Santa Barbara Advanced School of Quantitative Biology

Neurophysics of Sensory Navigation

Santa Barbara Advanced School of Quantitative Biology
Summer Research Course @ KITP, UCSB
July 30 - August 24, 2018


Online application coming soon.

Course fee $1250 (double room), $2000 (single room). Financial aid is available.

 

Course Directors: 

Matthieu Louis (UCSB) and Julie Simpson (UCSB)
 

Course Instructors (partial list):

Bill Kristan, Jr. (UCSD), Matthieu Louis (UCSB), Christopher Potter (Johns Hopkins), Pavan Ramdya (EPFL), Julie Simpson (UCSB), Marcus Stensmyr (Lund Univ.), and Claire Wyart (ICM Institute)

 

Course subject

Physics has become omnipresent in systems neuroscience: it plays a critical role in providing tools to record electrical activity of single neurons, monitor neural activity through multi-photon imaging, and model dynamical behavior of nonlinear systems of interconnected neurons. This KITP summer course aims to equip students with the conceptual and technical framework physics and quantitative biology offers to study the integrated function of neural circuits.

Experiments in systems neuroscience require a broad range of interdisciplinary techniques, necessitating collaboration between scientists with complimentary areas of expertise. Course participants will work in teams that include both theorists and experimentalists, building common vocabulary and learning the physics and neurobiology that will help them tackle challenging systems neuroscience questions: how do noisy sensors reliably capture multisensory sensory information? How is the integration of sensory signals transformed into decision making? How are behaviors such as locomotion planned and controlled by the motor system?

Students will attend morning research seminars with senior participants in the closely linked KITP program Neural computations for sensory navigation: mechanisms, models, and biomimetic applications. Afternoon research sessions will begin with a one-week boot camp on confocal and two-photon microscopy, rearing of small model organisms, and data analysis in Matlab and/or Python. The rest of the course will center around hands-on research projects covering two themes: (1) mapping and functional characterization of neural circuits and (2) quantitative description and sensory manipulations of elementary behaviors. Working under the guidance of instructors and teaching assistants, students will be encouraged to develop and pursue their own questions.


Apply online Submit a recommendation letter

Application deadline is March 12, 2017.

 

 

Course Directors: 

Richard Neher (Biozentrum, Basel), Paul Rainey (ESPCI, Paris), Boris Shraiman (KITP, UCSB)
 

Course Instructors:

R. Dutton (UCSD), D. Huson (U. Tübingen), T. Julou (Biozentrum, Basel), H. McCann (NZIAS), Richard Neher (Biozentrum, Basel), J. Quick (U. Birmingham), Paul Rainey (ESPCI, Paris), Boris Shraiman (KITP, UCSB), E. Toprak (UT Southwestern), E. Wilbanks (UCSB)


Guest Lecturers (partial list):

J. Bergelson (U. Chicago), O. Cordero (MIT), S. Copley (U. Colorado), S. De Monte (ENS, Paris), M. Desai (Harvard), M. Doebeli (UBC), I. Gordo (Gulbenkian Inst.), O. Hallatscheck (UC Berkeley), T. Hwa (UCSD), D. S. Fisher (Stanford), E. Koonin (NCBI), R. Lenski (MSU), B. Levin (Emory), A. Murray (Harvard/HHMI), F. Rohwer (SDSU), A. Spormann (Stanford), G. Suel (UCSD/HHMI), P. Turner (Yale), J. Weitz (Georgia Tech), N. Wingreen (Princeton)
 

Course subject

Microbial communities play a profoundly important role on all scales, from carbon cycling in the ocean to cellulose utilisation in the gut of a termite. Microbial communities provide the foundation for ecosystems on the planet and are increasingly recognized as a major factor in human health. While it is convenient to study separate microbial species as they interact with the environment, in reality the  environment experienced by any one microbe is shaped by its interaction with other members of the local ecological community. Behavior of an individual cell is intertwined with the behavior of other community members and many of the metabolic functions of a cell can be  “outsourced”, making the community the relevant physiological unit. Understanding the function of microbial communities is the current frontier of science at the interface of microbiology with ecology and evolution. What are the “forces” that bind these microbial communities? Is the community more than the sum of its parts? What are the “parts” and how do they interact? How do microbial communities respond and adapt to environmental challenges and how do they evolve? What is the role of horizontal gene transfer? What is the role of competition within a community and how is the diversity maintained?  

The course will introduce students to the fundamental questions and to the promising approaches towards the study of microbial communities. Students will get a hands-on introduction to microbial communities in the lab and will gain experience with new technologies for studying microbes. Students will be working in the groups of 4-5 (under the guidance of instructors and TAs) on different projects which will involve microbiology lab work, sequencing, bioinformatic analysis and modeling. Within the framework of the group projects  students will have an opportunity to define and pursue their own research questions. A number of short lecture series will introduce basic concepts and new developments. We expect that, even under the constraint of a 4 week course, students will be able to generate interesting “preliminary data” and perhaps make a discovery or two.

The course will run side by side with the KITP workshop Eco-Evolutionary Dynamics in Nature and the Lab. Students will attend the lectures and seminars and will have the opportunity to interact with numerous senior researchers that will be participating in the workshop.

 


Mini-Courses

  • Daniel Huson: Metagenomic sequence analysis (2 lectures + 3h “practical”)
  • Eugene Koonin and Richard Neher: Bioinformatics of microbial pan-genome (2 lectures + 3h “practical”)
  • Joshua Quick: Nanopore sequencing bootcamp
  • Alfred Spormann: Introduction to cellular and community metabolism (3 lectures)
  • Joshua Weitz and Forest Rohwer: Quantitative viral ecology (3 lectures)
  • Michael Doebeli and Daniel Fisher: Introduction into mathematical modeling of ecological and evolutionary dynamics (2 lectures+3h tutorial)

 


 

EXPERIMENTAL PROJECTS (preliminary list)

 

Rachel Dutton: Microbial communities on cheese

The microbial communities of cheese are relatively simple, easily culturable, rich in species interactions, and undergo reproducible dynamics of community assembly. They are hence ideal systems to study general properties of microbial communities. During the course, we will:

  • analyse time series sequence data of community dynamics
  • isolate species and sequence and assemble their genomes using NanoPore technology
  • study horizontal transfer within cheese communities.


