Tobias Bollenbach

Interstitial dendritic cell guidance by haptotactic chemokine gradients

A Well-Defined Path Although chemokines have long been thought to direct immune cell movements within tissues, a formal in vivo demonstration and detailed understanding are lacking. By tracking dendritic cell movements in the ears of mice, Weber et al. (p. 328) were able to provide both. Endogenous gradients of the chemokine CCL21 were observed in ear tissue and, at distances of up 90 µm, dendritic cells were able to use these gradients to migrate directionally toward lymphatic vessels. The CCL21 gradient was immobilized on heparan sulfates and disruption of the gradient inhibited dendritic cell migration. In mouse skin, immune cells migrate toward lymphatic vessels along an immobilized chemokine gradient. Directional guidance of cells via gradients of chemokines is considered crucial for embryonic development, cancer dissemination, and immune responses. Nevertheless, the concept still lacks direct experimental confirmation in vivo. Here, we identify endogenous gradients of the chemokine CCL21 within mouse skin and show that they guide dendritic cells toward lymphatic vessels. Quantitative imaging reveals depots of CCL21 within lymphatic endothelial cells and steeply decaying gradients within the perilymphatic interstitium. These gradients match the migratory patterns of the dendritic cells, which directionally approach vessels from a distance of up to 90-micrometers. Interstitial CCL21 is immobilized to heparan sulfates, and its experimental delocalization or swamping the endogenous gradients abolishes directed migration. These findings functionally establish the concept of haptotaxis, directed migration along immobilized gradients, in tissues.

Oct4 kinetics predict cell lineage patterning in the early mammalian embryo

Transcription factors are central to sustaining pluripotency, yet little is known about transcription factor dynamics in defining pluripotency in the early mammalian embryo. Here, we establish a fluorescence decay after photoactivation (FDAP) assay to quantitatively study the kinetic behaviour of Oct4, a key transcription factor controlling pre-implantation development in the mouse embryo. FDAP measurements reveal that each cell in a developing embryo shows one of two distinct Oct4 kinetics, before there are any morphologically distinguishable differences or outward signs of lineage patterning. The differences revealed by FDAP are due to differences in the accessibility of Oct4 to its DNA binding sites in the nucleus. Lineage tracing of the cells in the two distinct sub-populations demonstrates that the Oct4 kinetics predict lineages of the early embryo. Cells with slower Oct4 kinetics are more likely to give rise to the pluripotent cell lineage that contributes to the inner cell mass. Those with faster Oct4 kinetics contribute mostly to the extra-embryonic lineage. Our findings identify Oct4 kinetics, rather than differences in total transcription factor expression levels, as a predictive measure of developmental cell lineage patterning in the early mouse embryo.

Nonoptimal Microbial Response to Antibiotics Underlies Suppressive Drug Interactions

Suppressive drug interactions, in which one antibiotic can actually help bacterial cells to grow faster in the presence of another, occur between protein and DNA synthesis inhibitors. Here, we show that this suppression results from nonoptimal regulation of ribosomal genes in the presence of DNA stress. Using GFP-tagged transcription reporters in Escherichia coli, we find that ribosomal genes are not directly regulated by DNA stress, leading to an imbalance between cellular DNA and protein content. To test whether ribosomal gene expression under DNA stress is nonoptimal for growth rate, we sequentially deleted up to six of the seven ribosomal RNA operons. These synthetic manipulations of ribosomal gene expression correct the protein-DNA imbalance, lead to improved survival and growth, and completely remove the suppressive drug interaction. A simple mathematical model explains the nonoptimal regulation in different nutrient environments. These results reveal the genetic mechanism underlying an important class of suppressive drug interactions.

Precision of the Dpp gradient

Morphogen concentration gradients provide positional information by activating target genes in a concentration-dependent manner. Recent reports show that the gradient of the syncytial morphogen Bicoid seems to provide precise positional information to determine target gene domains. For secreted morphogenetic ligands, the precision of the gradients, the signal transduction and the reliability of target gene expression domains have not been studied. Here we investigate these issues for the TGF-β-type morphogen Dpp. We first studied theoretically how cell-to-cell variability in the source, the target tissue, or both, contribute to the variations of the gradient. Fluctuations in the source and target generate a local maximum of precision at a finite distance to the source. We then determined experimentally in the wing epithelium: (1) the precision of the Dpp concentration gradient; (2) the precision of the Dpp signaling activity profile; and (3) the precision of activation of the Dpp target gene spalt. As captured by our theoretical description, the Dpp gradient provides positional information with a maximal precision a few cells away from the source. This maximal precision corresponds to a positional uncertainly of about a single cell diameter. The precision of the Dpp gradient accounts for the precision of the spalt expression range, implying that Dpp can act as a morphogen to coarsely determine the expression pattern of target genes.

