Alistair Boettiger

All Publications

• Advances in Chromatin Imaging at Kilobase-Scale Resolution. Trends in genetics : TIG Boettiger, A., Murphy, S. 2020

  Abstract

  It is now widely appreciated that the spatial organization of the genome is nonrandom, and its complex 3D folding has important consequences for many genome processes. Recent developments in multiplexed, super-resolution microscopy have enabled an unprecedented view of the polymeric structure of chromatin - from the loose folds of whole chromosomes to the detailed loops of cis-regulatory elements that regulate gene expression. Facilitated by the use of robotics, microfluidics, and improved approaches to super-resolution, thousands to hundreds of thousands of individual cells can now be analyzed in an individual experiment. This has led to new insights into the nature of genomic structural features identified by sequencing, such as topologically associated domains (TADs), and the nature of enhancer-promoter interactions underlying transcriptional regulation. We review these recent improvements.

  View details for DOI 10.1016/j.tig.2019.12.010

  View details for PubMedID 32007290

• Atlas of Subcellular RNA Localization Revealed by APEX-Seq. Cell Fazal, F. M., Han, S., Parker, K. R., Kaewsapsak, P., Xu, J., Boettiger, A. N., Chang, H. Y., Ting, A. Y. 2019

  Abstract

  We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.

  View details for DOI 10.1016/j.cell.2019.05.027

  View details for PubMedID 31230715

• Visualizing DNA folding and RNA in embryos at single-cell resolution. Nature Mateo, L. J., Murphy, S. E., Hafner, A., Cinquini, I. S., Walker, C. A., Boettiger, A. N. 2019

  Abstract

  The establishment of cell types during development requires precise interactions between genes and distal regulatory sequences. We have a limited understanding of how these interactions look in three dimensions, vary across cell types in complex tissue, and relate to transcription. Here we describe optical reconstruction of chromatin architecture (ORCA), a method that can trace the DNA path in single cells with nanoscale accuracy and genomic resolution reaching two kilobases. We used ORCA to study a Hox gene cluster in cryosectioned Drosophila embryos and labelled around 30 RNA species in parallel. We identified cell-type-specific physical borders between active and Polycomb-repressed DNA, and unexpected Polycomb-independent borders. Deletion of Polycomb-independent borders led to ectopic enhancer-promoter contacts, aberrant gene expression, and developmental defects. Together, these results illustrate an approach for high-resolution, single-cell DNA domain analysis in vivo, identify domain structures that change with cell identity, and show that border elements contribute to the formation of physical domains in Drosophila.

  View details for PubMedID 30886393

• Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science (New York, N.Y.) Bintu, B., Mateo, L. J., Su, J. H., Sinnott-Armstrong, N. A., Parker, M., Kinrot, S., Yamaya, K., Boettiger, A. N., Zhuang, X. 2018; 362 (6413)

  Abstract

  The spatial organization of chromatin is pivotal for regulating genome functions. We report an imaging method for tracing chromatin organization with kilobase- and nanometer-scale resolution, unveiling chromatin conformation across topologically associating domains (TADs) in thousands of individual cells. Our imaging data revealed TAD-like structures with globular conformation and sharp domain boundaries in single cells. The boundaries varied from cell to cell, occurring with nonzero probabilities at all genomic positions but preferentially at CCCTC-binding factor (CTCF)- and cohesin-binding sites. Notably, cohesin depletion, which abolished TADs at the population-average level, did not diminish TAD-like structures in single cells but eliminated preferential domain boundary positions. Moreover, we observed widespread, cooperative, multiway chromatin interactions, which remained after cohesin depletion. These results provide critical insight into the mechanisms underlying chromatin domain and hub formation.

