New imaging and computational techniques to study vertebrate limb development

Lead Research Organisation: MRC Human Genetics Unit

Abstract

The human body, as a typical biological system, is incredibly complicated. While we may be able to understand small parts of it at a time (such as how a particular gene is regulated), we still do not understand how large collections of these components correctly function together. Nevertheless, if we are to properly understand how things go wrong in the diseased condition and eventually how to repair such problems, we will need a full understanding of how complex biological systems coordinate their thousands of interacting parts. ||Our lab aims to help us move towards such an understanding by combining computer modelling with real experiments - thereby creating a more holistic approach to the analysis of a well-known complex system: the development of the vertebrate limb. For the limb to correctly grow from a small ball of cells into a highly organised structure that includes bones, muscles, cartilage, nerves and skin, requires the complex interactions of a genetic regulatory network with the physics of cell movements inside the limb tissue. We are creating measurements and 3D computer models of this process in an attempt to unravel the mystery of how natures building blocks achieve this remarkable feat.

Technical Summary

There are at least three problems facing current attempts to understand complex biological systems. The first is the shear amount of information which may appear relevant to a particular biological problem. The observation that even an apparently simple developmental event (such as the outgrowth of the vertebrate limb bud) involves at least many tens of regulatory genes working in a co-ordinated fashion, provides a real problem for the scientist. To construct a concrete hypothesis of how the system works involves keeping in mind tens of genes and potentially hundreds of interactions. The second problem is that genetic networks which contain feedback loops can act in a non-intuitive manner even simple networks can sometimes display behaviour which cannot be predicted without the aid of computers. Real networks are certain to be more complex, and therefore in some cases even less intuitive. The third problem is that to predict the behaviours of complex networks often requires quantitative information, and many common molecular biology techniques do not generate quantitative data. These problems do not only apply to gene networks. Morphogenesis involves other important phenomena which are perhaps even less well understood, such as how physical forces, stresses, tensions etc. are coordinated within an organ to generate its correct shape. In the case of vertebrate limb bud outgrowth a number of alternative ideas have been proposed to explain the distally-oriented expansion of tissue, including: spatial gradients of proliferation rates, mechanical strength of the ectoderm, extracellular matrix structures, and active directional migration of the mesenchymal cells. Given that these ideas are clearly distinct mechanical phenomena, it is surprising that we still do not know which ones are important for shaping the limb. One solution to these problems is a more integrated, computational approach to studying complex biological systems, involving improvements to both: how data is captured and how it is subsequently used in hypothesis-generation. Our work is therefore focused on both of these areas: (a) further improvements to our own novel 3D optical imaging technique (OPT) to generate data about limb development, and (b) creating new computer simulations which allow us to explore various hypotheses. Given the issues raised above, we believe that models of tissue mechanics are as important as models of genetic patterning mechanisms, and we are therefore working on both types of simulation with the goal of integrating them in the near future.

Publications

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Description OPT Diagnostic Pathology Collaboration (Development Gap Funding of MRCT/Tayside NHS Trust/University of Dundee/University of Cambridge)
Amount £187,120 (GBP)
Organisation MRC-Technology 
Sector Private
Country United Kingdom
Start 01/2009 
End 01/2012
 
Title OPTICAL PROJECTION TOMOGRAPHY (NON FOCAL OPTICS FOR OPT SYSTEM) 
Description Apparatus for obtaining an image of a specimen (6) by optical projection tomography comprises a confocal microscope (1, 2, 3) which produces a light beam which scans the specimen (6) whilst the latter is supported in a rotary stage (7). Light passing through the specimen is passed through a convex lens (8) which directs, onto a central light detector of an array of detectors (9), light which exits or by-passes the specimen parallel to the beam incident on the specimen. 
IP Reference WO2004020997 
Protection Patent granted
Year Protection Granted
Licensed Yes
Impact This technology invented at the Human Genetics Unit continues to be developed for both research and potential diagnostic applications through a licence agreement with a major imaging company and development by MRC Technology. Four patent families continue to be supported, all of which are granted in at least one territory. This patent belongs to the family A813/1726 (Non focal optics for OPT system) and is also published as EP1520173 (B1), US2006093200 (A1), JP2005537476 (T), ES2257705 (T3), DE60303613 (T2), CN1675541 (A), CN100476430 (C), CA2490578 (A1), AU2003260751 (A1), AU2003260751 (B2), and AT317977 (T) MRC patents are licenced to Carl Zeiss MicroImaging GmbH, as the result of an agreement signed on 25/11/2008. Zeiss report good progress and their system should enter beta-test soon with an estimate that the system will be available in the first half of 2011.
 
