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LSM5194 Morphogens in biological development: Drosophila example Lecture 29 1 n LSM5194 The concept of morphogen gradients The concept of morphogens was proposed by L. Wolpert as a part of the positional information theory in 1969. Morphogen gradients can be very shallow and very sharp. In Xenopus, gradient of activin can be formed experimentally over 100 um in 1 hour. Teleman et al., Cell v 105, p 559, 2001 Several mechanisms have been proposed for the propagation of morphogens through the tissue: • diffusion • transcytosis – endocytic relay from cell to cell Tabata, Nat Rev Gen v 2, p 620, 2001 • cell-cell contact by cytonemes The main problem of morphogenesis can be formulated as one question. How do cells know what is their developmental fate? Early in the history of developmental biology it has become clear that for the cells to make a decision on choosing their future, they need to know their position in the developing tissue. This task to provide positional information to the cells was ascribed to the morphogens – diffusible substances able to form gradients in the tissue to enable cells to “read” both direction and the distance from the organizing centers. As opposed to Turing’s idea, these morphogens do not have to form any complex patterns themselves, only a system of long and short gradients whose interpretation by individual cells will eventually result in gradual creation of a complex pattern through the process of iterative refinement. In this lecture we consider an example of a very well studied developmental system – Drosophila embryo – which clearly demonstrates how such patterning does occur in nature. Despite earlier expectations, confirmed morphogens are almost all proteins and not low weight molecules. These are proteins from TGFb, hedgehog and wingless families. Interestingly most of morphogens require preprocessing, such as proteolytic cleavage, to become active. The pro-protein and active forms may also have very different life times ability to diffuse. 2 n LSM5194 The master plan of the Drosophila embryo The creation of the body plan of Drosophila by the gradient forming proteins is perhaps the best understood morphogenetic process. Formation of morphogenetic fields starts on the acellular level of syncytium. Unless specified otherwise, the illustrations are from Wolpert et al, Principles of development. 2002 The development of Drosophila is peculiar in a sense that during some 13 first cell divisions the cell-cell boundaries are not formed and the nuclei divide in one giant cell called syncytium. Only after the newly formed nuclei densely populate the near surface, cortex part of the syncytium the cellular membranes are formed to form one layer of cells that covers the original oocyte as a shell. The formation of pattern defining future body plan starts long before this cellularization process begins. Therefore, in case of early Drosophila development, the morphogen gradients are technically intracellular. The formation of the body plan is governed by five groups of genes which act in a strict sequence and order. The emergence of pattern is mediated by their complex logic of mutual activation and inhibition. 3 n LSM5194 Maternal genes define the body axes Both anterior-posterior and dorsal-ventral axes are defined by deposition of maternal mRNA which is transcribed after fertilization. The anterior end is defined by gene bicoid while the anterior end is defined by a group of 9 genes including nanos and caudal (red image below). For establishment of the gross axis alignment, Drosophila embryo relies on maternal genes. They are deposited as highly compact and highly localized stores of RNA which begin to be translated into protein shortly after fertilization and long before zygotic transcription begins. This maternal control is exerted by at least 50 different genes while from the theoretical viewpoint it would suffice only two genes to define the two axes. This proves highly redundant and thus robust character of the development. The anterior end of the embryo is defined by gene bicoid while posterior is defined by a group of genes with main genes being caudal and nanos. Bicoid as a transcription factor and acts directly as a morphogen by regulating downstream gap genes. Nanos, on the other hand exists to establish a gradient of another important gene – hunchback, as it suppresses its translation in the posterior end. 4 n LSM5194 Dorso-ventral axis uses different mechanism Specification of dorso-ventral axis occurs through activation of maternally deposited gene dorsal by means of extracellular ligand spatzle deposited on the ventral part of the egg shell through activation of Toll signaling pathway (analogue of the NF-kB pathway). Very different from the previous slide mechanism is used to specify dorsoventral axis. In this case the mRNA for the gene dorsal (NF-kB) is deposited homogeneously throughout the syncytium cortex. However, the inhomogeneity is achieved through spatially heterogeneous action of a signaling pathway which is a homolog of vertebrate NF-kB pathway. The ligand, spatzle, is deposited outside of the egg on the internal surface of the so-called viteline membrane that lines the inner surface of the egg shell. The ligand is deposited on the ventral surface and therefore the pathway is activated only there. 