  View details for PubMedID 30361340

• In Situ Super-Resolution Imaging of Genomic DNA with OligoSTORM and OligoDNA-PAINT SUPER-RESOLUTION MICROSCOPY: METHODS AND PROTOCOLS Beliveau, B. J., Boettiger, A. N., Nir, G., Bintu, B., Yin, P., Zhuang, X., Wu, C., Erfle, H. 2017; 1663: 231?52

  Abstract

  OligoSTORM and OligoDNA-PAINT meld the Oligopaint technology for fluorescent in situ hybridization (FISH) with, respectively, Stochastic Optical Reconstruction Microscopy (STORM) and DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) to enable in situ single-molecule super-resolution imaging of nucleic acids. Both strategies enable ?20 nm resolution and are appropriate for imaging nanoscale features of the genomes of a wide range of species, including human, mouse, and fruit fly (Drosophila).

  View details for PubMedID 28924672

  View details for PubMedCentralID PMC5919218

• Spatial organization shapes the turnover of a bacterial transcriptome ELIFE Moffitt, J. R., Pandey, S., Boettiger, A. N., Wang, S., Zhuang, X. 2016; 5

  Abstract

  Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Time-resolved RNA-sequencing revealed that degradation rates of inner-membrane-protein mRNAs are on average greater that those of the other mRNAs and that this selective destabilization of inner-membrane-protein mRNAs is abolished by dissociating the RNA degradosome from the membrane. Together, these results demonstrate that the bacterial transcriptome is spatially organized and suggest that this organization shapes the post-transcriptional dynamics of mRNAs.

  View details for DOI 10.7554/eLife.13065

  View details for Web of Science ID 000376881500001

  View details for PubMedID 27198188

  View details for PubMedCentralID PMC4874777

• Super-resolution imaging reveals distinct chromatin folding for different epigenetic states NATURE Boettiger, A. N., Bintu, B., Moffitt, J. R., Wang, S., Beliveau, B. J., Fudenberg, G., Imakaev, M., Mirny, L. A., Wu, C., Zhuang, X. 2016; 529 (7586): 418-?

  Abstract

  Metazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression. Here we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive or Polycomb-repressed states, and observed distinct chromatin organizations for each state. All three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed domains show the densest packing and most intriguing chromatin folding behaviour, in which chromatin packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins play an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that is directly relevant to genome regulation.

  View details for DOI 10.1038/nature16496

  View details for Web of Science ID 000368354800051

  View details for PubMedID 26760202

  View details for PubMedCentralID PMC4905822

• Chromatin topology is coupled to Polycomb group protein subnuclear organization. Nature communications Wani, A. H., Boettiger, A. N., Schorderet, P., Ergun, A., Münger, C., Sadreyev, R. I., Zhuang, X., Kingston, R. E., Francis, N. J. 2016; 7: 10291-?

  Abstract

  The genomes of metazoa are organized at multiple scales. Many proteins that regulate genome architecture, including Polycomb group (PcG) proteins, form subnuclear structures. Deciphering mechanistic links between protein organization and chromatin architecture requires precise description and mechanistic perturbations of both. Using super-resolution microscopy, here we show that PcG proteins are organized into hundreds of nanoscale protein clusters. We manipulated PcG clusters by disrupting the polymerization activity of the sterile alpha motif (SAM) of the PcG protein Polyhomeotic (Ph) or by increasing Ph levels. Ph with mutant SAM disrupts clustering of endogenous PcG complexes and chromatin interactions while elevating Ph level increases cluster number and chromatin interactions. These effects can be captured by molecular simulations based on a previously described chromatin polymer model. Both perturbations also alter gene expression. Organization of PcG proteins into small, abundant clusters on chromatin through Ph SAM polymerization activity may shape genome architecture through chromatin interactions.

  View details for DOI 10.1038/ncomms10291

  View details for PubMedID 26759081

  View details for PubMedCentralID PMC4735512

• Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes NATURE COMMUNICATIONS Beliveau, B. J., Boettiger, A. N., Avendano, M. S., Jungmann, R., Mccole, R. B., Joyce, E. F., Kim-Kiselak, C., Bantignies, F., Fonseka, C. Y., Erceg, J., Hannan, M. A., Hoang, H. G., Colognori, D., Lee, J. T., Shih, W. M., Yin, P., Zhuang, X., Wu, C. 2015; 6

  Abstract

  Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.

