ABSTRACT
The structure of the ureteric tree in developing mouse and rat kidneys has previously been quantified in two dimensions. While this type of analysis may provide evidence of changes in ureteric growth, these measurements are effectively inaccurate, as the ureteric tree is a three-dimensional (3D) object. Here we describe a method for measuring the ureteric tree in three dimensions. This technique involves (1) culture of the metanephric kidney at embryonic day 12 (mouse) or 14 (rat), (2) whole-mount immunofluorescence to selectively stain ureteric tree epithelium, (3) confocal microscopy to obtain a complete Z series through the ureteric tree, and (4) image analysis algorithms to binarize, skeletonize, and measure individual branch lengths in 3D. This method has been extended to analysis of the same ureteric tree over time (4D). The results obtained provide accurate and precise quantitation of ureteric tree growth in the developing mouse or rat kidney.
Subject(s)
Kidney/growth & development , Microscopy, Confocal/methods , Morphogenesis , Animals , Epithelium/ultrastructure , Imaging, Three-Dimensional , Kidney/ultrastructure , Mice , Organ Culture Techniques , Rats , Ureter/embryology , Ureter/ultrastructureABSTRACT
Early changes to branching morphogenesis of the prostate are believed to lead to enlargement of the gland in adult life. However, it has not been possible to demonstrate directly that alterations to branching during the developmental period have a permanent effect on adult prostate size. In order to examine branching morphogenesis in a quantitative manner in neonatal mice, a combination of imaging and computational technology was used to detect and quantify branching using bone morphogenetic protein 4 haplo-insufficient mice that develop enlarged prostate glands in adulthood. Accurate estimates were made of six parameters of branching, including prostate ductal length and volume and number of main ducts, branches, branch points, and tips. The results show that the prostate is significantly larger on day 3, well before the emergence of the phenotype in older animals. The ventral prostate is enlarged because the number of main epithelial ducts is increased; enlargement of the anterior prostate in mutant animals occurs because there are more branches. These lobe-specific mechanisms underlying prostate enlargement indicate the complex nature of gland pathology in mice, rather than a simple increase in weight or volume. This method provides a powerful means to investigate the aetiology of prostate disease in animal models prior to emergence of a phenotype in later life.
Subject(s)
Bone Morphogenetic Proteins/genetics , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Prostate/growth & development , Prostatic Hyperplasia/genetics , Animals , Animals, Newborn , Bone Morphogenetic Protein 4 , Bone Morphogenetic Proteins/deficiency , Homozygote , Male , Mice , Mice, Mutant Strains , Microscopy, Confocal , Morphogenesis/genetics , Prostate/metabolism , Prostate/pathology , Prostatic Hyperplasia/metabolism , Prostatic Hyperplasia/pathologyABSTRACT
The growth and branching of the epithelial ureteric tree is critical for development of the permanent kidney (metanephros). Current methods of analysis of ureteric branching are mostly qualitative. We have developed a method for measuring the length of individual branches, and thereby the total length of the ureteric tree in 3 dimensions (3D). The method involves confocal microscopy of whole-mount immunostained metanephroi and computer-based image segmentation, skeletonisation and measurement. The algorithm performs semi-automatic segmentation of a set of confocal images and skeletonisation of the resulting binary object. Length measurements and number of branch points are automatically obtained. The final representation can be reconstructed providing a fully rotating 3D perspective of the skeletonised tree. After 36 h culture of E12 mouse metanephroi, the total length of the ureteric tree was 6103 +/- 291 microm (mean +/- SD), a four-fold increase compared with metanephroi cultured for just 6 h (1522 +/- 149 microm). Ureteric duct length increased at a rate of 153 microm/h over the first 30 h period and was maximal between 18 and 24 h at 325 microm/h. The distribution of branch lengths at the six time points studied was similar, suggesting tight control of ureteric lengthening and branching. This method will be of use in analysing ureteric growth in kidneys cultured in the presence of specific molecules suspected of regulating ureteric growth. The method can also be used to analyse in vivo kidneys and to quantify branching morphogenesis in other developing organs.