Thomas Julou: Single cell phenotyping to study phage-bacteria interactions

In a complex microbial community, phages, nutrients, toxins, and other parameters are unpredictable. Bacteria respond heterogeneously to changing environments and this heterogeneous response can only be studied at the single cell level.  Using a “mother-machine” we will explore single cell responses to fluctuations in nutrients and phage. We will:

  • Use the mother machine to study the responses of bacteria to fluctuating nutrients
  • Explore dynamics of the interaction between phages and bacteria
  • Determine phage life history parameters

Joshua Quick: Nanopore boot camp

Sequencing technology is currently undergoing a second revolution from short-read to long-read sequencing. Nanopore Technologies has developed hand-held sequencers, the MinIon, with very short turn-around times that we will use during the course to sequence. We will:

  • use nanopore long read sequencing to assemble genomes from cheese and marine communities
  • metagenomic and environmental sequencing
  • quantify rearrangements and phage integrations using long reads


Honour McCann: Phylogeography and pangenomics of a kiwifruit canker pandemic

In this project, we will isolate bacteria from California-grown kiwifruit leaves using droplet technology, sequence a sample of these genomes, and analyze several hundred genomes of the kiwifruit pathogen Pseudomonas syringae obtained during the course of a recent global pandemic.  By comparing genomes in context of place and time of sample acquisition it is be possible to understand the evolutionary origins of the pathogen, identify the core genome and infer the core genome phylogeny.  A particularly dynamic component of the genome is marked by integrative and conjugative elements (ICEs) whose evolutionary origins are challenging to discern (but worth it). We will:
  • Use millifluidic technology [see / link to below] to isolate bacteria from leaves and process for sequencing in a single step.
  • Engage with new computational approaches for inferring biogeography and evolutionary origins of microbes
  • Study the population biology of ICEs


Paul Rainey with Maxime Ardré, Guilhem Dulcier and Laurent Boitard: Millifluidics

Millifluidic technologies offer new opportunities for studying and manipulating 1000s of microbial populations / communities.  Cultures are maintained in 250 nl droplets of media separated by a plug of air and oil, all within a 0.5 mm teflon tube.  Data on microbial growth / metabolism / phenotype are gathered in real time and can be coupled to downstream tasks such as DNA sequencing or high-throughput phenotyping tools. We will:
  • Use MilliDrop technology to isolate leaf colonising bacteria
  • Explore population dynamics of siderophore producers / non-producers
  • Analyse interactions between community members
  • Perform statistical analysis on population data using custom-built pipelines.


Paul Rainey with Steven Quistad: Phage and the evolution of microbial communities

Lateral gene transfer mediated by phage, plasmids, integrative conjugative elements and similar genetic parasites stand to effect -- fuel even -- adaptation of communities of microbes.  Communities arising from a year-long experiment will be established at KITP.  The communities were established from independent 1 g samples of compost and have been allowed to adapt to growth on minimal medium with cellulose as the sole carbon in the presence or absence of lateral gene transfer (mediated by phage).  The effect of lateral gene transfer on community level function will be determined through empirical tests.  Individual community members will be identified and sequenced.  Additionally, terabytes of DNA sequence data from multiple time points / treatments will be available for analysis. We will:
  • Isolate and characterise by DNA sequencing community members (phage and bacteria).
  • Determine the effect of selection and lateral gene transfer on the evolution of communities via phenotypic assay.
  • Delve into the dirty depths of DNA data and discover promiscuous phage, malicious microbes and culturable obscurities.


Erdal Toprak: Building and running a morbidostat

A morbidostat is a versatile computer controlled continuous culture device that adjusts the growth environment to maintain a specified growth rate, for example by adjusting the antibiotic concentration in the medium. We will:

  • build a morbidostat device
  • morbidostat experiments to study evolution of antibiotic resistance
  • analyse time series data from morbidostat experiments by phenotyping and genotyping evolved populations


Lizzy Wilbanks: Pink berry microbial consortia

The pink berries are photosynthetic microbial aggregates composed primarily of two closely associated species sulfur-metabolizing bacteria.  These species carry out a tight-coupled, syntrophic sulfur cycle within these macroscopic aggregates that found at the sediment-water interface of intertidal pools in the Sippewissett salt marsh (Falmouth, MA).  During the course, we will:

  • compare different long-read data for analysis and assembly of metagenomes
  • analyze strain-level biogeography between aggregates and along spatial transect
  • investigate the phages associated with pink berries and immunity profiles of different isolates.

Summer Course


 

Course Directors: 

 
Suzanne Eaton (MPI-CBG, Dresden), Andy Oates (MRC LMCB, London), Joel Rothman (MCDB, UCSB) and Boris Shraiman (KITP, UCSB)
 

Course Instructors:

S. Eaton (MPI-CBG, Dresden), C. Extavour (Harvard), N. Goehring (MRC LMCB, London), P. Lemaire (CRBM, Montpelier), M. Mani (NWU), A. Oates (MRC LMCB, London), A. Pavlopoulos (Janelia, HHMI), B. Shraiman (KITP, UCSB), S. Streichan (KITP, UCSB), A. Warmflash (Rice) and E. Wieschaus (Princeton)


Guest Lecturers (partial list):

A. Aulehla (EMBL, Heidelberg), Z. Bao (MSKCC, New York), B. Baum (MRC LMCB, London), Y. Bellaiche (Inst Curie, Paris), R. Carthew (NWU, Evanston), D. Devenport (Princeton), P. Francois (McGill U., Montreal), M. Gonzalez-Gaitan (U. Geneva), S. Grill (TU, Dresden), C.P. Heisenberg (IST, Vienna), K. Irvine (Rutgers), F. Julicher (MPI-PCS, Dresden), T. Lecuit (IBDM, Marseille), L. Mahadevan (Harvard), D. Montell (UCSB), E. Munro (U. Chicago), T. Schupbach (Princeton), S. Shvartsman (Princeton), E. Siggia (Rockefeller), D. Strutt (U. Sheffield)
 

Course subject

This course aims at bringing together the Biology and Physics perspectives on Morphogenesis - the process through which animals and plants acquire their physical form and biological function. Morphogenesis is a developmental program encoded in DNA and kick-started by “maternal factors” in the egg. Developmental Biology has made enormous progress in identifying the genes that specify developmental axes and the general body-plan as well as the numerous genes and biochemical signals that control growth and cell differentiation. Yet, even as we know the expression of what gene would cause a fruit-fly to grow a leg on its head instead of an antennae, we have little understanding of how controlled growth and cell differentiation actually generates the distinct shape and structure that make a leg rather than an antennae. A century ago, before the advent of developmental genetics, D’Arcy Thompson viewed Morphogenesis as an essentially physical process of controlled growth. Revisiting Thomson’s agenda, the challenge is to connect the macroscopic dynamics of morphogenesis to the underlying molecular genetic and cell-biological processes. Physics perspective in particular focuses on the dynamical aspect of morphogenesis. To use a metaphor, what are the “laws of motion” that define the developmental trajectory from maternal factors as “initial conditions” to embryonic structures and beyond? Physics perspective also brings to fore the role of intercellular interactions in coordinating development and, in particular, the role of mechanics alongside with biochemical signals governing collective and individual behavior of cells in tissues.

