A landmark-free morphometric staging system for the mouse limb bud.

We have created a 2D morphometric analysis of the developing mouse hindlimb bud. This analysis has provided two useful resources for the study of limb development. First, a temporally accurate numerical description of shape changes during normal mouse limb development. Second, a web-based morphometric staging system, which has the advantage of being easy to use, and with a reproducibility of about ±2 hours. It allows users to upload a dorsal-view photo of a limb bud, draw a spline curve and thereby stage the bud within a couple of minutes. We describe how the system is constructed, its robustness to user variation and illustrate one application: the accurate tracking of spatiotemporal dynamics of gene expression patterns.

The role of spatially controlled cell proliferation in limb bud morphogenesis.

Although the vertebrate limb bud has been studied for decades as a model system for spatial pattern formation and cell specification, the cellular basis of its distally oriented elongation has been a relatively neglected topic by comparison. The conventional view is that a gradient of isotropic proliferation exists along the limb, with high proliferation rates at the distal tip and lower rates towards the body, and that this gradient is the driving force behind outgrowth. Here we test this hypothesis by combining quantitative empirical data sets with computer modelling to assess the potential role of spatially controlled proliferation rates in the process of directional limb bud outgrowth. In particular, we generate two new empirical data sets for the mouse hind limb--a numerical description of shape change and a quantitative 3D map of cell cycle times--and combine these with a new 3D finite element model of tissue growth. By developing a parameter optimization approach (which explores spatial patterns of tissue growth) our computer simulations reveal that the observed distribution of proliferation rates plays no significant role in controlling the distally extending limb shape, and suggests that directional cell activities are likely to be the driving force behind limb bud outgrowth. This theoretical prediction prompted us to search for evidence of directional cell orientations in the limb bud mesenchyme, and we thus discovered a striking highly branched and extended cell shape composed of dynamically extending and retracting filopodia, a distally oriented bias in Golgi position, and also a bias in the orientation of cell division. We therefore provide both theoretical and empirical evidence that limb bud elongation is achieved by directional cell activities, rather than a PD gradient of proliferation rates.