Archive for January, 2012

Using physics to study life

My laboratory is interested in problems at the interface of physics and biology. The main thrust of our research is the design of new experimental approaches and the performance of high-precision physics-style measurements in living animals, such that our data allows for direct validation of mathematical models. This program is aimed at the generation of theories describing biological phenomena which are derived from general principles in the physics tradition. Currently our main interests lie in the collective behaviors of eukaryotic cells all the way from microbes to embryonic tissue, and in how the control of gene expression in early fly embryos leads to the formation of an animal body plan. We are making experimental and theoretical progress on the biological questions in each of these areas, the long-term goal, however, is to find theories inspired by experimental data that go beyond the specifics of the biological systems with the hope of finding in the living world some new physics, which has been hidden and cannot easily be revealed in the inanimate world. The approach outlined here is is an extension of the traditional physics approach, which my colleagues use to study the stars or inert matter on earth, to the complex processes underlying the living world.

1. Collective behaviors in living cell populations

One example of such new physics may lie in the emergence of collective behaviors in early developing cell populations and cell tissues. Cells are at the same time an ??active living agent?? while they are exposed to external chemical signaling and mechanical force fields. Combining these two properties, individuality and field dependence, is a fundamental issue across all the physical sciences that has not been resolved, neither theoretically nor experimentally. My laboratory approaches this question two fold: we study the emergence of multicellularity in starved cell populations of the social amoeba Dictyostelium, and we try to extract the mechanical tissue properties during the earliest stages of developing fly embryos.

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For the amoebae project, we have developed a novel optical sensor (based on Foerster-Resonance-Energy- Transfer technology), which allows for direct fluorescence measurements of intra-cellular signaling in develop- ing cell populations. We are currently designing cellular environments based on microfluidic technology to obtain a greater handle on the extracellular space (i.e. extracellular signaling molecule concentration measurements, generation of temporal and spatial concentration waves), allowing us to probe living cells at the single- and multi-cellular levels with great control. Further we are developing a phenomenological theoretical model in collaboration with Pankaj Mehta (Boston University/Physics) to bridge the gap between single cell properties and collective multicellular phenomena. In order to connect this top-down approach back to molecular detail of the cellular signaling cascade, we are measuring the single-cell signaling properties of various signaling mutants that display phenotypes at the macroscopic level. To this end, we are collaborating with Ted Cox in the Molecular Biology department in order to take advantage of his large mutagenesis database, which we combine with our optical sensors.

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The tissue mechanics project necessitates highly spatially and temporally resolved dynamic data of de- veloping fly embryos. My laboratory has set up microscopy to capture movies of living tissue in which individual nuclei are labeled optically. In collaboration with Andrea Liu (UPenn/Physics) and Lisa Manning (Syracuse/Physics) we are developing analysis tools to extract mechanical tissue properties from these movies. Preliminary re- sults generated by a novel cell tracking software suggest that mitotic divisions in Drosophila occur in a series of waves initiated in an excitable medium, i.e. one where waves are actively regenerated in the material. Our Dictyostelium work has also suggested that multicellular signaling proceeds by excitable, self-generated waves. The plan is to model both the mechanical and biochemical cell-cell (or nuclei-nuclei) interactions in these systems, to understand the mechanisms for wave propagation and tease apart the role of these excitable waves in multicellular organization.

 

2. Biological pattern formation

Currently, the most advanced topic in the lab is our work on biological pattern formation in developing fruit fly embryos; partly because this project evolves in an environment that has a long tradition at Princeton. How does an organism form a body axis, and how are the different body parts along this axis patterned and established is one of the most fundamental questions in developmental biology, and with current access to quantitative data the prospect of exposing general theoretical principles in the biological context, similar to our understanding of physical pattern formation, are conspicuous. My laboratory has chosen the earliest patterning events of the fly embryo for the conceptual reason that the blueprint for the future adult structure is determined entirely during this time, and for the practical reasons of ease of experimental access, and of the powerful scientific environment at Princeton in this field. Pattering at these early stages consists mainly in the differential expression of genes, where Princeton has been developing tools to quantify protein levels in living and fixed embryos since the early 2000s, both experimental and theoretical.

In the past 2.5 years my laboratory has focussed mainly on widening our tool chest to quantify patterning in the early embryo. The goal of these technical improvements is to obtain eventually a fully quantitative (absolute numbers) dynamic picture of the entire patterning process, and address issues such as reproducibility, precision or scaling of the patterning events. We have developed a method to count individual molecules of mRNA in whole fixed embryos, which gives us besides proteins an independent access into the transcriptional regulatory machinery and will hopefully allow us to quantify the ??central dogma?? at the molecular level in a natural multicellular context. We have made progress on expanding our investigation of patterning dynamics in living embryos (originally restricted to the maternal input gradient of Bicoid protein concentration) to other members of the regulatory network, which is formed by a cascade of interconnected genes that together with the input gradients determine the final body pattern within the first three hours of the embryo??s development. Using genetic engineering, we are in the process of labeling the gene products of this network with multicolored fluorescent proteins, for both mRNA and protein. In parallel we are developing microscopes that can track the concentrations of these differently colored molecules in living embryos, where we are facing challenges at the level of spectral unmixing, light exposure of the living specimen, and size constraints of our specimen that affect both spatial and temporal resolution of our data.