The course will be anchored by laboratory projects involving different morphogenetic processes in model organisms such as the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, zebra fish (D. rerio), a sea squirt (Ciona) and a crustacean (Parhyale hawaiensis). Experimental projects will aim to introduce quantitative approaches to the study of the dynamics of morphogenesis.

 


Course structure

This five-week course will consist of a first "bootcamp" week to be followed by two 2-week research project sessions. Each session will consist of three or four experimental projects led by instructors and TAs who will closely work with groups of 4-5 students. Each day will consist of a morning lecture and discussions followed by lab work late into the evening.

The bootcamp week will be aimed at introducing all students to a basic set of microscopy, quantitative image analysis and theoretical modeling tools that will be necessary for the later research projects. Bootcamp will also introduce students to different laboratory model systems of development.

The course will run side by side (and share review lectures and research seminars) with the KITP workshop "From Genes to Growth and Form", which will provide a fertile forum for discussions and the exchange of ideas with the workshop participants. The scientific program of this workshop will be anchored by the following intersecting themes, which are central to the subject of the course:

Role of time in patterning space:  will aim to reexamine the scenario of static spatial patterning by a gradient of morphogen concentration confronting it with the observations of an adaptive response and the hypothesis that cells respond only to the time derivative.
Interplay of global and local patterning signals: will focus on the relation between local polarization of cell and global anisotropy; examine the extent of global planar cell polarity order in tissues; reconcile observed local cell deformation with global distribution of mechanical stress.

Mechanical regulation of growth: consolidate evidence for mechanical regulation of growth of tissues in different systems. Evaluate different approaches to measuring stress in live tissues and monitoring its dynamics. What do we know about the molecular pathways of mechano-regulation?

Cellular “flows” and their generation: How can we determine the driving forces behind observed cellular flows? Comparison of cell-deformation, cell intercalation and cell proliferation as the drivers of global cell rearrangement.

Polarity and anisotropy in defining tissue morphology: Consolidate existing knowledge on the mechanisms of planar cell polarity (PCP and Fat/Ds pathways) and their role in defining clonal shapes and tissue anisotropy and controlling cellular flows and tissue rearrangement.
In toto organogenesis: will focus on the few model morphogenetic system, like Drosophila ovarian development, where the dynamics of morphogenesis could be examined in its entirety.

Comparative morphogenesis: will examine the similarity and differences between developmental mechanisms in different limbs and organs, e.g. wing vs leg and eye of Drosophila, and in different animals e.g. Drosophila vs wasp or beetle.

 


 

EXPERIMENTAL PROJECTS (preliminary list)

 

Suzanne Eaton: Drosophila wing development

 


Nathan Goehring: C. elegans embryonic development

 


Patrick Lemaire: Ciona embryonic development


Andy Oates:  Zebra fish somitogenesis

 


Aryeh Warmflash:  Stem cell differentiation patterns in vitro

       

 


Anastasios Pavlopoulos: Developmental morphogenesis in a crustacean

 


Sebastian Streichan and Eric Wieschaus: Cellular flow in Drosophila embryo

450

QBio 15


Application deadline is March 1, 2015.


Course Directors

Rob Phillips (Caltech) and Hernan Garcia (UC Berkeley)

Course Instructors

Mónica Bettencourt-Dias (Inst. Gulbenkian de Ciência), Cassandra Extavour (Harvard University), Michael Lynch (Indiana University), Jitu Mayor (NCBS Bangalore), Daniel Needleman (Harvard University), and José Pereira-Leal (Inst. Gulbenkian de Ciência)


Guest Lecturers (partial list)

Daniel Bolon (UMass Medical School), Irene Chen (UC Santa Barbara), Shelley Copley (University of Colorado), Allan Drummond (University of Chicago), Matt Good (University of Pennsylvania), Alexander Grosberg (New York University), Paul Higgs (McMaster University), KC Huang (Stanford University), Frank Jülicher (Max Planck Institute), Nicole King (UC Berkeley), Eugene Koonin (NCBI), Edo Kussell (New York University), Michael Lässig (University of Cologne), Wallace Marshall (UC San Francisco), Kimberly Reynolds (UT Southwestern), Sarah Teichmann (EMBL - European Bioinf. Inst.), and Mukund Thattai (NCBS Bangalore)
 

Course subject

Recent years have witnessed a revolution in our ability to quantitatively query and manipulate biological systems. Live imaging and genome editing techniques combined with quantitative approaches have made it possible to uncover the molecular basis of many cellular processes. Indeed, cell biology has made tremendous progress towards revealing the molecular mechanisms involved in a plethora of processes ranging from cellular maintenance to the coordinated cellular decisions involved in embryonic development. However, these studies on how cellular functions are realized have been mostly devoid of the question of how these same functions came to be through evolution and natural selection.

The quantitative study of evolution, on the other hand, has been primarily focused on a description of evolutionary dynamics at the scale of populations. Here, the evolutionary history of species in a population is quantified based on the natural selection afforded by pressures such as competition with other species and changing environmental conditions. These studies focus mostly on the effect of mutations on fitness but not on how these mutations, through their effect on cellular function, lead to a reshaping of evolutionary landscapes.

A gap has existed between cell and evolutionary biology. Namely, the uncovering of how novel cell biological function evolves as organisms adapt to different environmental conditions. The time is ripe to bridge this gap as the technology that has allowed for the cell biology revolution in model organisms can now be applied in the context of their fully sequenced non-model organism counterparts. The aim of this course is to make progress towards filling this gap by focusing on the evolution of cellular processes ranging from horizontal gene transfer, to the evolution of intracellular functions such as trafficking and mitosis, to the evolution of embryonic developmental programs. The course will combine state-of-the-art in vivo microscopy, bioinformatics analysis and theoretical modelling in a plethora of model and non-model systems aimed at uncovering the evolutionary basis of cellular function and behavior at these multiple scales.


Course structure

This five week course will consist of a first "bootcamp" week to be followed by two 2-week research project sessions. Each session will consist of three or four experimental threads led by instructors and TAs who will closely work with groups of 4-5 students. Each day will consist of a morning lecture and discussions followed by lab work late into the evening.

The bootcamp week will be aimed at introducing all students to a basic set of microscopy, quantitative image analysis using Matlab and theoretical modeling that will be necessary for the later research projects. The lectures will take place in parallel with the KITP workshop "Evolutionary Cell Biology" providing a fertile forum for discussions and the exchange of ideas with the workshop participants.

