Senior Scientist in Biology and Instructor in Bioengineering
California Institute of Technology
Beckman Institute 139-74
Pasadena, CA 91125 USA
Off: (626) 395-2004
Lab: (626) 395-2863
E: rusty@caltech.edu
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Research Interests
Dynamic analysis of forebrain and midbrain development, Interplay between genetics and hemodynamics in heart formation, DNA recombination in ES cells, Transgenic Avians, Bio-detection using Nanodevices, Advanced Optical Microscopy, Global Health & Poverty
Dynamic Visualization of Embryogenesis
My research aims to dynamically visualize and characterize avian embryonic development at sub-cellular resolution. My primary focus is determining how the brain and heart form and develop. Instead of labeling and following small numbers of cells at a time, I am have developed the means to optically record all or nearly all the cells within a developing embryo simultaneously. I typically try to place fluorescent tags within all avian embryonic cells using GFP-expressing retroviruses. Cell and tissue movements in the developing embryos are then recorded using multispectral, time-lapse fluorescent microscopy in 3D. Next the recorded data is analyzed using computers running sophisticated cell-tracking and color discrimination software capable of distinguishing the subtle movements that thousands of individual cells make and identifying a handful of genes that these cells express. The gene expression and cell migration data collected using laser microscopes is subsequently integrated within MRI collected datasets in order to understand the complex informational interactions that are occurring during development within the spatial and temporal context of the maturing embryo.
Production of transgenic GFP expressing quail
Developmental biologists have long used avians to study embryogenesis. Avians are often preferred to mice because the embryo is accessible at all stages of development. This accessibility makes them amenable to tissue transplantation and time-lapse videomicroscopy. However, the genetic techniques that have been so powerfully exploited in mice have not been developed for avians. So the crucial question is, is it easier to videorecord embryogenesis in mice or to introduce genetic techniques into avians?
It is my opinion that the best plan is to develop avian genetic techniques. Avian embryos are preferred because they are accessible to study at all stages of development without the heroic efforts that are required to study mouse embryos. Avians have long been the premier non-mammalian vertebrate model organism. The Japanese quail offers advantages in the small size of its egg, the moderate size of the breeding adults, and its short generation time. Because of these advantages, the proposed work permits molecular genetic experiments on a higher vertebrate embryo both more rapidly and less expensively than comparable work on the mouse.
Retroviral and expression vectors that co-express GFP and a selectable marker are being introduced into ES/PGC cells. Selection is arguably the most powerful force in biology. It governs species fitness and immune system B and T cell fitness. It can be used to isolate transfected cells that express resistance to certain drugs when grown in the presence of those drugs. Resistance to drug induced cell death imparts a selective advantage on the genetically modified cells. Ideally, the increased selective advantage will permit a higher degree of chimerism in the transfected embryos, which would lead to a higher frequency of germline transmission. Additionally, drug resistance can also be used to maintain gene expression is embryonic cells over time. By infecting blastoderm cells of stage X embryos (Hamburger and Hamilton 1951) with concentrated VSV-G pseudotyped GFP expressing retroviruses, I have been able to obtain >99% genetic chimerism in both quail and chick embryos. I am currently hatching putative transgenic quail to breed for germline transmission.
Tissue specific expression of GFP in avian embryos
(with Charlie Little, Univ. of Kansas Medical School)
We have developed and are presently making several new GFP-expressing MoLV and HIV retroviruses. These retroviruses are self-inactivating which means that we delete much of the transcriptional machinery from the 3'LTR U3 region that is often recognized as foreign by the host cells and inactivated by methylation. We are also incorporating lox/Cre recombinase technology in order to remove most of the retroviral provirus from the infected cells’ genome, leaving behind just the transgene. We are testing neural and cardiac specific transcription elements that display endogenous expression patterns when used as transgenes in mice. In addition, the transcriptional elements must be less than 2 kb is size in order to easily fit within the 9-10 kb size limitations of the retrovirus.
