Shaping BMP morphogen gradients through enzyme-substrate interactions

Bone morphogenetic proteins (BMPs) regulate dorsal/ventral (D/V) patterning across the animal kingdom; however, the biochemical properties of certain pathway components can vary according to species-specific developmental requirements. For example, Tolloid (Tld)-like metalloproteases cleave vertebrate BMP-binding proteins called Chordins constitutively, while the Drosophila Chordin ortholog, Short gastrulation (Sog), is only cleaved efficiently when bound to BMPs. We identified Sog characteristics responsible for making its cleavage dependent on BMP binding. Chordin-like variants that are processed independently of BMPs changed the steep BMP gradient found in Drosophila embryos to a shallower profile, analogous to that observed in some vertebrate embryos. This change ultimately affected cell fate allocation and tissue size and resulted in increased variability of patterning. Thus, the acquisition of BMP-dependent Sog processing during evolution appears to facilitate long-range ligand diffusion and formation of a robust morphogen gradient, enabling the bistable BMP signaling outputs required for early Drosophila patterning.     

Shaping BMP morphogen gradients in the Drosophila embryo and pupal wing

In the early Drosophila embryo, BMP-type ligands act as morphogens to suppress neural induction and to specify the formation of dorsal ectoderm and amnioserosa. Likewise, during pupal wing development, BMPs help to specify vein versus intervein cell fate. Here, we review recent data suggesting that these two processes use a related set of extracellular factors, positive feedback, and BMP heterodimer formation to achieve peak levels of signaling in spatially restricted patterns. Because these signaling pathway components are all conserved, these observations should shed light on how BMP signaling is modulated in vertebrate development.     

Scaling of morphogen gradients

Individuals of the same or closely related species can vary substantially in size. Still, the proportions within and between tissues are precisely kept. This adaptation of pattern with size termed scaling, is receiving a growing attention. We review experimental evidence for scaling, and describe theoretical models for mechanisms that scale morphogen gradients. We particularly note the Expansion-Repression mechanism, in which a diffusible molecule that positively regulates the morphogen gradient width is repressed by morphogen signaling. The Expansion-Repression circuit provides scaling in a robust manner and is readily implemented by a host of molecular mechanisms. We suggest means for identifying such a circuit in a system of interest.     

miRNAs and morphogen gradients

Morphogens induce biological diversity by operating in a dose-dependent manner. Here we review recent evidences indicating that microRNAs (miRNAs) are ideally suited to serve the morphogen cause. miRNAs regulate the establishment of morphogen gradients, including TGFβ, Wnt and other growth factors by acting on their secretion, distribution and clearance. miRNA are also critical in receiving cells, establishing context-dependency and threshold responses. Moreover, miRNAs contributes to gene networks that transform the graded activity of a morphogen into robust cell fate decisions. Finally, we discuss in the perspective section the implication of the new ceRNA hypothesis for morphogen biology.     

Genetics of morphogen gradients

The organization of cells and tissues is controlled by the action of 'form-giving' signalling molecules, or morphogens, which pattern a developmental field in a concentration-dependent manner. As the fate of each cell in the field depends on the level of the morphogen signal, the concentration gradient of the morphogen prefigures the pattern of development. In recent years, molecular genetic studies in Drosophila melanogaster have allowed tremendous progress in understanding how morphogen gradients are formed and maintained, and the mechanism by which receiving cells respond to the gradient.     

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.     

Travelling gradients in interacting morphogen systems

Morphogen gradients are well known to play several important roles in development; however the mechanisms underlying the formation and maintenance of these gradients are often not well understood. In this work, we investigate whether the presence of a secondary morphogen can increase the robustness of the primary morphogen gradient to perturbation, thereby providing a more stable mechanism for development. We base our model around the interactions of Fibroblast Growth Factor 8 and retinoic acid, which have been shown to act as morphogens in many developmental systems. In particular, we investigate the formation of opposing gradients of these morphogens along the antero-posterior axis of vertebrate embryos, thereby controlling temporal and spatial aspects of axis segmentation and neuronal differentiation.     

Dynamics of maternal morphogen gradients in Drosophila

The first direct studies of morphogen gradients were done in the end of 1980s, in the early Drosophila embryo, which is patterned under the action of four maternally determined morphogens. Since the early studies of maternal morphogens were done with fixed embryos, they were viewed as relatively static signals. Several recent studies analyze dynamics of the anterior, dorsoventral, and terminal patterning signals. The results of these quantitative studies provide critical tests of classical models and reveal new modes of morphogen regulation and readout in one of the most extensively studied patterning systems.     

Elucidating mechanisms underlying robustness of morphogen gradients

Morphogen gradients play a pivotal role in most phases of developmental patterning. To ensure proper patterning, reproducible gradients are established under diverse environmental conditions and genetic backgrounds. We refer to the capacity to buffer fluctuations in gene dosage or environmental conditions as 'robustness'. By theoretical analysis of mechanisms that facilitate robustness, it is possible to unravel the machinery responsible for generating the spatial distribution of morphogens.     