 


EXPERIMENTAL PROJECTS (preliminary)

 

The Evolutionary Cell Biology of Gene Transfer. Instructor: Rob Phillips.

Microbial populations live in a fertile soup of foreign genetic information. Whether by viral infections, conjugative transfer, or direct uptake of the DNA through transformation, bacteria frequently share genetic information with their neighbors through horizontal gene transfer. Despite its deep evolutionary importance, the mechanistic details about how such gene transfer works (both at the molecular and systematic level) remain enigmatic. In our laboratory rotation, we will use microscopy to systematically explore how bacteria take up DNA.  By using fluorescently labeled proteins that bind to specific sequences on the transferred DNA, we will develop a real-time picture of DNA uptake and dynamics and consider its effect on evolution across many timescales.

 


Vesicle Transport Evolution in the Ciliated Protozoan Paramecium. Instructor: Michael Lynch.

Vesicle Transport Evolution in the Ciliated Protozoan Paramecium

In eukaryotic membrane trafficking, both soluble cargo and membrane-bound components must travel efficiently and specifically between organelles via lipid-bound vesicles. These vesicles are directed specifically to their target membranes via sets of interacting protein determinants that are themselves tightly controlled in space and time. These protein determinants are members of large gene families, whose duplication and diversification have enabled trafficking pathways in different eukaryotic lineages to diversify and take on new roles. We will focus on one trafficking pathway, endocytosis, whose associated protein determinants have shown remarkable plasticity over evolutionary time. We will identify the molecular determinants involved in one endocytic trafficking step, trace their evolutionary relationships, and then track them at the subcellular level. We will look particularly for instances of functional change, or hints of functional diversification occurring in these determinant subfamilies, and test our hypotheses of functional change gleaned from targeted sequence analysis.

This work will employ members of the Paramecium “aurelia” complex, a cryptic species complex of ciliates that diverged several hundreds of millions of years ago following two complete genome duplications. As outgroup comparators, several pre-duplication species are available. Having a large macronucleus, ciliates are uniquely amenable to rapid transformation by injection of chromosomal constructs with key genes fused to GFP, which can then be used to visualize subcellular localizations of gene products.

 

The evolution of gene regulation and pattern formation in development. Instructor: Hernan Garcia.

The evolution of embryonic development is the substrate for the emergence of new body plans. The general features of these developmental programs are highly conserved between closely-related species. Indeed, in the Drosophila family, of which the fruit fly D. melanogaster is its most prominent member, the same transcription factors lead to the expression of genes in similar patterns in the early embryo. Despite phenotypic similarities in developmental programs, homologous regulatory decisions are realized by different arrangements of binding sites for repressors and activators in each species. For example, the regulatory region leading to the famed stripe two of the eve gene has endured significant rearrangements as binding sites have been deleted, appeared or changed in their affinity and in their spacing. Still, all of these regulatory regions lead to stripes that are qualitatively comparable and that can even rescue development when inserted into different species.

Using novel imaging techniques that allow for the quantification of transcription in single cells in living embryos we will determine how different species create the “same” pattern of gene expression. We will insert regulatory regions for the eve stripe 2 from different species as well as proposed ancestral reconstructions into D. melanogaster and some of its allied species. These experiments will open the door to determining the role of regulatory drift in these highly conserved programs as well as generate a “phylogeny of strategies for pattern formation”. This phylogeny will then be compared to the more familiar phylogeny of the DNA regulatory sequence in order to develop theoretical models of how regulatory architecture determines function and how both evolve in tandem. Finally, in order to explore the limits of what evolution could have created we will use synthetic biology to rewire this network by creating the same stripe in response to engineered, exogenous transcription factors and comparing the dynamics of formation of this genetic rescue to those dynamics of the wild-type stripes.

The evolution of gene regulation and pattern formation in development

Evolution of pattern formation strategies. (A) Different fly species realize a stripe of expression of the same gene by using different regulatory architectures. Namely, the placement, number and affinity for activators and repressors in the vicinity of the gene. (B) Live imaging techniques give access to the dynamics of stripe formation in different species and to contrasting this “phylogeny of pattern formation strategies” to the more familiar DNA sequence phylogeny.

 


The evolution of an active membrane composite. Instructor: Jitu Mayor with Thomas van Zanten

The outer membrane of the living cell is the interface that demarcates the cell and its environment. Its chemistry is largely build up from lipids and proteins. Local organization of this chemistry primes the cell to adapt and react to the external milieu and communicate information about its internal state. In a range of metazoan cells the membrane organization is not simply passively constituted, but it is actively maintained away from thermodynamic equilibrium. This characteristic emerges from an interplay between the constituents of the membrane closely juxtaposed to a dynamic actin-based cortical layer, together forming an active composite. Cells consume energy to mechanically restructure this composite that consists out of, amongst others, the evolutionary conserved proteins such as actin, actin binding proteins and myosins. Perturbing the non-equilibrium state of cells has a dramatic effect on essential functions such adhesion or endocytosis.

Do the different cellular organisms we find in the local marine or soil environment display an active organised plasma membrane? Using an experimental scheme that was originally used for studying the membranes of metazoan cells we will explore the organisational capacity of membranes across the phylogenetic tree. This approach will consist of doping the membranes of protists with fluorescence lipid dyes and imaging FRET between the doped lipid probes to evaluate their local organization at the nanometer scale. Another way to explore this question is take a more bio-informatics approach and trace back different proteins over evolutionary time using available genome data.


Combined projects of Monica Bettencourt-Dias, Cassandra Extavour, Jose Pereira Leal, and Dan Needleman.

 

Within-Species Variation in Cell Biology in Drosophila

Everyone is unique.  We have different appearances, different behaviors, and different talents.  But to what extent are the dynamics and organization of our cells different?  The answer is currently unknown.  Variations in cellular behaviors between different people likely underlie many of our obvious external differences and might be responsible for differences in susceptibility to disease. Understanding the extent and nature of the genetic basis of within species variations in cell biology will also help to understand the evolution of variations in cell biology.

Recent work in the nematode Caenorhabditis elegans has shown that there is extensive within-species variation for the structure and dynamics of the mitotic spindle.  We will extend this work by investigating the extent of within-species variation for a variety of cell biological features in the fruit fly Drosophila melanogaster.  We will use quantitative approaches to characterize this variation and we will attempt to map the genetic basis of any differences we discover.

