Recent work on Drosophila limb development has indicated that short-range interactions between distinctly specified populations of cells (compartments) establish organizing centers at compartment boundaries. These organizing centers direct pattern formation and growth in the developing limbs. In the Drosophila wing imaginal disc, there are at least two such organizing centers, located at the anterior/posterior (A/P) and dorsal/ventral (D/V) compartment boundaries. The genetic hierarchies which establish these organizers are starting to be understood, and it also appears that the key mediators of some of the organizers have been identified. Thus Decapentaplegic (Dpp, a secreted signalling molecule of the TGF-B family) is the mediator of the A/P organizer, while Wingless (DWnt-l, a secreted molecule of the Wnt family) is a key mediator of the D/V organizer. In this thesis, several aspects of Wingless function in the wing imaginal disc are examined. Two regulatory mutations, spadeflag (spdfg) and Sternopleural (Sp), that affect Wingless expression in the wing imaginal disc are characterized. The analysis of the mutation spdfg, together with other data, identifies a role of Wingless as a localized mitogen in the developing wing hinge, and also indicates that cells in different regions of the wing disc respond very differently to the Wingless signal. The mutations spdfg and Sp are also among the tools used to examine the position of Wingless in the genetic hierarchy that establishes and mediates the activity of the D/V organizer. These experiments extend the evidence suggesting that Wingless mediates both short-range and long-range effects of the D/V organizer. Wg does so by controlling the expression domains of different target genes, including the acheate-scute genes, Distal-less and vestigial. Finally, the mechanism by which Wingless mediates the activity of the D/V organizer is examined. The results obtained suggest that Wingless functions as a long-range morphogen.

PhD Thesis / Morphogens (often acting as transcription activators or repressors) govern pattern formation and cell differentiation during early embryogenesis. Abnormal distributions of morphogens can result in developmental defects or even death. Oftentimes, thresholds of concentrations of morphogens behave like an ON/OFF switch for the activation or repression of downstream genes. Accurate measurements of morphogen concentration and mobility in space and time can help tackle the puzzle of how exactly cascades of hundreds of morphogens coordinate their targets precisely and promptly amidst crowded and complicated cellular environments. The research question at the centre of my thesis is that of the concentration and dynamics of two morphogens with opposite functions in the early fly embryo. In the work presented in this thesis, we use Fluorescence Correlation Spectroscopy (FCS) and confocal imaging to achieve extremely low ($\sim$ nM) concentration measurements in live \textit{Drosophila} embryos expressing recombinant fluorescent morphogens, by carefully taking into account background noise and photobleaching effects. The dynamics of both Bicoid (Bcd) and Capicua (Cic), an activator and a repressor morphogens, were further studied using FCS, Fluorescence Recovery After Photobleaching (FRAP) and Monte Carlo simulation. We found that both types of morphogens are very mobile in nuclei, explaining how they are able to turn on or off gene expression in only a few minutes. However, these two morphogens with opposite functions have drastically different nucleo-cytoplasmic transport behaviours, where the activator can pass through the nuclear envelop (NE) relatively freely while the repressor is jailed inside nuclei during interphase. These findings can provide clues to distinguish between several hypothetical models (including the newly proposed hub hypothesis) trying to explain the mechanisms of target gene search and transcription regulation. In this thesis, a background introduction on transcription factors and morphogens is given in Chapter 1, with a focus on the two transcription factors (the activator Bicoid and the repressor Capicua) studied in this thesis. Next, experimental details such as fruit fly maintenance, and fluorescent techniques used to measure concentration and mobility are described in Chapter 2. From Chapter 3 to Chapter 5, three manuscripts from the thesis author, either published or in preparation for submission are presented in sequence. Chapter 3 introduces a new method to accurately measure protein concentration in the presence of noise and photobleahing in early \textit{Drosophila} embryos using FCS. Chapter 4 contains the results of concentration and mobility measurements for Cic which contribute to the finding that Cic acts like a fast brake in transcription repression. Chapter 5 compares the similarities and differences of the dynamics of Bcd and Cic through multiple lenses. Finally, a conclusion and future outlook are given in Chapter 6. / Thesis / Doctor of Philosophy (PhD) / Have you ever wondered how a single fertilized egg turns miraculouly into a beautifully organized living being, be it an insect, a cat, or a human? It turns out that an important group of molecules called morphogens govern the formation of body pattern. These molecules (usually proteins) form concentration gradients along the different body axes of that organism and influence gene expression. Abnormal distribution of morphogen can result in defects in embryo development and even death. Thus knowing how much morphogen is present in the early developing embryo, as well as how it forms gradients and how the morphogen concentration is translated into a pattern can help us better understand early embryo development. My thesis focuses on accurate measurements of morphogen concentrations and dynamics using fluorescence techniques. We were able to obtain concentration maps for two morphogens, the activator Bicoid and the repressor Capicua, in early developing fruit fly embryos. We also found that despite having opposite functions, the activator and the repressor have similar intranuclear dynamics, but drastically different internuclear mobility. Our findings provide clues to distinguish between multiple hypothetical models scientists have put forward to explain the mechanisms of transcription regulation.