Discovery of a Small Molecule that Blocks Wall Teichoic Acid Biosynthesis in Staphylococcus aureus

Both Gram-positive and Gram-negative bacteria contain bactoprenol-dependent biosynthetic pathways expressing non-essential cell surface polysaccharides that function as virulence factors. Although these polymers are not required for bacterial viability in vitro, genes in many of the biosynthetic pathways are conditionally essential: they cannot be deleted except in strains incapable of initiating polymer synthesis. We report a cell-based, pathway-specific strategy to screen for small molecule inhibitors of conditionally essential enzymes. The screen identifies molecules that prevent the growth of a wildtype bacterial strain but do not affect the growth of a mutant strain incapable of initiating polymer synthesis. We have applied this approach to discover inhibitors of wall teichoic acid (WTA) biosynthesis in Staphylococcus aureus. WTAs are anionic cell surface polysaccharides required for host colonization that have been suggested as targets for new antimicrobials. We have identified a small molecule, 7-chloro-N,N-diethyl-3-(phenylsulfonyl)-[1,2,3]triazolo[1,5-a]quinolin-5-amine (1835F03), that inhibits the growth of a panel of S. aureus strains (MIC = 1-3 microg mL(-1)), including clinical methicillin-resistant S. aureus (MRSA) isolates. Using a combination of biochemistry and genetics, we have identified the molecular target as TarG, the transmembrane component of the ABC transporter that exports WTAs to the cell surface. We also show that preventing the completion of WTA biosynthesis once it has been initiated triggers growth arrest. The discovery of 1835F03 validates our chemical genetics strategy for identifying inhibitors of conditionally essential enzymes, and the strategy should be applicable to many other bactoprenol-dependent biosynthetic pathways in the pursuit of novel antibacterials and probes of bacterial stress responses.

Robust formation of morphogen gradients

We discuss the formation of graded morphogen profiles in a cell layer by nonlinear transport phenomena, important for patterning developing organisms. We focus on a process termed transcytosis, where morphogen transport results from the binding of ligands to receptors on the cell surface, incorporation into the cell, and subsequent externalization. Starting from a microscopic model, we derive effective transport equations. We show that, in contrast to morphogen transport by extracellular diffusion, transcytosis leads to robust ligand profiles which are insensitive to the rate of ligand production.

Dpp gradient formation by dynamin-dependent endocytosis: receptor trafficking and the diffusion model

Developing cells acquire positional information by reading the graded distribution of morphogens. In Drosophila, the Dpp morphogen forms a long-range concentration gradient by spreading from a restricted source in the developing wing. It has been assumed that Dpp spreads by extracellular diffusion. Under this assumption, the main role of endocytosis in gradient formation is to downregulate receptors at the cell surface. These surface receptors bind to the ligand and thereby interfere with its long-range movement. Recent experiments indicate that Dpp spreading is mediated by Dynamin-dependent endocytosis in the target tissue, suggesting that extracellular diffusion alone cannot account for Dpp dispersal. Here, we perform a theoretical study of a model for morphogen spreading based on extracellular diffusion, which takes into account receptor binding and trafficking. We compare profiles of ligand and surface receptors obtained in this model with experimental data. To this end, we monitored directly the pool of surface receptors and extracellular Dpp with specific antibodies. We conclude that current models considering pure extracellular diffusion cannot explain the observed role of endocytosis during Dpp long-range movement.

Coordination of progenitor specification and growth in mouse and chick spinal cord