 

Between-Species Variation in Cell Biology in Insects

Between-Species Variation in Cell Biology in Insects

Above is a “t-projection” of a 26-hour whole Gryllus (cricket) embryo time lapse of the H2B-GFP cricket transgenic line, taken from 5 to 31 hours after egg laying (AEL). Each time point is colored in a different color along a gradient lookup table and then stacked (with dark warm colors at the beginning and yellow to white at the end of the time lapse). This shows the general course of the time lapse. (Taro Nakamura & Seth Donoughe)

Drosophila melanogaster has served as the exemplar for molecular and quantitative understanding of insect development for nearly half a century. However, this fly belongs to the lineage furthest derived from the last common insect ancestor, and its developmental and cell biological characteristics are unlikely to be representative of the most species-rich animal group, the insects. Working with not only Drosophila, but also the beetle Tribolium castaneum and the cricket Gryllus bimaculatus, students will be able to perform cell biological comparisons between lineages that diverged hundreds of millions of years ago, yet still share a highly conserved adult body plan.

Because early insect embryos undergo syncytial cleavage, in which nuclear cleavage takes place without cytokinesis, these embryos are essentially huge multinucleate cells. Moreover, although all of these insects reproduce sexually, their haploid, unfertilized eggs can be induced to begin cleavage and start to proceed through development. This provides an opportunity to compare the dynamics of nuclear spacing, synchrony and number of nuclear divisions, sexual versus asexual development, and nuclear dynamics across a broad phylogenetic range.

For both Tribolium and Gryllus, we will provide stable transgenic lines expressing fluorescently labeled cell components, enabling live imaging and quantitative analysis of image data.  In addition, we will be able to manipulate embryogenesis in these animals by injecting mRNAs encoding proteins of interest, including fluorescent tags for cell biological components, pharmacological agents, and inducing RNA interference against genes of interest.

A zoomed in view of nuclei at the mid-blastoderm stage of Gryllus

A zoomed in view of nuclei at the mid-blastoderm stage of Gryllus, with four successive time points 10 minutes apart superimposed in different colors. For some nuclei, there is no overlap between neighboring time points. (Seth Donoughe)

Cancer cells show supernumerary centrosome numbers

Visualization of centrosome duplication using labelled tubulin in embryos.

 

Cellular Evolution In Cancer- Does It Provide New Therapeutic Opportunities?

Cancer cells show several modifications in cellular characteristics, that allow them to survive, proliferate and conquer new environments. Those modifications are often specific of cancer cells and can be used for selectively killing them.

DNA).The centrosome, the major microtubule-organizing center in animal cells, participates in processes such as cell division, migration and establishment of cell polarity. Centrosome aberrations are commonly observed in cancer. Those changes might lead to increased genomic diversity and ability to invade and conquer new environments. However, those changes can also lead to very abnormal divisions that result in cell death. Mechanisms that allow cancer cells to survive in the presence of centrosome aberrations have been recently described and are being used to selectively kill cancer.

We have recently conducted a phenotypic screen for centrosome changes in a panel of 60 human cell lines that reflect different tumor types. We have seen that centrosome aberrations are a hallmark of cancer cells. While there are a few major mechanisms used to cope with those changes, we have observed that cancer cells “are creative” and can adapt in multiple ways. We will extend this work by investigating the strategies cancer cells use to adapt to centrosome changes. We will do so by using quantitative analyses of cell cycle progression and mitotic division, while monitoring chromosomes, mitotic spindle and centrosomes in live cell populations. Information on gene expression and proteome is available for the different cell lines, so ultimately cellular mechanisms might be linked to molecules. We can further investigate whether mechanisms of adaptation are already present within the population as standing variation, or arise after the appearance of centrosome changes. We will do that by acutely manipulating centrosome and spindle structural properties in normal and cancer cells, and investigating how they adapt to them.

We can also model those processes to predict whether drugs that target them would be effective in eliminating cancer cells. This project will involve microscopy, tissue culture, genetic manipulation of cells, quantitative imaging, bionformatic analysis and modeling.

 

Natural variation in the cell biology of marine micro-organisms

Life started in the sea, and the sea is still a source of breathtaking cellular diversity. This includes serial endosymbiotic systems where we find stable associations of cells nested within cells. For example, some dinoflagelates are found in algal blooms, where they co-exist alongside cells that have a silica-based cell-wall with amazing diversity and beauty under the microscope. The two types of organisms are examples of microalgae, primary biomass and oxygen producers in the ocean. They include many toxic species that experience explosive growths, giving rise to algal blooms that cause untold economic costs to a variety of industries worldwide, for example including shellfish farming and fisheries.

Marine micro-organisms are a good system to study natural variation of cells “in the wild,” particularly algal blooms where high concentration of diverse species co-exist. While a lot of attention has been devoted to inter-species variation and to species composition of marine micro-algae, little attention has been devoted to within-species variation. For example, do all individuals in a population for a given species multiply at the same rate? This is an important cell biology question with an impact in the dynamic of algal blooms.

In this project we will sample the local marine environment and characterize its species diversity using different techniques. We will isolate species and expand them in culture for cell biological characterization, studying natural variation of division rates, cell size, etc. We will pay particular attention to characterizing microtubule-derived organelles and structures, such as centrosomes and mitotic spindles, as proposed in the other projects in this session.

We will also explore the genetic variation and the basis of phenotypic variation using a genomics and bioinformatics approach. We will analyze publicly available genome sequence data, using bioinformatics and molecular evolution approaches, to detect for signatures of selection, expansion of protein families, variation of biophysical properties of proteins etc., in relation to phenotypic variation.

Students will be able to choose to focus more on the computational or on the wet-lab side of the project, or a hybrid of both.

Microbial Strategies for Survival and Evolution

 

Course Directors: 

Paul Rainey (NZIAS, MPI for Evolutionary Biology), Joel Rothman (MCDB, UCSB) and Boris Shraiman (KITP, UCSB)
 

Course Instructors:

Josep Casadesús (Seville), Michael Elowitz (Caltech), Christopher Hayes (UCSB), Sanna Koskimiemi (Uppsala), David Low (UCSB), Silvia De Monte (ENS Paris), Andrew Murray (Harvard), and Nathalie Questembert-Balaban (Hebrew University)
 

Guest Lecturers (partial list):

Bonnie Bassler (Princeton), Daniel Fisher (Stanford), Isabel Gordo (Gulbenkian Inst), Terry Hwa (UCSD), Roy Kishony (Harvard Medical School), Edo Kussel (NYU), Michael Lässig (Cologne), Michael Laub (MIT), Richard Lenski (MSU), Bruce Levin (Emory), Richard Losick (Harvard), Richard Neher (MPI Tubingen), Alan Perelson (LANL), Paul Sniegowski (U. Pennsylvania), Paul Turner (Yale), Daniel Weinreich (Brown), David Weitz (Harvard), and Ned Wingreen (Princeton)

 

 

COURSE SUBJECT

 

