NPTEL Biotechnology - Systems Biology Drosophila melanogaster- Morphogen Gradient Dr. M. Vijayalakshmi School of Chemical and Biotechnology SASTRA University Joint Initiative of IITs and IISc Funded by MHRD Page 1 of 10
NPTEL Biotechnology - Systems Biology Table of Contents 1 INTRODUCTION... 3 1.1 MECHANISTIC ISSUES IN MORPHOGEN GRADIENT... 6 1.2 QUANTITATIVE MODELS OF (MORPHOGEN) GRADIENT FORMATION... 6 1.3 CONSIDERATIONS OF THE CRICK S MODEL... 7 1.4 MORPHOGEN GRADIENTS DUE TO DIFFUSION... 7 2 REFERENCES... 10 2.1 TEXT BOOK... 10 2.2 LITERATURE REFERENCES... 10 2.4 VIDEO LINK... 10 Joint Initiative of IITs and IISc Funded by MHRD Page 2 of 10
1 Introduction Challenges during the developmental phases of multi cellular organisms from a single fertilized cell to a development of a matured organism have been a fascination for developmental biologists. Such concepts have evolved even from the time of Aristotle who first related the complexity and organization of the embryo to the phenomenon of development. This was followed by observations in the 19 th century that cell proliferation and differentiation were two key events in development. Driesch s experiments on sea urchin blastomeres (Driesh 1891, 1908) clearly indicated that cell proliferation and differentiation should be controlled at the level of whole organism in order to achieve the correct morphological pattern and size in organisms. This led to a postulate that the phenomenon of regeneration is influenced by gradients of formative substances (Morgan 1901). Boveri and Horstadius s experiments on embryo patterning established patterning by gradients of form providing substances in the sea urchin embryo while the Spemann organizer - a group of dorsal cells (to gastrula) induce a secondary body access when grafted to the opposite ventral pore of a host gastrula. Spemann hypothesized that morphogenesis could result from the action of signals released from localized group of cells to induce differentiation of surrounding cells (De Robertis 2006). Experiments in 1941 by Child established that these patterning signals might be representative metabolic gradients. But the mechanisms of formation of these gradients, their regulation and emergence into distinctive patterns were poorly understood. Turing s reaction diffusion model of 1952 demonstrated that chemical substances (morphogens) would self organize into spatial patterns starting from homogenous distributions. His model clearly showed that two or more morphogens can generate spatial patterns of morphogen concentration if they differ slightly in their diffusion properties and when they react through auto or cross catalysis or through inhibition of their production. Turing was the first to term these chemical substances morphogens to convey the meaning form producers. Joint Initiative of IITs and IISc Funded by MHRD Page 3 of 10
Fig 1. A schematic of Morphogen concentrations with varying distances Lewis Wolpert s French flag model in Fig 2. (Wolpert 1969) explained how positional information is generated inside the embryo. This model suggested that morphogen is secreted from a group of source cells and forms a concentration gradient in the target tissues. The different target genes are expressed when their expression levels cross distinct concentration thresholds at varying distances from the source. This therefore generates a spatial pattern of gene expression. Joint Initiative of IITs and IISc Funded by MHRD Page 4 of 10
Morphogen concentration Tissue patterning by morphogen gradients was evident from experiments performed in the 1970s, the most striking of them being Sander s experiment which showed that a Morphogen released from the posterior cytoplasm of an insect egg specifies its anterior posterior position (Sander 1976). Experiments from Nusslein-Volhard laboratory identified the function of the bicoid gene in the Drosophila melanogaster embryo and its gradient through antibody staining (Nusslein-Volhard and Wieschaus 1980, Nusslein-Volhard 1986, 1987,1988). T 1 T 2 Step 1 Step 2 Fig 2. French Flag model demonstrating morphogen gradients These models clearly establish that the signal produced from the localized source spreads as a concentration gradient through surrounding tissues and the gradient signal acts directly on cells dependent on the signal concentration and specifies changes in gene expression. Both vertebrate and invertebrate genes signalling at the tissue level are dictated by a variety of molecules that function as graded signals. Such signals are involved in establishing initial polarities in the embryo, providing tissue specific cellular identity especially in the limb appendages and in the nervous systems in vertebrates as well as in Drosophila melanogaster (Gorden and Bourillot, 2001; Tabata and Takei, 2004). Joint Initiative of IITs and IISc Funded by MHRD Page 5 of 10
1.1 Mechanistic issues in Morphogen gradient The concept of morphogen gradient provides a robust framework for understanding pattern formation in Drosophila and vertebrates. But the mechanical understanding of this phenomenon remains poorly interpreted. The mechanism by which a gradient morphogen signal is transformed into alterations in gene expression programs and the accurate spatial pattern of cellular differentiation needs to be addressed with a systematic approach. The questions to be addressed involve 1. How is graded information transmitted at the intracellular level to control concentration dependent differential gene expression? 2. How do discrete changes in gene expression influence cell fate decisions? 3. How does the signal gradient treat fluctuations in biological conditions to make the system robust for accurate developmental patterning? 