Publication: Dev Dyn (2006, in press)
Dynamic Analysis of Blood Flow and Heart Development in Avian Embryos
(with Mauri Gharib, Dept of Bioengineering and Aeronautics at CIT and Kent Thornberg, Univ. of Oregon)
Our research is aimed at determining interacting roles of genetic programming and hemodynamic physical forces in regulating cardiovascular development in the early embryo. To capture blood flow in the developing avian we fluorescently label cells using various methods. Conventional laser microscopes are too slow to be able to collect XY images of blood flow or to resolve the cells within a beating heart without blurring. Digital particle image velocimetry (DPIV), a non-invasive, laser-based technique that enables accurate measurements of velocity in a plane, requires images without motion blurring. Test experiments show that frame rates of >300 frames/s are essential to reliable DPIV. We have designed and integrated a novel high speed imaging system in order to capture images of these fast-moving fluorescently labeled cells and tissues at frame rates of 300-500 frames/s.
We recently succeeded in recording and analyzing the first microscale cardiac electrical activity from an early embryonic (pre-looping) vertebrate heart. We monitored electrocardiograms from E2.5 quail embryos. A single recording electrode was positioned at various points adjacent to the heart (ventricle, A-V constriction, and atrium) with a second reference electrode in the egg albumen. Software tools we are now refining allow us to use the recorded ECG to assemble the individual images from the high speed imaging system into a full 4D image of flow patterns in the heart as well as the position/movements of the labeled cells that comprise the heart.
MRI of Quail Embryogenesis
(with Seth Ruffins, Melanie Martin, and Russ Jacobs, Caltech)
Numerous experiments have been carried out with an 11.7 Tesla magnetic resonance imager (MRI) to determine which parameters are the most useful to optimize image contrast in developing avian and mouse embryos. In MRI, signal intensity is obtained from water protons and is a function of the concentration/environment of water. MRI contrast results from variations in the environment that changes the characteristics of proton relaxation times. The T2-weigted 3D multi-spin echo routine used to collect images of murine embryos at different developmental stages (Dhenain et al., 2001) has also worked for quail embryos. Currently, the acquired 3D quail datasets have 20-50 um voxel resolution. We are able to take images of quail embryos in ovo and in vitro.
Digital Three-Dimensional Atlas of Quail Development Using High-Resolution MRI
Link to MRI Atlas of Quail Embryogenesis
We have developed a Quail Developmental Atlas (QDA) that provides a perfectly registered volumetric reconstruction of the developing quail embryos using a magnetic resonance imaging (MRI) microscope. We are collaborating with curators at the California Science Center and the San Francisco Exploratorium to design and develop interactive exhibits that will bring the QDA to a broader lay audience and around which we intend to develop more formal educational programming. We are in the process of publishing our ex ovo (E5-E10) and then in ovo (E7-E15) data with TheScientificWorld in an online format for the developmental biology and anatomy research community. We have annotated the gross anatomical structures within the classic spatial compartments comprising the embryo using Amira software. Anatomy will also be used as primary framework for incorporating specific patterns, such as gene expression, the distribution of receptors and their ligands, and cell migration routes.
The QDA project has been greatly advanced with high school students helping to annotate the MRI data sets using Amira software and to produce exhibit quality movies using Final Cut Pro movie editing software. We are updating on a weekly basis a website has been established to disseminate the QDA (http://atlasserv.caltech.edu/Quail/Start_Quail.html). Our use of high school students also provides several young potential scientists with their first exposure to ‘cutting edge’ research and direct mentoring from research scientists.
Under current consideration, we propose to develop a publicly accessible educational exhibit. This exhibit would be replicated in at least two institutions and would supplement and enhance existing displays of embryo development with high-resolution interactive imagery. In addition to these public displays, we are collaborating with the Teacher Institute of the San Francisco Exploratorium to bring the QDA to high school class biology, math, and physics classes. The students will be able to carry out ‘virtual dissections’ of the quail embryos in the biology modules, run MRI based simulations in the physics modules, and work on numerous “EggMath” and embryo growth math problems in the math modules.