Microbial life is considerably more complex than is commonly appreciated.  Far from merely proliferating exponentially until nutrients are exhausted, microbes engage in a wide range of adaptive behaviors. For example, the diauxie phenomenon associated with the nutrient induced shift of bacterial metabolism was at the heart of Monod and Jacob’s discovery of regulated gene expression. Much like cells in the multicellular organisms, microbes – bacteria and yeast for the purposes of this course – can exist in different phenotypic states. Microbes can switch between different phenotypes and the phenotypic states can persist through cell division thanks to the existence of epigenetic memory.  The mechanisms of the epigenetic memory are remarkably diverse, from hysteretic transcriptional regulation circuits to metabolic circuits to DNA methylation and are a subject of much current research, focusing in particular on the role of inductive signals and stochastic fluctuations in switching between states. Interestingly, epigenetic switching coexists with mechanisms of genetic switching via genomic rearrangement (phase variation) or recurrent mutations bridging in a very interesting way the phenotypic dynamics of microbial cells and evolutionary dynamics of microbial colonies. Quantitative exploration of these phenomena will be the central themes of the course.

   
The adaptive utility of phenotypic diversity of microbial colonies sometimes seems transparent: sporulation of a small fraction of a B. subtilis colony or a small number of slow growing E. coli “persisters” serve as insurance policies for the survival of respective genotypes, should the environment suddenly turn inhospitable. Transition, under conditions of stress, into a “mutator” state or a transition into the competence state, which allows active import of DNA from outside, may also seem like good ideas: increasing the rate of genetic innovation increases the probability of a finding an evolutionary solution to the stress problem. Yet, the balance between the uncertain benefit and the clear cost of increased mutational load is not obvious and requires a fully quantitative analysis.  The study of these questions must combine quantitative experiments and models – an approach the course will serve to foster.
   
Another fascinating aspect of microbial phenotypic variation is the interaction between cells that it often involves. Bacteria are known to obtain information from their environment via “quorum sensing” systems, but the adaptive significance and full scope of the behavioral consequences are not yet known. Some of the interactions are interspecies competition, like the microbial production and secretion of antibiotics, yet other interactions are more complex. For example, bacteria expressing a Contact Dependent Inhibition system inhibit the growth of neighbors, unless they are of the same phenotypic state. This is capable of generating local collective behavior. The locally collective behavior is particularly interesting from the point of view of the possibility of cooperation. Interesting recent experiments clearly demonstrate the benefits of “sticking together” – quite literally! – as a way of sharing secreted “common goods” upon which competing types that do not contribute to the common good can gain advantage.  Once again understanding this behavior requires quantitative approaches.
 
Tools, methods and technologies.
The course will emphasize direct engagement between experiment, analysis and quantitative modeling.  In addition to classical methods of genetic and phenotypic analysis, students will obtain hands-on experience with a range of state-of-the-art tools, including fluorescent microscopy for imaging of phenotypic diversity and single cell tracking, microfluidics and genomics (including deep sequencing).  Live imaging will focus on the dynamics of epigenetic switching of single cells within the context of growing populations, which together with image segmentaiton and lineage tracking has proven invaluable in the study of microbial behaviour.  To enable quantitative analysis of low frequency (switching) phenotypes the course will employ various microfluidic devices including droplet-microfluidics coupled to strategies for cell sorting, including FACS.  Real-time evolution experiments of mutation-driven switching will be dissected with the help of deep sequencing and computational analysis.   
 
Course structure.
The 5 week course will consist of a one week "bootcamp" followed by two 2-week research project sessions, each consisting of 3 experiments performed by groups of 4-5 students led by instructors and TAs. The daily schedule will consist of two morning lectures and/or discussions, followed by the afternoon and evening of work in the lab. The intensive bootcamp week will aim to bring everyone up to speed on basic microbiology lab techniques and provide a hands-on introduction to microfluidics. The course will also provide tutorials on microscopy and image analysis as well as computational modeling and data analysis (in Matlab). The lecture and discussion component of the course will provide an in-depth coverage of central concepts in microbial population and evolutionary biology with the focus on the genetic and epigenetic mechanisms underlying phenotypic diversity and its role in long term survival. The course will run alongside the KITP workshop on "Evolution of Drug Resistance" with the students attending its seminars and interacting with workshop participants.
 
 

EXPERIMENTAL PROJECTS (preliminary)

 

Bet-hedging and rapid phenotypic switching
Instructors: Paul Rainey and Silvia De Monte

Project Description: All organisms are faced with the challenge of maintaining fitness in diverse and changing environments.  Under predictable conditions, coordinated regulation of gene expression allows organisms to modify aspects of behavior, morphology or phenotype to match prevailing circumstances. In fluctuating, or unpredictable, environments, such strategies fail: survival requires that organisms hedge their evolutionary bets.  In bacteria bet hedging is achieved by mechanisms that promote rapid stochastic phenotype switching.  We will take advantage of two real-time evolution experiments using bacteria, out of which genotypes have evovled that stochastically switch between distinct cellular states.  Using a combination of theory, modeling, single cell analyses and deep-sequencing we will explore cellular and mutaitonal details of these switches in order to understand evolutionary parameters affecting the success of populations. For additional background please see Beaumont et al, Nature 2009 and Libby&Rainey, Proc. R. Soc. B (2011) 278, 3574–3583
 
 
____________________________________________________________________________________

Mutational and non mutational resistance to bacteriophage.
Instructor: Josep Casadesús

Classical studies showed that bacterial resistance to phages can be acquired by mutation. In certain cases, however, bacteriophage resistance can be also acquired by non-mutational mechanisms. For instance, the Salmonella enterica opvAB operon encodes cytoplasmic membrane proteins that alter lipopolysaccharide O antigen chain length, and confer resistance to bacteriophage P22. Because expression of opvAB undergoes phase variation, S. enterica populations contain a mixture of opvAB-ON and opvAB-OFF cells. Infection of Salmonella enterica with the virulent phage P22 H5 kills the major opvAB-OFF subpopulation and selects the opvAB-ON subpopulation. Cessation of phage challenge permits the appearance of a novel opvAB-OFF subpopulation generated by opvAB phase variation. Epigenetic resistance thus pre-adapts S. enterica to survive phage challenge in a reversible manner and without the fitness costs associated with mutation. However, the S. enterica opvAB-ON subpopulation is avirulent in mice and unable to proliferate in macrophages. Phase variation of opvAB expression may thus be viewed as a tradeoff between virulence and phage resistance. We will characterize phage and bacterial populations using fluctuation tests, assays of fitness costs associated with individual mutants and flow cytometry to determine rates of switching between ON and OFF states.
____________________________________________________________________________________

Quantitative study of “persistor” behavior
Instructor: Nathalie Questembert-Balaban

Persister cells are a minority of phenotypically distinct cells that evade antibiotic treatments. Typically, those cells 
manage to persist under transient antibiotic treatments as long as they stay dormant during the exposure to the antibiotic. They may stochastically switch to normal growth and re-create the original population.  We will evolve bacterial populations under transient exposure to antibiotics. We will quantify the growth arrest of bacterial population and determine the relation, both experimentally and by modelling, between the distribution of growth arrest times of the evolved strains and the time scale of the antibiotic treatment.
 