  View details for DOI 10.1038/ncomms8147

  View details for Web of Science ID 000355533700006

  View details for PubMedID 25962338

  View details for PubMedCentralID PMC4430122

• RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science (New York, N.Y.) Chen, K. H., Boettiger, A. N., Moffitt, J. R., Wang, S., Zhuang, X. 2015; 348 (6233)

  Abstract

  Knowledge of the expression profile and spatial landscape of the transcriptome in individual cells is essential for understanding the rich repertoire of cellular behaviors. Here, we report multiplexed error-robust fluorescence in situ hybridization (MERFISH), a single-molecule imaging approach that allows the copy numbers and spatial localizations of thousands of RNA species to be determined in single cells. Using error-robust encoding schemes to combat single-molecule labeling and detection errors, we demonstrated the imaging of 100 to 1000 distinct RNA species in hundreds of individual cells. Correlation analysis of the ~10(4) to 10(6) pairs of genes allowed us to constrain gene regulatory networks, predict novel functions for many unannotated genes, and identify distinct spatial distribution patterns of RNAs that correlate with properties of the encoded proteins.

  View details for DOI 10.1126/science.aaa6090

  View details for PubMedID 25858977

  View details for PubMedCentralID PMC4662681

• Analytic Approaches to Stochastic Gene Expression in Multicellular Systems BIOPHYSICAL JOURNAL Boettiger, A. N. 2013; 105 (12): 2629-2640

  Abstract

  Deterministic thermodynamic models of the complex systems, which control gene expression in metazoa, are helping researchers identify fundamental themes in the regulation of transcription. However, quantitative single cell studies are increasingly identifying regulatory mechanisms that control variability in expression. Such behaviors cannot be captured by deterministic models and are poorly suited to contemporary stochastic approaches that rely on continuum approximations, such as Langevin methods. Fortunately, theoretical advances in the modeling of transcription have assembled some general results that can be readily applied to systems being explored only through a deterministic approach. Here, I review some of the recent experimental evidence for the importance of genetically regulating stochastic effects during embryonic development and discuss key results from Markov theory that can be used to model this regulation. I then discuss several pairs of regulatory mechanisms recently investigated through a Markov approach. In each case, a deterministic treatment predicts no difference between the mechanisms, but the statistical treatment reveals the potential for substantially different distributions of transcriptional activity. In this light, features of gene regulation that seemed needlessly complex evolutionary baggage may be appreciated for their key contributions to reliability and precision of gene expression.

  View details for DOI 10.1016/j.bpj.2013.10.033

  View details for Web of Science ID 000328597400008

  View details for PubMedID 24359735

  View details for PubMedCentralID PMC3909422

• Rapid Transcription Fosters Coordinate snail Expression in the Drosophila Embryo CELL REPORTS Boettiger, A. N., Levine, M. 2013; 3 (1): 8-15

  Abstract

  Transcription is commonly held to be a highly stochastic process, resulting in considerable heterogeneity of gene expression among the different cells in a population. Here, we employ quantitative in situ hybridization methods coupled with high-resolution imaging assays to measure the expression of snail, a developmental patterning gene necessary for coordinating the invagination of the mesoderm during gastrulation of the Drosophila embryo. Our measurements of steady-state mRNAs suggest that there is very little variation in snail expression across the different cells that make up the mesoderm and that synthesis approaches the kinetic limits of Pol II processivity. We propose that rapid transcription kinetics and negative autoregulation are responsible for the remarkable homogeneity of snail expression and the coordination of mesoderm invagination.