Dresden, Techn. Univ., Diss., 2005.

The expression of the Drosophila melanogaster morphogen Hedgehog (Hh) plays a key role in co-ordinating proliferation and differentiation during animal development. Tight spatial and temporal regulation of Hh expression is essential for its correct function in these essential processes. Both mis-expression of its mammalian orthologue Sonic Hedgehog (Shh) and aberrant stimulation of the associated signalling pathway occur in a wide range of human tumours. Although there is extensive knowledge of the signal transduction pathway that is activated in a Hh-stimulated cell, very little is known about pathways governing the expression of the morphogen itself. The Drosophila tumour suppressor protein Hyperplastic Discs (Hyd), an E3 ubiquitin ligase, negatively regulates hedgehog (hh) expression and Hh pathway activity by independent mechanisms in the developing Drosophila eye. Genetically generated hyd mutant clones in the eye mis-express hh and the transcriptional activator of Hh target genes, Cubitus interruptus (Ci), and cause overgrowth of the surrounding wildtype tissue. However, the underlying molecular mechanism(s) by which Hyd regulates these morphogen regulatory pathways is not known. Hyd may be involved in ubiquitylating target proteins in these pathways, which could have degradative or non-degradative outcomes. In order to elucidate Hyd’s molecular role in potential morphogen regulatory pathways, I applied a proteomics-based approach to identify novel Hyd binding partners and ubiquitylated substrates. Tandem affinity purification in combination with mass spectrometry was used to purify and identify Hyd and its complexed binding partners from Drosophila cells. Binding and ubiquitylation assays were subsequently used to verify and characterize the interactions. In addition, a biased, literature-guided approach was applied to identify likely Hyd binding partners based on their involvement in morphogen signalling and conservation across species. Finally, to assess the functional consequences of a newly identified interaction, I used a Drosophila in vivo model to determine whether the novel binding partner was capable of modifying the hyd mutant phenotype. For this purpose, the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique was used to generate hyd mutant clones in the developing larval eye, which were expressing transgenes resulting in either the over-expression or RNAi-mediated knockdown of the gene of interest. My results indicate that Hyd is involved in regulating both Hh and Wg morphogen signalling in the Drosophila eye, and that the molecular mechanism of action may, at least in part, involve the protein kinase Shaggy (Sgg). Hyd interacts with the Hh and Wg transcriptional activator proteins Ci and Armadillo, respectively, as well as the Sgg kinase. Sgg is a negative regulator of both the Hh and Wg pathways, and acts to direct the proteolytic processing or degradation of the transcriptional effectors of these morphogen pathways. Sgg and its mammalian orthologue GSK3β were ubiquitylated in vitro, and GSK3β ubiquitylation was negatively regulated by the mammalian homologue of Hyd, EDD. Knockdown of sgg in eye disc cells mutant for hyd resulted in a dramatic rescue of the overgrowth phenotype. Loss of hyd in clones located in the anterior region of the eye disc resulted in low levels of the full-length Hh transcriptional activator protein Ci. This effect was reversed completely as a result of sgg knockdown. Furthermore, loss of hyd in eye disc clones resulted in elevated Hh and Wg morphogen expression. Mis-expression of hh in hyd mutant clones was significantly reduced upon over-expression of a constitutively active Sgg kinase. Hence sgg appears to genetically act downstream of hyd to regulate hh gene expression and Ci expression. In summary my results identify Sgg as a novel regulator of hh gene expression, whose activity may be regulated by ubiquitylation, and which may be acting downstream of Hyd in a ubiquitin-regulated manner to control both hh gene expression and Hh pathway activity in the developing Drosophila eye. Hyd may also regulate Hh pathway activity by directly interacting with Ci and affecting its activity. The results also indicate that Hyd may be a master regulator of both Hh and Wg morphogen signalling during Drosophila development.