Fluorescence induction of growth arrested cells in a microfluidic device,  Gefen O. et al., PNAS (2014)
 
 
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Stochasticity in phenotypic switching: Stochastic Pulse Regulation in Bacterial Stress Response
Instructor: Michael Elowitz

Gene regulatory circuits can use dynamic, and even stochastic, strategies to respond to environmental conditions.  Using individual Bacillus subtilis cells we will explore the general stress response mediated by alternative sigma factors and characterize effects due to energy stress.  Models based on minimal genetic circuitries will be constructed to which data from single cell dynamics will be fitted.  The overall goal is to understand how  how bacteria encode signals using stochastic pulse frequency modulation through a compact regulatory architecture.

 

SigmaB activation, from Locke et al, Stochastic Pulse Regulation in Bacterial Stress Response, Science 2011: Vol. 334 no. 6054 pp. 366-369

 

To watch a movie see http://www.elowitz.caltech.edu/graphics/1208144s1.mov

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Evolution of cooperation in S. cerevisiae
Instructor: Andrew Murray

Recent experimental evolution using yeast resulted two phenotypes that depend on cells sticking together. One is the formation of multicellular clumps to successfully exploit the nutrients harvested by a secreted common good and the other is a circadian oscillator that is detected by its ability to regulate intercellular adhesion. Students will study these

strains to try and understand the mechanisms that regulate clump size.  In particular we will ask how the nutrient-harvesting clumps control their size: whether connections between cells break at random or whether the probability of breaking increases with age or with clump size.  Models will be developed to predict the distribution of clump sizes.  For one of the nutrient-harvesting strains clump size varies depending on the nutrient it receives: wewill determine how this control works.  We will also analyze the oscillator.  This type requires precise temporal control of cellular adhesion.  We will determine whether it is possible to monitor adhesion directly and perform experiments to unravel causal factors.

Clumpiness in the evolved clone of S. cerevisiae due to failure to separate (see Koschwanez et al eLife 2, pp. e00367 (2013)

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Rapid evolution in bacterial contact inhibition system

Instructors: Sanna Koskiniemi, Christopher Hayes and David Low

Contact-dependent growth inhibition (CDI) is a phenomenon in which one bacterium inhibits the growth of another upon direct cell-to-cell contact. During CDI, a large stick like exoprotein known as CdiA with a highly divergent C-terminal end containing the toxin domain is transported to the inhibitor cell surface. Upon cell-to-cell contact the toxic C-terminus is delivered to target cells. To protect inhibition from self, a small CdiI protein binds specifically to its cognate toxin and prevents inhibition. The interactions between inhibitor and target cells are intricate and delivery of toxin to target cells requires multiple steps, including binding of a receptor on target cell surface, transfer through the inner and outer-membrane as well as binding of the toxin to an intra-cellular protein required for toxic activity. Disruption in any of these steps allows the target cells to survive and thus resistance development should occur frequently. However, the protein players identified so far consist mostly of essential proteins where even small substitutions might have large detrimental effects on the cell fitness. We have recently carried out a long-term evolution experiment using Salmonella in which derived bacteria evolved the capacity to contact-inhibit the ancestral type.  We will explore the adaptive significance of this behavior through construction of simple models and will subject strains from the experiment to genetic (genomic) and phenotypic analyses in order to understand the basis of derived contact-dependent killing of ancestral types.

For additional background please see Hayes et al, Cold Spring Harb Perspect Med 2014

 

 

 

Summer Course 2013Course Directors:  

Thomas Lecuit (IBDML, Marseille), Joel Rothman (MCDB, UCSB), Ewa Paluch (MRC LMCB, UCL London), Boris Shraiman (KITP, UCSB).

Course Instructors:

Otger Campas (UCSB), Thomas Gregor (Princeton U), Lars Hufnagel (EMBL Heidelberg), Denise Montell (UCSB), Pierre Neveu (EMBL Heidelberg), Bill Smith (UCSB)

Guest Lecturers (partial list):

D. Axelrod (Palo Alto), B.Baum (London), Y. Bellaiche (Paris), J. Briscoe (London), A. Boudaoud (Lyon), E. Davidson (Pasadena), S. Eaton (Dresden), M. Gonzalez-Gaitan (Geneva), S. Grill (Dresden), CP. Heisenberg (Vienna), K. Irvine (New Brunswick), F. Julicher (Dresden), A. Maddox (Montreal), E. Munro (Chicago), J. Nelson (Palo Alto), A. Oates (London), K. Oegema (San Diego), P. O’Farrell (San Francisco), O. Pourquie (Strasbourg), J. Prost (Paris), E. Siggia (New York), D. Sprinzak (Tel-Aviv), J. Spudich (Palo Alto), D. Strutt (Sheffield),  J-P. Vincent (London),  E. Wieschaus (Princeton).

 

 

 

Course Subject:

This Course will focus on the fundamental problem of understanding the dynamics of morphogenesis: the process that converts the genetic blueprint of a multicellular organism into the complex physical structure. How does the morphogenetic "program" of animal development convert genetic information into shape, form and function? To answer this question one needs to understand the dynamics of controlled growth and differentiation, which is directed by intercellular signals and unfolds on the mesoscopic scale of growing tissues. Morphogenesis involves numerous interacting processes that result in phenotypes of great complexity, disentanging which requires quantitative models and quantitative measurements. Thus the goal of the Course is to advance the D'Arcy Thompson's agenda of quantitative description of "Growth and Form" using the full power of modern imaging and molecular genetics which makes the field ready for rapid progress.

On the technical level, the course will introduce several model organisms including D. melanogaster, C. elegans and Ciona and provide instruction on live imaging, micro-manipulation, and genetic and chemical perturbations as quantitative tools to study developmental dynamics. Experimental work will be complemented by theoretical and computational modeling and analysis and involve collaboration with the participants of the concurrent KITP workshop on "New Quantitative Approaches to Morphogenesis".

Course structure:

The 5 week Course will provide an intensive laboratory experience, involving research level projects in small teams of "students" guided by Instructors and TAs.  In addition the students will attend and participate in research seminars at the  KITP workshop on "New Quantitative Approaches to Morphogenesis".