  View details for DOI 10.1016/j.celrep.2012.12.015

  View details for Web of Science ID 000321891400002

  View details for PubMedID 23352665

  View details for PubMedCentralID PMC4257496

• Multiple enhancers ensure precision of gap gene-expression patterns in the Drosophila embryo PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Perry, M. W., Boettiger, A. N., Levine, M. 2011; 108 (33): 13570-13575

  Abstract

  Segmentation of the Drosophila embryo begins with the establishment of spatially restricted gap gene-expression patterns in response to broad gradients of maternal transcription factors, such as Bicoid. Numerous studies have documented the fidelity of these expression patterns, even when embryos are subjected to genetic or environmental stress, but the underlying mechanisms for this transcriptional precision are uncertain. Here we present evidence that every gap gene contains multiple enhancers with overlapping activities to produce authentic patterns of gene expression. For example, a recently identified hunchback (hb) enhancer (located 5-kb upstream of the classic enhancer) ensures repression at the anterior pole. The combination of intronic and 5' knirps (kni) enhancers produces a faithful expression pattern, even though the intronic enhancer alone directs an abnormally broad expression pattern. We present different models for "enhancer synergy," whereby two enhancers with overlapping activities produce authentic patterns of gene expression.

  View details for DOI 10.1073/pnas.1109873108

  View details for Web of Science ID 000293895100046

  View details for PubMedID 21825127

  View details for PubMedCentralID PMC3158186

• Inferring ecological and behavioral drivers of African elephant movement using a linear filtering approach ECOLOGY Boettiger, A. N., Wittemyer, G., Starfield, R., Volrath, F., Douglas-Hamilton, I., Getz, W. M. 2011; 92 (8): 1648-1657

  Abstract

  Understanding the environmental factors influencing animal movements is fundamental to theoretical and applied research in the field of movement ecology. Studies relating fine-scale movement paths to spatiotemporally structured landscape data, such as vegetation productivity or human activity, are particularly lacking despite the obvious importance of such information to understanding drivers of animal movement. In part, this may be because few approaches provide the sophistication to characterize the complexity of movement behavior and relate it to diverse, varying environmental stimuli. We overcame this hurdle by applying, for the first time to an ecological question, a finite impulse-response signal-filtering approach to identify human and natural environmental drivers of movements of 13 free-ranging African elephants (Loxodonta africana) from distinct social groups collected over seven years. A minimum mean-square error (MMSE) estimation criterion allowed comparison of the predictive power of landscape and ecological model inputs. We showed that a filter combining vegetation dynamics, human and physical landscape features, and previous movement outperformed simpler filter structures, indicating the importance of both dynamic and static landscape features, as well as habit, on movement decisions taken by elephants. Elephant responses to vegetation productivity indices were not uniform in time or space, indicating that elephant foraging strategies are more complex than simply gravitation toward areas of high productivity. Predictions were most frequently inaccurate outside protected area boundaries near human settlements, suggesting that human activity disrupts typical elephant movement behavior. Successful management strategies at the human-elephant interface, therefore, are likely to be context specific and dynamic. Signal processing provides a promising approach for elucidating environmental factors that drive animal movements over large time and spatial scales.

  View details for Web of Science ID 000293459100011

  View details for PubMedID 21905431

• Transcriptional Regulation: Effects of Promoter Proximal Pausing on Speed, Synchrony and Reliability PLOS COMPUTATIONAL BIOLOGY Boettiger, A. N., Ralph, P. L., Evans, S. N. 2011; 7 (5)