No description available.

Morphogens are signaling molecules that play a key role in animal development. They spread from a restricted source into an adjacent target tissue forming a concentration gradient. The fate of cells in the target tissue is determined by the local concentration of such morphogens. Morphogen transport through the tissue has been studied in experiments which lead to the suggestion of several transport mechanisms. While diffusion in the extracellular space contributes to transport, recent experiments on the morphogen Decapentaplegic (Dpp) in the fruit fly Drosophila provide evidence for the importance of a cellular transport mechanism that was termed "planar transcytosis". In this mechanism, morphogens are transported through cells by repeated rounds of internalization and externalization. Starting from a microscopic theoretical description of these processes, we derive systems of nonlinear transport equations which describe the interplay of transcytosis and passive diffusion. We compare the results of numerical calculations based on this theoretical description of morphogen transport to recent experimental data on the morphogen Dpp in the Drosophila wing disk. Agreement with the experimental data is only achieved if the parameters entering the theoretical description are chosen such that transcytosis contributes strongly to transport. Analyzing the derived transport equations, we find that transcytosis leads to an increased robustness of the created gradients with respect to morphogen over-expression. Indications for this kind of robustness have been found in experiments. Furthermore, we theoretically investigate morphogen gradient formation in disordered systems. Here, an important question is how the position of concentration thresholds can be defined with high precision in the noisy environment present in typical developing tissues. Among other things, we find that the dimensionality of the system in which the gradient is formed plays an important role for the precision. Comparing gradients formed by transcytosis to those formed by extracellular diffusion, we find substantial differences that may result in a higher precision of gradients formed by transcytosis. Finally, we suggest several experiments to test the theoretical predictions of this work.

Morphogens are signaling molecules that play a key role in animal development. They spread from a restricted source into an adjacent target tissue forming a concentration gradient. The fate of cells in the target tissue is determined by the local concentration of such morphogens. Morphogen transport through the tissue has been studied in experiments which lead to the suggestion of several transport mechanisms. While diffusion in the extracellular space contributes to transport, recent experiments on the morphogen Decapentaplegic (Dpp) in the fruit fly Drosophila provide evidence for the importance of a cellular transport mechanism that was termed "planar transcytosis". In this mechanism, morphogens are transported through cells by repeated rounds of internalization and externalization. Starting from a microscopic theoretical description of these processes, we derive systems of nonlinear transport equations which describe the interplay of transcytosis and passive diffusion. We compare the results of numerical calculations based on this theoretical description of morphogen transport to recent experimental data on the morphogen Dpp in the Drosophila wing disk. Agreement with the experimental data is only achieved if the parameters entering the theoretical description are chosen such that transcytosis contributes strongly to transport. Analyzing the derived transport equations, we find that transcytosis leads to an increased robustness of the created gradients with respect to morphogen over-expression. Indications for this kind of robustness have been found in experiments. Furthermore, we theoretically investigate morphogen gradient formation in disordered systems. Here, an important question is how the position of concentration thresholds can be defined with high precision in the noisy environment present in typical developing tissues. Among other things, we find that the dimensionality of the system in which the gradient is formed plays an important role for the precision. Comparing gradients formed by transcytosis to those formed by extracellular diffusion, we find substantial differences that may result in a higher precision of gradients formed by transcytosis. Finally, we suggest several experiments to test the theoretical predictions of this work.

