsal ectoderm of the Drosophila embryo into amnioserosa and dorsal epidermis,. The proteins Short gastrulation (Sog) and Tolloid (Tld) are required to shape this gradient. Sog has been proposed to form an inhibitory complex with either Dpp or the related ligand Screw,, and is subsequently processed by the protease Tld. Paradoxically, Sog appears to be required for amnioserosa formation, which is specified by peak Dpp signalling activity,. Here we show that the misexpression of sog using the even-skipped stripe-2 enhancer redistributes Dpp signalling in a mutant background in which dpp is expressed throughout the embryo. Dpp activity is diminished near the Sog stripe and peak Dpp signalling is detected far from this stripe. However, a tethered form of Sog suppresses local Dpp activity without augmenting Dpp activity at a distance, indicating that diffusion of Sog may be required for enhanced Dpp activity and consequent amnioserosa formation. The long-distance stimulation of Dpp activity by Sog requires Tld, whereas Sog-mediated inhibition of Dpp does not. The heterologous Dpp inhibitor Noggin inhibits Dpp signalling but fails to augment Dpp activity. These results suggest an unusual strategy for generating a gradient threshold of growth-factor activity, whereby Sog and its protease specify peak Dpp signalling far from a localized source of Sog.

Brat promotes stem cell differentiation via control of a bistable switch that restricts BMP signaling.

Summary Drosophila 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.

Extensive polymerase pausing during Drosophila axis patterning enables high-level and pliable transcription

Cascades of zygotic gene expression pattern the anterior-posterior (AP) and dorsal-ventral (DV) axes of the early Drosophila embryo. Here, we used the global run-on sequencing assay (GRO-seq) to map the genome-wide RNA polymerase distribution during early Drosophila embryogenesis, thus providing insights into how genes are regulated. We identify widespread promoter-proximal pausing yet show that the presence of paused polymerase does not necessarily equate to direct regulation through pause release to productive elongation. Our data reveal that a subset of early Zelda-activated genes is regulated at the level of polymerase recruitment, whereas other Zelda target and axis patterning genes are predominantly regulated through pause release. In contrast to other signaling pathways, we found that bone morphogenetic protein (BMP) target genes are collectively more highly paused than BMP pathway components and show that BMP target gene expression requires the pause-inducing negative elongation factor (NELF) complex. Our data also suggest that polymerase pausing allows plasticity in gene activation throughout embryogenesis, as transiently repressed and transcriptionally silenced genes maintain and lose promoter polymerases, respectively. Finally, we provide evidence that the major effect of pausing is on the levels, rather than timing, of transcription. These data are discussed in terms of the efficiency of transcriptional activation required across cell populations during developmental time constraints.

Cease and desist: modulating short-range Dpp signalling in the stem-cell niche

Drosophila ovarian germline stem cells (GSCs) are maintained by the extracellular BMP2/4 orthologue Dpp, which is produced from the surrounding somatic niche. The Dpp signal has a short range; it induces a response in GSCs within the niche, but is rapidly extinguished in their progeny only one cell‐diameter away. To ensure the correct balance between stem‐cell maintenance and differentiation, several regulatory mechanisms that modulate the Dpp signal at many stages of the pathway have been described. Here, we discuss the nature of the ovarian Dpp signal and review the catalogue of mechanisms that regulate it, demonstrating how the exquisite modulation of Dpp signalling in this context can result in precise and robust control of stem‐cell fate. This modulation is applicable to other stem‐cell environments that use BMPs as a niche signal, and the regulatory mechanisms are conceptually relevant to several other stem‐cell systems.

Smad Affinity Can Direct Distinct Readouts of the Embryonic Extracellular Dpp Gradient in Drosophila

BACKGROUND The TGF-beta signaling molecule Decapentaplegic (Dpp) is an essential morphogen that patterns many tissues during Drosophila development, including the embryonic dorsal ectoderm and larval wing imaginal disk. An activity gradient of Dpp specifies distinct cell fates in the dorsal ectoderm of the embryo through the activation of different transcriptional threshold responses. RESULTS We have analyzed the gene Race, which is expressed in response to peak levels of Dpp signaling in gastrulating embryos. We show that the Smad transcription factors, which are intracellular transducers of Dpp signaling, are essential activators of Race in vivo. Furthermore, increasing the affinity of the Smad binding sites in the Race enhancer broadens the expression pattern of a linked reporter gene and alters its behavior in mutant embryos to that characteristic of a distinct threshold response. CONCLUSIONS Smad activator affinity is a critical determinant of the threshold response to the extracellular Dpp gradient in the embryo. Our results identify a mechanism for interpreting the Dpp gradient in the embryo which is different to the reciprocal repressor gradient model proposed for the wing disk. We suggest that transcription factor binding site affinity will be a general strategy used in the interpretation of other extracellular morphogen gradients.

Medea SUMOylation restricts the signaling range of the Dpp morphogen in the Drosophila embryo

Morphogens are secreted signaling molecules that form concentration gradients and control cell fate in developing tissues. During development, it is essential that morphogen range is strictly regulated in order for correct cell type specification to occur. One of the best characterized morphogens is Drosophila Decapentaplegic (Dpp), a BMP signaling molecule that patterns the dorsal ectoderm of the embryo by activating the Mad and Medea (Med) transcription factors. We demonstrate that there is a spatial and temporal expansion of the expression patterns of Dpp target genes in SUMO pathway mutant embryos. We identify Med as the primary SUMOylation target in the Dpp pathway, and show that failure to SUMOylate Med leads to the increased Dpp signaling range observed in the SUMO pathway mutant embryos. Med is SUMO modified in the nucleus, and we provide evidence that SUMOylation triggers Med nuclear export. Hence, Med SUMOylation provides a mechanism by which nuclei can continue to monitor the presence of extracellular Dpp signal to activate target gene expression for an appropriate duration. Overall, our results identify an unusual strategy for regulating morphogen range that, rather than impacting on the morphogen itself, targets an intracellular transducer.

Multistep molecular mechanism for Bone morphogenetic protein extracellular transport in the Drosophila embryo

In the Drosophila embryo, formation of a bone morphogenetic protein (BMP) morphogen gradient requires transport of a heterodimer of the BMPs Decapentaplegic (Dpp) and Screw (Scw) in a protein shuttling complex. Although the core components of the shuttling complex—Short Gastrulation (Sog) and Twisted Gastrulation (Tsg)—have been identified, key aspects of this shuttling system remain mechanistically unresolved. Recently, we discovered that the extracellular matrix protein collagen IV is important for BMP gradient formation. Here, we formulate a molecular mechanism of BMP shuttling that is catalyzed by collagen IV. We show that Dpp is the only BMP ligand in Drosophila that binds collagen IV. A collagen IV binding–deficient Dpp mutant signals at longer range in vivo, indicating that collagen IV functions to immobilize free Dpp in the embryo. We also provide in vivo evidence that collagen IV functions as a scaffold to promote shuttling complex assembly in a multistep process. After binding of Dpp/Scw and Sog to collagen IV, protein interactions are remodeled, generating an intermediate complex in which Dpp/Scw-Sog is poised for release by Tsg through specific disruption of a collagen IV–Sog interaction. Because all components are evolutionarily conserved, we propose that regulation of BMP shuttling and immobilization through extracellular matrix interactions is widely used, both during development and in tissue homeostasis, to achieve a precise extracellular BMP distribution.