 

Current projects

The following paragraphs summarize the projects that are currently progressing in the lab to contribute to the execution of the program outlined above:

  1. Originally instigated in the summer of 2009 by an undergraduate project, we developed a novel mRNA quantification method in fly embryos, the core concept of which was published last year as a proof of principle. We essentially tag individual mRNA particles with fluorescent probes in fixed tissue and use diffraction-limited confocal microscopy to image and count. The real challenge of this method, however, was to convince ourselves that we are indeed looking at individual molecules (and not spots or aggregates of molecules) and that we can indeed count all of them. A further challenge was to go beyond simple spot identification, and to quantify the actual intensities that we obtain per spot. Intensities at the DNA location of active native transcription reveal the actual transcriptional state of that site at the moment of specimen fixation. Using multiple colors on the same site we will be able to even extract dynamic parameters such as binding rates and lifetimes from our data. We have resolved most of these challenges over the last year in a very talented team composed of a postdoc, a graduate student and an undergraduate. Using this powerful method and some of the other tools currently developed in the laboratory I am convinced that we will be able to reveal many of the molecular underpinnings of transcriptional regulation in the early fly embryo over the next few years.
  2. The wealth of approaches developed in molecular biology over the past decades gives us the luxury in this field to do the same experiment twice, but with a completely different set of systematic errors. Consequently, over the past two years we have developed an independent way to measure the mean number of mRNA molecules in single embryos. Our method exploits a combination of polymerase-chain-reactions (PCR) with techniques typically used by biochemists, and by making sure at each step we understand what we do to our absolute numbers of initial mRNA molecules. The key is then to compare the numbers we get from single embryos with a precisely quantified molecular standard. The means of the two methods match within less than either of the two standard deviations.
  3. Again in the spirit of an alternative approach to the same problem, we are also developing a direct handle on transcriptional dynamics by directly labeling mRNA strands with fluorescent proteins. This technique has been pioneered in bacteria to trace individual mRNA molecules and in fly embryos to trace mRNA aggregates. We are extending this application to reveal the dynamics of nascent mRNA molecules as they are assembled at the DNA transcription site in living fly embryos. By simultaneously measuring fluorescently labeled input transcription factors (as we have done in the past) and mRNA output dynamics we will be able to extract dynamic input-output relations of transcription in a natural, which has never been done in any biological system. We thus will have a fine tool at hand to put models of transcriptional regulation to a real quantitative test.
  4. We are collaborating with Bill Bialek (Princeton/Physics) and Eric Wieschaus (Princeton/Molecular Biology) to measure the amount of information transmitted through the gene network, testing the idea whether optimization principles govern and apply during early fly patterning, i.e. is there an optimal matching between the distribution of transcription factor concentrations and the noise levels in the control mechanism? What would be the structure of of genetic regulatory networks that maximize information transmission, if they optimize their regulatory power and precision while using a limited number of molecules? We are currently measuring information that is transmitted through the gene network that controls the early patterning events in the fly embryo, and initial results are about to be disclosed in full.
  5. Perturbation experiments have a long tradition in both the physical and biological sciences to understand complex processes and networks. In biology, however, it is customary to perform such perturbations in terms of a mutation analyses which result in the partial or complete disruption of the function of a particular protein. In reality, such a mutation is much more than a simple perturbation, as it leads to a transformation of the original network to a new and completely different network that has one less node. To avoid such a disruptive perturbation, my laboratory has developed a system in which we have a dial knob for the network??s input concentration. We genetically generated a set of fly lines that express the input protein Bicoid at dosages that differ from its wild type expression level in increments of 10-20% up to factors of 2.5-3 in either direction. We are currently in the process to carefully measure the response of the network to these subtle absolute input concentration changes. Our preliminary results indicate that the traditional picture of a local concentration readout may no longer be in agreement with our observations.

Recent and future projects

Besides these well-advanced projects above there are a number of exciting recently initiated projects:

  1. We started a collaboration with Steve Small (NYU/Biology) to measure the structure-function relationship of one particular enhancer (small piece if DNA that regulates a gene). How is the actual string of code (nucleotides) affecting the final output (mRNA transcripts)? We are genetically engineering enhancer variants that we express in both embryonic cell lines and in living embryos and we are measuring their transcriptional activity by using our single molecule mRNA counting technology.
  2. We are in the process of generating flies that simultaneously express up to three fast-folding/fast-maturing fluorophores attached to different members of the patterning network, and we are building custom microscopes for live imaging that can cope a) with spectral unmixing for multi-color measurements, b) with the fast evolving expression level dynamics, and c) with resolving the full three-dimensional structure of an entire embryo.
  3. In an effort to link our expression level measurements back to evolutionary constraints, we are exploring three interconnected paths: 1) What are the variations of expression levels in natural wild type Drosophila populations from different parts of the world? 2) What are the expression level variations in isogenic fly populations, and how do these variations respond to environmental fluctuations? 3) We are generating tools to quantify phenotypes in the adult organism, such as left-right symmetry of fly wings, to ask how this symmetry is affected if we perturb embryonic expression levels or environmental conditions such as temperature or food supply, and whether perturbances in the embryo and in the adult are correlated.

 

Quantifying the Bicoid morphogen gradient in living fly embryos

Alexander H. Morrison, Martin Scheeler Julien O. Dubuis and Thomas GregorCold Spring Harb Protoc. 2012(4): 398-406 (2012).
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