 

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

Week 1

Boot-camp

Safety training

Introduction to model organisms

Introduction

to model organisms

Introduction

to model organisms

Introduction

to model organisms

Principles of microscopy.

Demos

Image

processing:

Ideas and

tools

Week 2

1st project

Week 4

2nd project

Supervised

experiments

Supervised

experiments

Supervised

experiments

Supervised

experiments

Supervised

experiments

Supervised

experiments

Week 3

1st project Week 5

2nd project

Data Analysis/

Additional experiments/

Modeling

Analysis/

Additional experiments/

Modeling

Analysis/

Additional experiments

Modeling

Analysis/

Additional experiments

/ Modeling

Analysis/

Additional experiments/ Modeling

Project presentations

 

Boot-camp:  Daily schedule:

9:30am-noon:

2 tutorial lectures

1pm – 6pm

Demonstrations/basic lab skills development

Project SessionsDaily schedule:

9:30am-noon:

Lectures/discussions at KITP

12-1pm 

Lunch

1pm – 6:30pm

Project supervision; Students divided into three groups (~ 4 per group) working in parallel with instructor teams.

6:30-8pm

Dinner (UCSB dining services)

8pm-midnight:

Open Lab time

Sunday Social activities:  Beach BBQ, whale-watching, hiking, sports activities. 

Topics covered by the boot-camp:

  • Introduction to model organisms (Drosophila, C. elegans, Ciona, etc.) and system-specific tools (basic genetics; developmental characteristics; sample preparation)
  • Principles of microscopy. Basics optics and theory and practice of biphotonics
  • Image segmentation, particle tracking; optical flow and PIV.

Preliminary description of specific projects :     

Project 1: Quantifying transcription and pattern formation in developing fly embryos (Thomas Gregor, Princeton). The pattern blueprint of the adult fly is determined by only a handful of genes within the first 3 hours of embryogenesis. Qualitatively, this system is extremely well understood, and is amenable to quantitative analysis leading to a comprehensive mathematical description of early transcription and patterning processes. We propose two specific goals: 1) live imaging and modeling of gene expression patterns, and 2) measurement and modeling of gene regulation at the single molecule level. For 1) we have transgenic fly constructs that express GFP fusions with various genes of the early patterning cascade, and propose to image, quantify, and model their spatio-temporal dynamics by confocal microscopy. For 2) we will follow transgenic fly constructs expressing alterations of a specific enhancer element that is crucial for early patterning. Using a novel mRNA labeling technique that allows us to optically count individual mRNA molecules in whole embryos, we propose to quantify the effect of the alterations and use stochastic models of transcription to study structure-function relationships of this enhancer element.

Project (2) Building a Single Plane Illumination Microscope (SPIM). (Lars Hufnagel, EMBL). Embryonic development is a highly dynamic process involving many spatial and temporal scales. Novel light microscopy methods are needed to elucidate fundamental morphological processes and to enable close contact with theoretical modeling. The light-sheet concept has proven to be high suitable to image developmental processes. During the 1st week of this project, students will assemble a light-sheet microscope on an optical table. Students will receive hands-on training in basic optics concepts and learn how to program the microscope control software in LabView. During the 2nd week they will quantify/calibrate optical properties of the setup by imaging fluorescent beads (2 days). We will teach students (2 days) light-sheet microscopy specific mounting, image processing and analysis techniques, image stack fusion algorithms and de-convolution, and image the dynamics of (GFP-labeled) nuclei in a Drosophila syncytial blastoderm.

Project (3)  SPIM imaging and morphometric analysis of marine invertebrates (Bill Smith, UCSB, and L. Hufnagel, EMBL). Embryos of marine invertebrates such as ascidians and sea urchins, are attractive models for studying developmental dynamics owing to their transparency and ease of culture. This project will avail of the world-class marine laboratory at UCSB and the SPIM built in Project (2). Expression constructs driving GFP fusion proteins of morphogenetically important proteins will be introduced into embryos by microinjection or electroporation. Using image analysis tools we will focus on quantifying and correlating dynamic changes in cell shape and positions with changes in subcellular localization of the fusion proteins. This will tie in with the (vertex-type) modeling of tissue mechanics, that are being developed in Hufnagel’s and in Shraiman’s labs, both of whom will provide theory supervision.

Project (4) Analyzing variation, robustness and compensation during embryogenesis. (Joel Rothman, UCSB). Variant initial conditions, molecular noise, and environmental conditions mean that every individual embryo in a complex animal follows a unique developmental pathway that nevertheless culminates in a similar final product. This profound variation seen between individuals has recently also been observed in simpler animals, such as C. elegans, that were renowned for their developmental constancy. Natural variation in C. elegans will be studied in developing embryos and individual cells and cellular substructures tagged with fluorescent proteins. Genetic alterations (e.g., mutants defective for buffering systems), laser-microsurgery, temperature gradients, and chemical perturbants, will be used to test the plasticity of individual cellular events (cell division timing, position, shape, and organelle distribution). Computational modeling will be performed to identify at least one step or process that drives deviations in cellular geometry toward a normalized state, resulting in reproducible output of embryogenesis.

Project (5)  Mechanics of tissue morphogenesis in Drosophila (Otger Campas, UCSB). Morphogenesis involves tissue growth and remodeling in space and time. This project will introduce the students to the cutting-edge techniques developed for quantitatively measuring mechanics and collective cellular movements in living tissues. We will focus on early Drosophila embryogenesis to study cellular motions and cell shape changes during gastrulation. Students will characterize tissue flow quantitatively by analyzing correlations in cellular movements that define their collective behavior.  Tissue flows will be perturbed using UV laser pulses to disrupt cellular junctions. Quantitative analysis of the tissue flow after laser ablations provides quantitative information about local tissue mechanics, which can be interpreted with the help of the vertex-type models. This project will provide the students the necessary quantitative methods to analyze tissue mechanics in other systems.

Project (6) Single cell studies of stem cell differentiation dynamics. (Pierre Neveu, EMBL). This project will study differentiation of mouse embryonic stem cell into a neural lineage. We will use ESC lines expressing fluorescent proteins reporting expression of miRNAs (excellent markers of cell states) and transcription factors associated with pluripotency and neurogenesis. To probe the differentiation landscape, we will use ESC lines where we can perturb the expression of key neurogenesis regulators. Students will perform automated quantitative single cell live imaging of neural differentiation under various conditions. Movies will be segmented and tracked to extract the behavior of single cells. The data will be used to differentiate between two quantitative models: one based on the assumption of exchange of stability between two attractor fixed points; the other based on bistability.