  Abstract

  Recent whole genome polymerase binding assays in the Drosophila embryo have shown that a substantial proportion of uninduced genes have pre-assembled RNA polymerase-II transcription initiation complex (PIC) bound to their promoters. These constitute a subset of promoter proximally paused genes for which mRNA elongation instead of promoter access is regulated. This difference can be described as a rearrangement of the regulatory topology to control the downstream transcriptional process of elongation rather than the upstream transcriptional initiation event. It has been shown experimentally that genes with the former mode of regulation tend to induce faster and more synchronously, and that promoter-proximal pausing is observed mainly in metazoans, in accord with a posited impact on synchrony. However, it has not been shown whether or not it is the change in the regulated step per se that is causal. We investigate this question by proposing and analyzing a continuous-time Markov chain model of PIC assembly regulated at one of two steps: initial polymerase association with DNA, or release from a paused, transcribing state. Our analysis demonstrates that, over a wide range of physical parameters, increased speed and synchrony are functional consequences of elongation control. Further, we make new predictions about the effect of elongation regulation on the consistent control of total transcript number between cells. We also identify which elements in the transcription induction pathway are most sensitive to molecular noise and thus possibly the most evolutionarily constrained. Our methods produce symbolic expressions for quantities of interest with reasonable computational effort and they can be used to explore the interplay between interaction topology and molecular noise in a broader class of biochemical networks. We provide general-purpose code implementing these methods.

  View details for DOI 10.1371/journal.pcbi.1001136

  View details for Web of Science ID 000291015800010

  View details for PubMedID 21589887

  View details for PubMedCentralID PMC3093350

• Shadow Enhancers Foster Robustness of Drosophila Gastrulation CURRENT BIOLOGY Perry, M. W., Boettiger, A. N., Bothma, J. P., Levine, M. 2010; 20 (17): 1562-1567

  Abstract

  Critical developmental control genes sometimes contain "shadow" enhancers that can be located in remote positions, including the introns of neighboring genes [1]. They nonetheless produce patterns of gene expression that are the same as or similar to those produced by more proximal primary enhancers. It was suggested that shadow enhancers help foster robustness in gene expression in response to environmental or genetic perturbations [2, 3]. We critically tested this hypothesis by employing a combination of bacterial artificial chromosome (BAC) recombineering and quantitative confocal imaging methods [2, 4]. Evidence is presented that the snail gene is regulated by a distal shadow enhancer located within a neighboring locus. Removal of the proximal primary enhancer does not significantly perturb snail function, including the repression of neurogenic genes and formation of the ventral furrow during gastrulation at normal temperatures. However, at elevated temperatures, there is sporadic loss of snail expression and coincident disruptions in gastrulation. Similar defects are observed at normal temperatures upon reductions in the levels of Dorsal, a key activator of snail expression (reviewed in [5]). These results suggest that shadow enhancers represent a novel mechanism of canalization whereby complex developmental processes "bring about one definite end-result regardless of minor variations in conditions" [6].

  View details for DOI 10.1016/j.cub.2010.07.043

  View details for Web of Science ID 000281941100034

  View details for PubMedID 20797865

  View details for PubMedCentralID PMC4257487

• Morphogen Gradients: Limits to Signaling or Limits to Measurement? CURRENT BIOLOGY Bothma, J. P., Levine, M., Boettiger, A. 2010; 20 (5): R232-R234

  Abstract

  In Drosophila embryos, a concentration gradient of nuclear Dorsal protein controls pattern formation along the dorsal-ventral axis. Recent quantitative studies agree on the temporal dynamics of the gradient, but disagree on its spatial limits.

  View details for DOI 10.1016/j.cub.2010.01.040

  View details for Web of Science ID 000275476900008

  View details for PubMedID 20219171

• Emergent complexity in simple neural systems. Communicative & integrative biology Boettiger, A. N., Oster, G. 2009; 2 (6): 467-470

  Abstract

  The ornate and diverse patterns of seashells testify to the complexity of living systems. Provocative computational explorations have shown that similarly complex patterns may arise from the collective interaction of a small number of rules. This suggests that, although a system may appear complex, it may still be understood in terms of simple principles. It is still debatable whether shell patterns emerge from some undiscovered simple principles, or are the consequence of an irreducibly complex interaction of many effects. Recent work by Boettiger, Ermentrout and Oster on the biological mechanisms of shell patterning has provided compelling evidence that, at least for this system, simplicity produces diversity and complexity.

