Automated micro-aspiration of mouse embryo limb bud tissue

Abstract

Mechanical force is an integral part of tissue morphogenesis and patterning. We have developed an automated micro-aspiration system to investigate how mouse limb bud tissue responds to extrinsic forces in order to understand whether tissue-generated forces can be a part of the mechanism causing oriented cell behaviors observed in mouse limb bud morphogenesis. The system is capable of performing automated micropipette tracking, tissue-tip contact detection, pressure control, and prolonged application of constant pressure. A three-dimensional tissue tracking algorithm is developed based on the processing of time-lapsed confocal Z-stack images. 3D visual feedback from confocal microscopy imaging, for the first time, is used to realize 3D visual servoing to control the micropipette position to compensate for tissue movement. This enables stable force application in a biologically relevant time scale (e.g., 60 minutes) during which cell remodeling occurs. Experimental results demonstrate that micro-aspiration on mouse limb bud is capable of creating tension anisotropy which causes force-responsive cells to dynamically remodel through polarized cell division and rosette resolution.