4. How many thresholds does a particular morphogen control? Aligning with the definition of a morphogen, a signal gradient should be capable of directing generation of at least two different types of distinct cell types at different concentrations. Though theoretical evidence supports control up to 30 concentration gradients (Louis et al., 1977) empirical evidence promotes between 3 to 7 distinct threshold gradient signals for patterning. The dorsal gradient has been shown to specify between 4 and 7 distinct thresholds along the dorsoventral axis of Drosophila embryos (Stathopoulos and Levin, 2008). Xenopus blastula cells have been shown to respond to 5 cellular states to a concentration gradient of activin (Green et al., 1992). 1.2 Quantitative models of (Morphogen) gradient formation Several quantitative models of formation of the Morphogen gradient have been proposed to explain the process of pattern formation in organisms. Crick s model of 1970 demonstrated that freely diffusing morphogen produced in a source cell and destroyed in a sink cell at a distance develops a linear gradient over developmentally relevant time scales. This concept is not relevant to today s Joint Initiative of IITs and IISc Funded by MHRD Page 6 of 10
thinking that a gradient formation does not require a localized sink and that gradients can form if all cells act as sinks and degrade or not degrade morphogen. 1.3 Considerations of the Crick s model Crick s model presents the diffusion of morphogen over a row of cells. This one dimensional model can be generalized to two or three dimensional epithelial or mesenchymal tissues. This model is evolved on the basis that a Morphogen produced in the source spreads to the target tissue and is degraded. The spatio temporal changes in the concentration of the morphogen c due to morphogen production, spreading and degradation can be used to describe the formation of gradients. A steady state is reached when the processes of production and degradation are equilibrated so that the gradient is un altered. Changes in the concentration of Morphogen in tissues can be explained using cellular processes like extra cellular diffusion, internalization, intra and extra cellular degradation. These models vary with underlying transport mechanisms specific for different morphogens and tissues. Gradient formation can be explained by continuum models, introducing random walk inclusive of diffusion coefficients in effective degradation rates. These models also capture the biophysical behavior of the system that regulates morphogen transport and dictates gradient shapes. 1.4 Morphogen gradients due to diffusion Let us assume an ideal condition which does not involve degradation or depleting effects. A non directional spreading of the Morphogen through random walk and the consequent gradient formation in the target can now be described by Fick s second law of diffusion. Joint Initiative of IITs and IISc Funded by MHRD Page 7 of 10
Fig 3. Spatial Diffusion model of morphogen gradients (1) where one observes that the rate of change of concentration c/ t is proportional to the second derivative of the concentration with respect to space. Here D refers to the diffusion coefficient. Joint Initiative of IITs and IISc Funded by MHRD Page 8 of 10
The derivation for the same has been presented in page no 9 in Ortrud et al., Morphogen gradient formation Since this model assumes no depleting effects on molecules, the equation 1 does not contain a negative term. Due to constant secretion of molecules from the source to the target, obeying the boundary conditions, the concentration of the tissue can be shown to constantly increase with distance Fig 3. Such diffusion and morphogen production events lead to Gaussian gradients which do not define steady states (Berg 1993). Work by Coppey et al has shown under no degradation, the formation of quasi stable gradients of the transcription factor bicoid in the syncytium of the Drosophila embryo. The diffusivity of the bicoid gradient is decreased with the increase in the nuclear density caused by the nuclear divisions in the syncytium, hence stabilizing the nuclear bicoid concentration over several nuclear divisions. When cell fate decisions are made before the gradient reaches the steady state, the pattern formation is decided by the threshold gene expression of the target and the time in which cells respond to the morphogen gradient. The signalling time window influences cellular response and as shown to be effective in regulating Shhs in the neural tube (Dessaud et al., 2007) and active in Xenopus mesoderm formation.(gurdon and Bourillot 2001). Once target gene domains are assigned, these depend on downstream signalling cascade across different target genes, besides its dependence on the morphogen gradient as shown by (Bergmann et al., 2007) for the Drosophila melanogaster Gap genes. Such dependence might enhance robustness in patterning against fluctuations in morphogen s production. Joint Initiative of IITs and IISc Funded by MHRD Page 9 of 10
2 References 2.1 Text Book 1. Lewis Wolpert, Principles of Development, 2/e, Oxford University Press, (2002). 2.2 Literature references 1. Nusslein-Volhard C, Wieschaus E, Mutations affecting segment number and polarity in Drosophila, Nature, 287, 795-801. 2. Coppey M et al., Modelling the bicoid gradient: diffusion and reversible nuclear trapping of a stable protein, Developmental Biology, (2007), 15, 623-630. 3. Gurdon JB et al., Morphogen gradient interpretation, Nature, (2001), 413, 797-803. 4. Nusslein-Volhard C, The bicoid protein determines position in the Drosophila embryo in a concentration dependent manner,cell,(1998), 54, 95-104. 2.4 Video Link 1. Eric Wieschaus part 1: Patterning development in the embryo- Youtube. 2. Eric Wieschaus part 2: Stability of Morphogen gradients and movement of molecules- Youtube. Joint Initiative of IITs and IISc Funded by MHRD Page 10 of 10