  View details for PubMedID 20195452

  View details for PubMedCentralID PMC2829821

• Synchronous and Stochastic Patterns of Gene Activation in the Drosophila Embryo SCIENCE Boettiger, A. N., Levine, M. 2009; 325 (5939): 471-473

  Abstract

  Drosophila embryogenesis is characterized by rapid transitions in gene activity, whereby crudely distributed gradients of regulatory proteins give way to precise on/off patterns of gene expression. To explore the underlying mechanisms, a partially automated, quantitative in situ hybridization method was used to visualize expression profiles of 14 developmental control genes in hundreds of embryos. These studies revealed two distinct patterns of gene activation: synchronous and stochastic. Synchronous genes display essentially uniform expression of nascent transcripts in all cells of an embryonic tissue, whereas stochastic genes display erratic patterns of de novo activation. RNA polymerase II is "pre-loaded" (stalled) in the promoter regions of synchronous genes, but not stochastic genes. Transcriptional synchrony might ensure the orderly deployment of the complex gene regulatory networks that control embryogenesis.

  View details for DOI 10.1126/science.1173976

  View details for Web of Science ID 000268255100055

  View details for PubMedID 19628867

  View details for PubMedCentralID PMC4280267

• The neural origins of shell structure and pattern in aquatic mollusks PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Boettiger, A., Ermentrout, B., Oster, G. 2009; 106 (16): 6837-6842

  Abstract

  We present a model to explain how the neurosecretory system of aquatic mollusks generates their diversity of shell structures and pigmentation patterns. The anatomical and physiological basis of this model sets it apart from other models used to explain shape and pattern. The model reproduces most known shell shapes and patterns and accurately predicts how the pattern alters in response to environmental disruption and subsequent repair. Finally, we connect the model to a larger class of neural models.

  View details for DOI 10.1073/pnas.0810311106

  View details for Web of Science ID 000265506800074

  View details for PubMedID 19351900

  View details for PubMedCentralID PMC2672551

• Evolution of insect dorsoventral patterning mechanisms. Cold Spring Harbor symposia on quantitative biology Perry, M. W., Cande, J. D., Boettiger, A. N., Levine, M. 2009; 74: 275-279

  Abstract

  The dorsoventral (DV) patterning of the early Drosophila embryo depends on Dorsal, a maternal sequence-specific transcription factor related to mammalian NF-kappaB. Dorsal controls DV patterning through the differential regulation of approximately 50 target genes in a concentration-dependent manner. Whole-genome methods, including ChIP-chip and ChIP-seq assays, have identified approximately 100 Dorsal target enhancers, and more than one-third of these have been experimentally confirmed via transgenic embryo assays. Despite differences in DV patterning among divergent insects, a number of the Dorsal target enhancers are located in conserved positions relative to the associated transcription units. Thus, the evolution of novel patterns of gene expression might depend on the modification of old enhancers, rather than the invention of new ones. As many as half of all Dorsal target genes appear to contain "shadow" enhancers: a second enhancer that directs the same or similar expression pattern as the primary enhancer. Preliminary studies suggest that shadow enhancers might help to ensure resilience of gene expression in response to environmental and genetic perturbations. Finally, most Dorsal target genes appear to contain RNA polymerase II (pol II) prior to their activation. Stalled pol II fosters synchronous patterns of gene activation in the early embryo. In contrast, DV patterning genes lacking stalled pol II are initially activated in an erratic or stochastic fashion. It is possible that stalled pol II confers fitness to a population by ensuring coordinate deployment of the gene networks controlling embryogenesis.

