2013 Course Description

Course 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):

 

 

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 Sessions:  Daily 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.