Dresden, Techn. Univ., Diss., 2005.

The correct patterning of embryos requires the exchange of information between cells. This is in part achieved by the proper distribution of signaling molecules, many of which exert their function by establishing gradients of concentration. Because of this property they were named "morphogens", or "form giving" substances. Among these, proteins belonging to the Hedgehog (Hh) family have received much attention, owing to their unusual double lipid modification and their involvement in human disease, causing congenital birth defects and cancer. Great efforts have been made in order to elucidate the mechanisms by which Hh molecules are propagated in the embryo. However, no conclusive evidence exists to date to which structures these molecules localize and how they, despite their membrane association, establish a gradient of concentration. Therefore, I decided to study the distribution of the vertebrate Hh homolog, Sonic Hedgehog (Shh) in developing zebrafish embryos. By fluorescently tagging Shh proteins, I found that these localize to discrete punctate structures at the membranes of expressing cells. These were often regions from which filopodial protrusions emanated from the cells. Puctate deposits of Shh were also located outside of expressing cells. In dividing cells, Shh accumulated at the cleavage plane. Furthermore, by making use of confocal microscopy and time lapse analysis, I visualized Shh proteins moving in filopodial extensions present between cells. This suggests a novel mechanism of Shh distribution, which relies on the direct contact of cells by filopodia for the exchange of signaling proteins. In a second part of my thesis, I characterized genes implicated in regulating Shh protein distribution and signaling function. I cloned three zebrafish genes belonging to the Ext1 (exostosin) family of glycosyltransferases required for the synthesis of Heparan Sulfate Proteoglycans and established a tentative link of these genes to somitic Hh signaling. In addition, I characterized the developmental expression and function of zebrafish Rab23, a small GTPase, which acts as a negative regulator of the Shh signaling pathway. Performing knock-down experiments of zebrafish Rab23, I found that Rab23 functions in left-right axis specification, a process previously shown to depend on proper Shh signaling.

The correct patterning of embryos requires the exchange of information between cells. This is in part achieved by the proper distribution of signaling molecules, many of which exert their function by establishing gradients of concentration. Because of this property they were named "morphogens", or "form giving" substances. Among these, proteins belonging to the Hedgehog (Hh) family have received much attention, owing to their unusual double lipid modification and their involvement in human disease, causing congenital birth defects and cancer. Great efforts have been made in order to elucidate the mechanisms by which Hh molecules are propagated in the embryo. However, no conclusive evidence exists to date to which structures these molecules localize and how they, despite their membrane association, establish a gradient of concentration. Therefore, I decided to study the distribution of the vertebrate Hh homolog, Sonic Hedgehog (Shh) in developing zebrafish embryos. By fluorescently tagging Shh proteins, I found that these localize to discrete punctate structures at the membranes of expressing cells. These were often regions from which filopodial protrusions emanated from the cells. Puctate deposits of Shh were also located outside of expressing cells. In dividing cells, Shh accumulated at the cleavage plane. Furthermore, by making use of confocal microscopy and time lapse analysis, I visualized Shh proteins moving in filopodial extensions present between cells. This suggests a novel mechanism of Shh distribution, which relies on the direct contact of cells by filopodia for the exchange of signaling proteins. In a second part of my thesis, I characterized genes implicated in regulating Shh protein distribution and signaling function. I cloned three zebrafish genes belonging to the Ext1 (exostosin) family of glycosyltransferases required for the synthesis of Heparan Sulfate Proteoglycans and established a tentative link of these genes to somitic Hh signaling. In addition, I characterized the developmental expression and function of zebrafish Rab23, a small GTPase, which acts as a negative regulator of the Shh signaling pathway. Performing knock-down experiments of zebrafish Rab23, I found that Rab23 functions in left-right axis specification, a process previously shown to depend on proper Shh signaling.