  View details for DOI 10.1101/sqb.2009.74.021

  View details for PubMedID 19843594

  View details for PubMedCentralID PMC4280271

• Nuclear trapping shapes the terminal gradient in the Drosophila embryo CURRENT BIOLOGY Coppey, M., Boettiger, A. N., Berezhkovskii, A. M., Shvartsman, S. Y. 2008; 18 (12): 915-919

  Abstract

  Patterning of the terminal regions of the Drosophila embryo relies on the gradient of phosphorylated ERK/MAPK (dpERK), which is controlled by the localized activation of the Torso receptor tyrosine kinase [1-4]. This model is supported by a large amount of data, but the gradient itself has never been quantified. We present the first measurements of the dpERK gradient and establish a new intracellular layer of its regulation. Based on the quantitative analysis of the spatial pattern of dpERK in mutants with different levels of Torso as well as the dynamics of the wild-type dpERK pattern, we propose that the terminal-patterning gradient is controlled by a cascade of diffusion-trapping modules. A ligand-trapping mechanism establishes a sharply localized pattern of the Torso receptor occupancy on the surface of the embryo. Inside the syncytial embryo, nuclei play the role of traps that localize diffusible dpERK. We argue that the length scale of the terminal-patterning gradient is determined mainly by the intracellular module.

  View details for DOI 10.1016/j.cub.2008.05.034

  View details for Web of Science ID 000257151900032

  View details for PubMedID 18571412

  View details for PubMedCentralID PMC2500156

• Modeling the bicold gradient: Diffusion and reversible nuclear trapping of a stable protein DEVELOPMENTAL BIOLOGY Coppey, M., Berezhkovskii, A. M., Kim, Y., Boettiger, A. N., Shvartsman, S. Y. 2007; 312 (2): 623-630

  Abstract

  The Bicoid gradient in the Drosophila embryo provided the first example of a morphogen gradient studied at the molecular level. The exponential shape of the Bicoid gradient had always been interpreted within the framework of the localized production, diffusion, and degradation model. We propose an alternative mechanism, which assumes no Bicoid degradation. The medium where the Bicoid gradient is formed and interpreted is very dynamic. Most notably, the number of nuclei changes over three orders of magnitude from fertilization, when Bicoid synthesis is initiated, to nuclear cycle 14 when most of the measurements were taken. We demonstrate that a model based on Bicoid diffusion and nucleocytoplasmic shuttling in the presence of the growing number of nuclei can account for most of the properties of the Bicoid concentration profile. Consistent with experimental observations, the Bicoid gradient in our model is established before nuclei migrate to the periphery of the embryo and remains stable during subsequent nuclear divisions.

  View details for DOI 10.1016/j.ydbio.2007.09.058

  View details for Web of Science ID 000251734300013

  View details for PubMedID 18001703

  View details for PubMedCentralID PMC2789574

• Role of boundary conditions in an experimental model of epithelial wound healing AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Nikolic, D. L., Boettiger, A. N., Bar-Sagi, D., Carbeck, J. D., Shvartsman, S. Y. 2006; 291 (1): C68-C75

  Abstract

  Coordinated cell movements in epithelial layers are essential for proper tissue morphogenesis and homeostasis, but our understanding of the mechanisms that coordinate the behavior of multiple cells in these processes is far from complete. Recent experiments with Madin-Darby canine kidney epithelial monolayers revealed a wave-like pattern of injury-induced MAPK activation and showed that it is essential for collective cell migration after wounding. To investigate the effects of the different aspects of wounding on cell sheet migration, we engineered a system that allowed us to dissect the classic wound healing assay. We studied Madin-Darby canine kidney sheet migration under three different conditions: 1) the classic wound healing assay, 2) empty space induction, where a confluent monolayer is grown adjacent to a slab of polydimethylsiloxane and the monolayer is not injured but allowed to migrate upon removal of the slab, and 3) injury via polydimethylsiloxane membrane peel-off, where an injured monolayer migrates onto plain tissue culture surface, as in the case of empty space induction allowing for direct comparison. By tracking the motion of individual cells within the sheet under these three conditions, we show how the dynamics of the individual cells' motion is responsible for the coordinated migration of the sheet and is coordinated with the activation of ERK1/2 MAPK. In addition, we demonstrate that the propagation of the waves of MAPK activation depends on the generation of reactive oxygen species at the wound edge.

  View details for DOI 10.1152/ajpcell.00411.2005

  View details for Web of Science ID 000238287500009

  View details for PubMedID 16495370