5 n LSM5194 Zygotic genes do actual segmentation work Upon establishment of the gradient of dorsal, the rest of the dorso-ventral pattern consisting of the 6 segments is done by a group of 7 proteins that pattern ventral side (rhomboid, twist and snail) separately from dorsal (dpp, tolloid and zernknullt). The final result is achieved through a complex activation-inhibition pattern of gene expression. Once the initial symmetry breaking gradients are established throughout the syncytium, the more detailed patterning begins. This process is however already defined by zygotically transcribed genes. The dorso-ventral pattern of six segments is a typical example. In the ventral part, the established gradient of dorsal activates twist, rhomboid and snail. The three genes are related by complex pattern of activation and inhibition which results in spatio-temporal pattern of gene expression which is eventually responsible for definition of cell fates. On the dorsal side, the show is run by the gene product of decapentaplegic or simply “dpp”. This is a homolog of BMP4, one of the most important morphogens in the TGFb family. Its gradient is created by complex interaction with other proteins, for example sog (short gastrulation) which we will encounter in the next lecture again. Note that this part of the embryo patterning occurs already on the cellular phase. 6 n LSM5194 Gap genes pattern antero-posterior axis After establishment of the antero-posterior gradient of maternal genes, the zygotic gap genes switch on. Hunchback is directly induced by bicoid while giant, kruppel and knirps are induced downstream of hunchback. All gap genes are transcription factors necessary for the following tissue patterning. The shown spatial patterns of expression result from complex pattern of mutual activation and inhibition. While on the stage of syncytium, the gap genes are induced by the maternal genes whose gradients have already been established. Bicoid directly induces hunchback. As shown on the slide there is low critical concentration of bicoid below which hunchback expression is not activated. The location of this threshold in the embryo defines the position of sharp hunchback boundary. Multistriped patterns of giant, kruppel and knirps are results of similar transformation of smooth gradients into binary outcome by use of thresholds. As shown on the slide, the stripe of kruppel forms on the decaying gradient of hunchback under the existence of two thresholds – for activation and inhibition. 7 n LSM5194 Segmentation and pair-rule genes Shortly after the gap genes, pair-rule genes eve and fushi tarazu create 14 stripes of alternating expression which will become after some modification the segments of the larval body. Each strip has its own genetic control and is not a result of a periodic process! Emergence of periodic stripes of expression of genes even skipped (eve) and fushi tarazu is a magnificent pattern formation phenomenon. Various hypothesis and models were proposed to explain periodic stripes. To the great surprise of both experimentalists and theorists, each stripe is individually controlled by a specific combination of activation and inhibition by the gap genes. Not surprisingly, the regulation regions of eve gene are the most explored and the best understood gene control units up to date. Shown here as an example is the anatomy of the second stripe of eve with the corresponding details of the regulatory element on the eve gene. Overall, the expression of eve (in this stripe) is activated by bicoid and hunchback while giant and kruppel inhibit it. The group of pair-rule genes includes already known to you gene hairy. 8 n LSM5194 Segment polarity genes finalize segmentation Segment polarity genes finally mark the boundaries of the body segments and provides them with antero-posterior direction. The genes of this group code for such important morphogens like wingless, engrailed and hedgehog and they are connected by complex interaction network. 9 n LSM5194 Selector and homeotic genes define future of segments Finally, the future of the Drosophila segments is defined by homeotic and selector genes which are homologous to the Hox genes of vertebrates. These genes can change the whole organ into another organ. For example, mutation antennapedia results in substitution of antennae by legs. The relationship to simple gradients can hardly be followed on this level of development. The complexity on this level exceeds the scope of this lecture. Just remember that these genes define which segments will turn into which parts of a body. 10 n LSM5194 What to take home • Morphogenesis in real biological systems is controlled by complex networks of morphogens • Each of them forms only simple gradient, yet the combination of these simple prepatterns being superimposed on each other can define layout of any complexity • The relative importance of morphogen gradients diminishes with the size of embryo and with the level of development • To achieve necessary robustness and scale independence, morphogenetic networks must possess high redundancy and non-linearity achieved through multiple positive and negative feedback loops 11