Understanding the mechanisms that regulate vertebrate limb development
We tackle this problem by looking at two fundamental events of limb formation. We study the earliest event in limb formation in which a cohort of cells from specific regions of the embryo flank are recruited to become the progenitors of the mature limb tissues. We define this event as as limb bud initiation. We also study the later events of tissue morphogenesis, in which progenitors specified for particular cell fates are organised into functional, interconnected tissue units, such as muscle bundles and tendons of the limb.
Limb Tissue formation and morphogenesis
Initiation of the limb bud
The first step in limb formation is an inductive signal from the main body axis acting on adjacent cells at specific regions along the flank of the main body. For the prospective limb-forming regions to respond to an instructive signal they must be competent to receive such cues. We are dissecting the mechanism that make these regions competent to respond to instructive cues, how these regions of competence are correctly positioned along the main body axis and the combination of signals that convert mesodermal cells of the embryo into progenitors of the forelimb and hindlimb.
Many of the genetic programmes that determine cell fates are now known, for example the cascade of myogenic factors that convert progenitors to myoblasts, through myocytes to differentiated muscle fibres. Very little is known, however, about how differentiating and differentiated cell types are organised into functional tissues, for example, in the case of muscle, how individual muscle fibres are organised into discrete muscle bundles. And, furthermore how distinct nascent tissues such as muscle and tendon communicate with one another to form a precise interconnected array that is an essential step in forming a functional musculoskeletal unit.
We aim to decipher the molecular and cellular events that control formation of limb tissues and to use this knowledge to develop regenerative strategies to build limb tissues. We are combining knowledge gained from our studies of the recruitment and expansion of limb progenitors with that of how limb tissues are constructed to develop methods that will enable us to manipulate ES cells and/or multipotential progenitors to produce mature tissues that could be used therapeutically.
Developing 3D imaging techniques to study limb development
We have developed methods that enable us to extensively phenotype the mutant animal models we generate. We use 3D imaging techniques to analyse our experimental animal models and have produced a Moouse 3D Limb Anatomy database, hosted at the eMouse Atlas project, emap.
We have developed protocols using Optical Projection Tomography (OPT) that enabled us to describe to a previously unobtainable level of resolution. We also use High resolution Episcopic microscopy (HREM) in combination with OPT to characterise limb soft tissue morphogenesis and patterning defects.
The developing mouse limb is widely used as a model system for studying tissue patterning. Despite this, few references are available that can be used for the correct identification of developing limb structures, such as muscles and tendons. Existing textual references consist of two-dimensional (2D) illustrations of the adult rat or mouse limb that can be difficult to apply when attempting to describe the complex three-dimensional (3D) relationship between tissues.
To improve the resources available in the mouse model, we generated a free, web-based, interactive reference of limb muscle, tendon, and skeletal structures. The Atlas was produced using mouse forelimb and hindlimb specimens stained using immunohistochemistry to detect muscle and tendon. Limbs were scanned using Optical Projection Tomography (OPT), reconstructed to make 3D models and annotated using computer-assisted segmentation tools. Users click on the names of structures and view their shape, position and relationship with other structures within the 3D model and also in 2D virtual sections.
The E14.5 mouse forelimb (EMAP ID EMA108) and hindlimb (EMAP ID EMA109) models are accessible by clicking on the 3D hypertext link. This opens a viewer window that lists all anatomy structures that have been segmented and that can be viewed in isolation or together by selecting on the +/- buttons. 'All' items or 'none' can also be selected using the appropriate buttons.
To see the selected items in a 3D window select '3d view' button in the top right-hand corner. This opens a new window. The 3D datasets can be moved freely using your cursor to preferred orientation.
The Mouse Limb Anatomy Atlas provides a novel and valuable tool for researchers studying limb development and can be applied to a range of research areas, including the identification of abnormal limb patterning in transgenic lines and studies of models of congenital limb abnormalities. By using the Atlas for "virtual" dissection, this resource also offers an alternative to animal dissection. The techniques we have developed and employed are also applicable to many other model systems and anatomical structures.