Title OPTICAL PROJECTION TOMOGRAPHY APPARATUS WITH ROTARY STAGE FOR IMAGING A SPECIMEN 
Description A rotary stage (10) for use in optical projection tomography includes a stepper motor (42) with a rotatable vertical shaft (44) the lower end of which carries a specimen (28) to be imaged so that the specimen is rotated about a substantially vertical axis. The stepper motor (42) is mounted on a table (34) the position of which is accurately adjustable in tilt and in vertical position to ensure that the rotational axis of the specimen is perpendicular to the optical axis (29). The specimen (28) rotates within a stationary chamber (26) and the rotary stage is used with a microscope which provides a three-dimensional image of the specimen. 
IP Reference EP1530073 
Protection Patent granted
Year Protection Granted 2007
Licensed Yes
Impact This technology invented at the Human Genetics Unit continues to be developed for both research and potential diagnostic applications through a licence agreement with a major imaging company and development by MRC Technology. Four patent families continue to be supported, all of which are granted in at least one territory. This patent belongs to the family A813/1617 (Optical Stage) and is also published as WO02095476 (A2) WO02095476 (A3) US2004207840 (A1) US7218393 (B2) JP2004531729 (T) HK1067412 (A1) ES2294583 (T3) ES2248548 (T3) EP1530073 (A1) EP1530073 (B1) EP1410090 (A2) EP1410090 (B1) DK1530073 (T3) DE60223728 (T2) DE60206388 (T2) CA2445780 (A1) CA2445780 (C) AT305623 (T) MRC patents are licenced to Carl Zeiss MicroImaging GmbH, as the result of an agreement signed on 25/11/2008. Zeiss report good progress and their system should enter beta-test soon with an estimate that the system will be available in the first half of 2011.
 
Title TREATMENT OF TISSUE SPECIMENS 
Description Apparatus for treating tissue specimens comprises a stationary outer casing (2) in the top of which is positioned an annular trough (3) for holding a liquid. Rotatably mounted in the outer casing (2) is a central hub (5) supporting a disc-like lid (6) the underside of which carries a magnet (7). In use, the magnets hold, in a detachable manner, tissue specimens (12) and the trough (3) is filled with a liquid for treating the specimens. The hub (5) is capable of vertical translational movement with respect to the casing (2) to enable the specimens to be lowered into and subsequently lifted out of the trough (3). A method of treating tissue specimens is also provided. 
IP Reference WO2004003129 
Protection Patent granted
Year Protection Granted
Licensed Yes
Impact This technology invented at the Human Genetics Unit continues to be developed for both research and potential diagnostic applications through a licence agreement with a major imaging company and development by MRC Technology. Four patent families continue to be supported, all of which are granted in at least one territory. This entry relates to the patent family A813/1724 (Treatment of Tissue Specimens) and is also published as WO2004003129 (A3) US2006105332 (A1) US7677197 (B2) PT1516183 (E) JP2005531005 (T) ES2285147 (T3) EP1516183 (A2) EP1516183 (B1) DK1516183 (T3) DE60313170 (T2) CA2489096 (A1) AU2003280409 (A1) AU2003280409 (A2) AU2003280409 (B2) AT359507 (T) The potential diagnostic applcations of this technology have led to MRC support for work to examine use in cancer pathology (described under further funding).
 
Title USES OF OPTICAL PROJECTION TOMOGRAPHY METHODS AND APPARATUS 
Description Uses of optical projection tomography methods and apparatus in a variety of analyses and procedures is disclosed. The apparatus is a rotary stage (10) which includes a stepper motor (42) with a rotatable vertical shaft (44) the lower end of which carries a specimen (28) to be imaged so that the specimen is rotated about a substantially vertical axis. The stepper motor (42) is mounted on a table (34) the position of which is accurately adjustable in tilt and in vertical position to ensure that the rotational axis of the specimen is perpendicular to the optical axis (29). The specimen (28) rotates within a stationary chamber (26) and the rotary stage is used with a microscope which provides a three-dimensional image of the specimen. 
IP Reference WO2004048970 
Protection Patent granted
Year Protection Granted
Licensed Yes
Impact This technology invented at the Human Genetics Unit continues to be developed for both research and potential diagnostic applications through a licence agreement with a major imaging company and development by MRC Technology. Four patent families continue to be supported, all of which are granted in at least one territory. This patent also published as AU2003285523 The patent family is A813/1725 (laser scanning opt system) and the application has been abandoned in most territories as a result of poor examination process.
 
Title OPT Microscopes 
Description Since 2005 MRC Technology has provided 39 OPT microscope systems to laboratories around the world. These are fully supported by Quality Assurance systems in place on the Edinburgh site. MRC Technology also supports extensive training on the system. This is done mainly on-site in Edinburgh where 29 courses have been provided but also at the laboratories who have bought the system. 
Type Support Tool - For Fundamental Research
Current Stage Of Development Small-scale adoption
Year Development Stage Completed 2006
Development Status Under active development/distribution
Impact The system has also been a feature at the EMBO Practical Course on 3D Developmental Imaging in 2009 and 2010.