The Role of Spatially Controlled Cell Proliferation in Limb Bud Morphogenesis

Oriented cell behaviors likely have a more important role in limb bud elongation during development than previously suggested by the “growth-based morphogenesis” hypothesis.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.Author Summary: Although the vertebrate limb bud has been studied for decades as a classical model system for the spatial control of cell fates, the question of how the limb bud physically elongates has been much less studied. One particular hypothesis has been dominant in the field, known either as the proliferation gradient hypothesis or growth-based morphogenesis. This states that elongation is achieved by distal cells (furthest from the body) being stimulated to divide faster than proximal cells. Importantly, this hypothesis does not propose any kind of oriented or directional cell behaviours—high distal rates of non-oriented proliferation are considered to be sufficient—and indeed several 2D computer simulations have reproduced this concept in silico. However, thus far quantitative data from the limb bud has not been incorporated into these models. Here, we extended computer simulations into 3D and incorporated quantitative data on both shape changes and proliferation rates. These new simulations demonstrated that gradients of non-oriented proliferation are unable to explain limb bud elongation. We thus experimentally tested for evidence of oriented cell behaviours and indeed found that the cell shape, Golgi orientation, and cell divisions all display a non-random bias during limb bud outgrowth. Our data run contrary to the proliferation gradient hypothesis, indicating instead that oriented cell behaviours are important for driving elongation.