Self-enhanced ligand degradation underlies robustness of morphogen gradients

Morphogen gradients provide long-range positional information by extending across a developing field. To ensure reproducible patterning, their profile is invariable despite genetic or environmental fluctuations. Common models assume a morphogen profile that decays exponentially. Here, we show that exponential profiles cannot, at the same time, buffer fluctuations in morphogen production rate and define long-range gradients. To comply with both requirements, morphogens should decay rapidly close to their source but at a significantly slower rate over most of the field. Numerical search revealed two network designs that support robustness to fluctuations in morphogen production rate. In both cases, morphogens enhance their own degradation, leading to a higher degradation rate close to their source. This is achieved through reciprocal interactions between the morphogen and its receptor. The two robust networks are consistent with properties of the Wg and Hh morphogens in the Drosophila wing disc and provide novel insights into their function.     

Theoretical and experimental approaches to understand morphogen gradients

Morphogen gradients, which specify different fates for cells in a direct concentration‐dependent manner, are a highly influential framework in which pattern formation processes in developmental biology can be characterized. A common analysis approach is combining experimental and theoretical strategies, thereby fostering relevant data on the dynamics and transduction of gradients. The mechanisms of morphogen transport and conversion from graded information to binary responses are some of the topics on which these combined strategies have shed light. Herein, we review these data, emphasizing, on the one hand, how theoretical approaches have been helpful and, on the other hand, how these have been combined with experimental strategies. In addition, we discuss those cases in which gradient formation and gradient interpretation at the molecular and/or cellular level may influence each other within a mutual feedback loop. To understand this interplay and the features it yields, it becomes essential to take system‐level approaches that combine experimental and theoretical strategies.     

Perturbations in morphogen gradients induce budding in hydra.

Lateral grafting of small pieces of midregion tissue into different levels of the hydra body column was done to assess the influence of the host hypostome and basal disc (or, of the underlying morphogenetic gradients) in inducing secondary structures in the transplanted tissue; and also to identify the role, if any, of the induced secondary structures (or, perturbed morphogen gradients) on the pattern of the host. The same midpiece tissue differentiated to a basal disc when grafted near the host hypostome, and to a small hypostome with tentacles when grafted near the host basal disc. Chimeras with induced secondary basal discs showed a phenomenal increase in budding compared to the controls and to the chimeras having induced hypostomes. These results indicate a positive cross-reaction between both organizing regions during patterning in hydra.     

Understanding morphogen gradients: a problem of dispersion and containment

Protein morphogens are instructive signals that regulate growth and patterning of tissues and organs. They form long-range, dynamic gradients by moving from regions of high concentration (producing cells) to regions of low concentration (the adjacent, nonproducing developmental field). Since morphogen activity must be limited to the adjacent target field, we want to understand both how signaling proteins move and how their dispersion is restricted. We consider the variety of settings for long-range morphogen systems in Drosophila. In the early embryo, morphogens appear to disperse by free diffusion, and impermeable membranes physically constrain them. However, at later stages, containment is achieved without physical barriers. We argue that in the absence of constraining barriers, gradient-generating dispersion of morphogens cannot be achieved by passive diffusion and that other mechanisms for distribution must be considered.     

Morphogenetic apoptosis: a mechanism for correcting discontinuities in morphogen gradients

Smooth gradients of the morphogens Hh, Dpp, and Wg are required for proper development of Drosophila imaginal discs. Here, it is reported that, when a discontinuity is generated between two adjacent cells in the reception of either the Dpp or Wg signal, then cells on either side of the discontinuity boundary undergo apoptosis by activating the c-Jun N-terminal Kinase (JNK) pathway. Furthermore, in the medial region of the wing imaginal disc, the JNK pathway is also activated if cells do not receive the proper levels of Dpp and Hh signals. These observations suggest that cells within a developing field have the ability to access their spatial positions by comparing the level of morphogen signal they receive with that of their neighbors. This phenomenon is likely related to the process of cell competition, and we suggest that it is an evolutionarily important mechanism that helps prevent abnormal tissue specification and growth during development.     

Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans

The signaling molecules Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) function as morphogens and organize wing patterning in Drosophila. In the screen for mutations that alter the morphogen activity, we identified novel mutants of two Drosophila genes, sister of tout-velu (sotv) and brother of tout-velu (botv), and new alleles of tout-velu (ttv). The encoded proteins of these genes belong to an EXT family of proteins that have or are closely related to glycosyltransferase activities required for biosynthesis of heparan sulfate proteoglycans (HSPGs). Mutation in any of these genes impaired biosynthesis of HSPGs in vivo, indicating that, despite their structural similarity, they are not redundant in the HSPG biosynthesis. Protein levels and signaling activities of Hh, Dpp and Wg were reduced in the cells mutant for any of these EXT genes to a various degree, Wg signaling being the least sensitive. Moreover, all three morphogens were accumulated in the front of EXT mutant cells, suggesting that these morphogens require HSPGs to move efficiently. In contrast to previous reports that ttv is involved exclusively in Hh signaling, we found that ttv mutations also affected Dpp and Wg. These data led us to conclude that each of three EXT genes studied contribute to Hh, Dpp and Wg morphogen signaling. We propose that HSPGs facilitate the spreading of morphogens and therefore, function to generate morphogen concentration gradients.