The Teachers Institute at the San Francisco Exploratorium will lead efforts to develop a QDA workshop for high school teachers that will be taught first at the San Francisco Exploratorium in Summer 2007 and then at the California Science Center. We also plan to develop and freely distribute self-contained interactive DVDs that contain QuickTime movies of the QDA that students can easily use, along with the physics and math modules. Our long-term goal is that the website, interactive DVDs and museum exhibits will have a nationwide audience within three years.
Past Research Projects
BioNEMS
(with Michael Roukes, Scott Fraser, and the BioNEMS team)
Our goal is to draw upon the advances and insights from a variety of fields to create a modern suite of rapid biochemical assays with sufficient sensitivity to assay the contents of single cells. Recent work from several labs has shown that Chemical Force Microscopy can be realized with straightforward modifications of an Atomic Force Microscope. This has resulted in measurements of the force of single receptor-ligand bonds, antibody-antigen interactions, and even single hydrogen bonds with surprising accuracy. These impressive advances pose the challenge that must now be realized if one it to establish a single cells assay. The assay must be capable of responding to the ~10-1000 copies of a given molecular species in the volume of a single cell (~1pL), and must have the temporal resolution to follow the binding kinetics of single biomolecules (&Mac178;1usec). Recent advances in Nano Electro Mechanical Systems (NEMS) suggest that these design goals can be achieved. Our goal is to build a device with a large array of NEMS cantilevers (&Mac179;500) in a small volume (~100pL) capable of harvesting and analyzing the contents of a single cell.
My group has designed and synthesized alkanethiol molecules that are specifically attached in the form of self-assembled monolayers to gold pads located at the cantilever tips. We have developed the protocols to bind bacteria, virus, proteins, and nucleic acids to these molecules in a specific manner. We hope to be able to qualitatively and quantitatively compare the cellular components of individual cells within one another using this approach.
Publications: Lab Chip. (2006) 6, 289-295; Tetrahedron Letters.(2005) 46, 4813-4816
Multispectral imaging to resolve numerous fluorescent markers
(with Greg Bearman (JPL) and Scott Fraser)
We developed a new image detector that permits a plurality of fluorescent markers to be collected simultaneously instead of sequentially (Patent issued (US #6,403,332)). The approach (META) is now commercially available from Carl Zeiss, Inc.
2002 R&D 100 Award and the 2003 NASA Space Award
Publication: J Biomed Optics (2001) 6, 311-318.
Automated quantitative analysis of cell behaviors
(with Jerry Solomon and Steve Speicher, CIT)
We created a computer cell tracking program, XVTrack, that can track and analyze individual cells or populations of cells in 3D from time-lapse data.
Publication: Current Biology 1997, 7:571–580.
Using retroviruses to fluorescently mark cells
We generated over forty replication-defective, fluorescent protein expressing retroviruses to infect avian embryos. The fluorescent proteins have been tagged to direct their localization to specific cellular organelles, thereby allowing these structures to be specifically imaged, cell divisions to be followed, and morphological changes to be dynamically observed.
Publication: Exp. Neurol. (1999)156, 394-406; Nature Neuroscience (2003) 6(5), 507 – 518.
Blood island-derived stem cells contribute to intraembryonic vasculogenesis.
(with Mandy LaRue and Chris Drake, Medical University of South Carolina)
We showed that blood islands, the embryonic equivalent of the adult bone marrow, are a source of endothelial precursors that contribute to intraembryonic blood vessel formation (vasculogenesis).
Publication: Developmental Biology (2003) 262,162-72.
Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration
(with Catherine Krull, Scott Fraser, Marianne Bronner-Fraser (CIT) and Nicholas Gale, George Yancopoulos (Regeneron))
We demonstrated that Eph-related receptor tyrosine kinases and their ligands are essential for the segmental migration of trunk neural crest cells through the somites.
Publication: Current Biology 1997, 7:571–580.