Introduction The hallmarks of development are tissue growth and the generation of cell diversity, resulting in reproducibly patterned animals. Yet, how growth and cell fate specification are coordinated to determine the variety of cell types and the proportions of their populations is not well understood. The specification of cell types can be controlled by long-range signals, called morphogens. In some tissues, the shape of morphogen gradients is controlled to match the rate at which the tissue grows, which may keep the pattern proportional to the overall tissue size. To understand whether a similar strategy applies to the patterning of the spinal cord, we measured growth and cell fate specification in chick and mouse embryos of normal size, as well as mutant mice of smaller size. Growth and patterning of the spinal cord. Progenitors are organized in a striped pattern, revealed by their gene expression. In animals of different size, the pattern changes in the same way as the spinal cord grows. This is controlled sequentially, first by progenitor specification by opposing morphogen gradients (bottom left), then by cell-type–specific differentiation resulting in differently shaped clones (green, bottom right). Rationale Morphogen gradients emanating from the dorsal and ventral sides of the spinal cord establish a striped pattern of gene expression in neural progenitors along the dorsoventral axis. Ventrally, Sonic Hedgehog (Shh) is the key morphogen. As the tissue grows in size, the levels of Shh activity in progenitors decrease and are not constant at progenitor domain boundaries. This prompted us to examine whether growth contributed to pattern formation. To this end, we measured the four parameters that control the number of progenitors in a domain: (i) cell proliferation, (ii) cell death, (iii) terminal differentiation of progenitors into postmitotic neurons, and (iv) switches in cell identity—morphogen-driven changes in gene expression respecifying one progenitor type into another. Results Unlike systems in which pattern is proportional to size, we found that the relative dorsoventral sizes of neural progenitor domains change continuously during development . These changes in proportions are conserved between mouse, chick, and smaller mouse mutants. Because the proliferation rate of progenitors was spatially uniform and cell death was negligible, neither of these processes could account for the dynamics of pattern formation. Instead, the data revealed two distinct phases of spinal cord development. Initially, the influence of the morphogens dominates, and switches in cell identity establish pattern. During the second phase, domain-specific differentiation rates emerge, causing changes in the relative proportions of progenitor cell populations. Clonal analysis indicated that this effect is anisotropic: Differentiation affects dorsoventral and apicobasal, but not anterioposterior, domain growth. The outcome is that different domains grow in register along the anterioposterior axis. Consistent with the two-phase model, the switches in cell fate and the sensitivity to changes in morphogen signaling decrease as development proceeds. Conversely, experimentally flattening the difference in differentiation rate between domains during the second phase alters pattern. Conclusion The data reveal two phases of neural tube development and show that sequential control of progenitor cell specification and differentiation elaborates pattern without requiring signaling gradients to expand as tissues grow. Control of the differentiation rate is likely to contribute to pattern formation in other tissues. Furthermore, the domain-specific regulation of the differentiation rates could suggest a means to achieve reproducible development despite differences in individual size. Differentiation rates regulate pool sizes Even though a basketball player is bigger than a gymnast, their neural tubes are organized in the same way. Studying chick and mouse embryos, Kicheva et al. show that rates of cell differentiation are key (see the Perspective by Pourquie). In a two-phase process, signaling sweeps through the neural tube early on to establish some aspects of cell fate, but later, pools of progenitor cells take on their own regulation. A progenitor that differentiates is no longer a progenitor, and thus the rate of differentiation determines the size of the progenitor pool. The relative sizes of progenitor pools shift as development progresses, to build the spinal cord so that everyone, large or small, has the right proportion of each component. Science, this issue p. 10.1126/science.1254927; see also p. 1565 Embryonic chick and mouse reveal how neural tube differentiation takes on new strategies as development progresses. [Also see Perspective by Pourquie] Development requires tissue growth as well as cell diversification. To address how these processes are coordinated, we analyzed the development of molecularly distinct domains of neural progenitors in the mouse and chick neural tube. We show that during development, these domains undergo changes in size that do not scale with changes in overall tissue size. Our data show that domain proportions are first established by opposing morphogen gradients and subsequently controlled by domain-specific regulation of differentiation rate but not differences in proliferation rate. Regulation of differentiation rate is key to maintaining domain proportions while accommodating both intra- and interspecies variations in size. Thus, the sequential control of progenitor specification and differentiation elaborates pattern without requiring that signaling gradients grow as tissues expand.

Morphogen transport in epithelia

We present a general theoretical framework to discuss mechanisms of morphogen transport and gradient formation in a cell layer. Trafficking events on the cellular scale lead to transport on larger scales. We discuss in particular the case of transcytosis where morphogens undergo repeated rounds of internalization into cells and recycling. Based on a description on the cellular scale, we derive effective nonlinear transport equations in one and two dimensions which are valid on larger scales. We derive analytic expressions for the concentration dependence of the effective diffusion coefficient and the effective degradation rate. We discuss the effects of a directional bias on morphogen transport and those of the coupling of the morphogen and receptor kinetics. Furthermore, we discuss general properties of cellular transport processes such as the robustness of gradients and relate our results to recent experiments on the morphogen Decapentaplegic (Dpp) that acts in the wing disk of the fruit fly Drosophila.

Investigating the principles of morphogen gradient formation: from tissues to cells.

Morphogen gradients regulate the patterning and growth of many tissues, hence a key question is how they are established and maintained during development. Theoretical descriptions have helped to explain how gradient shape is controlled by the rates of morphogen production, spreading and degradation. These effective rates have been measured using fluorescence recovery after photobleaching (FRAP) and photoactivation. To unravel which molecular events determine the effective rates, such tissue-level assays have been combined with genetic analysis, high-resolution assays, and models that take into account interactions with receptors, extracellular components and trafficking. Nevertheless, because of the natural and experimental data variability, and the underlying assumptions of transport models, it remains challenging to conclusively distinguish between cellular mechanisms.