Tomographic quantification of branching morphogenesis and renal development

Abstract

Branching morphogenesis is a central process in renal development, but imaging and quantifying this process beyond early organogenesis presents challenges due to growth of the kidney preventing ready imaging of the complex structures. Current analysis of renal development relies heavily on explant organ culture and visualization by confocal microscopy, as a more developmentally advanced native tissue is too thick for conventional microscopic imaging. Cultured renal primordia lack vascularization and a supportive matrix for normal growth, resulting in tissue compression and distortion of ureteric branching. To overcome this, we used optical projection tomography to image and reconstruct the branching ureter epithelium of ex vivo embryonic kidneys and developed software to quantify these three-dimensional (3D) data. Ureteric branching was assessed by measuring tree and terminal branch length, tip number, branching iterations, branch angles, and inter-tip distances in 3D space. To validate this approach for analyzing genetic influences on renal development, we assessed branching and organ morphology in Tgfbeta2(+/-) embryos from E12.5 through E15.5. We found decreased branching, contrary to previous findings using organ culture, and quantified a primary defect in renal pelvic formation. Our approach offers many advantages from improved throughput, analysis, and observation of in vivo branching states, and has demonstrated its utility in studying the basis of renal developmental disease.

Spatial mapping and quantification of developmental branching morphogenesis

“…Foetal mouse kidneys (n=4) and lungs (n=4) were stained as previously published (Metzger et al, 2008) (Short et al, 2010) using pan-cytokeratin (Sigma) and E-cadherin (Invitrogen) antibodies, respectively. Embryonic day (E) 9 chicken lungs were dissected in PBS, fixed in 4% paraformaldehyde for 1 hour at 4°C, then stained using the same protocol as for mouse lungs.…”

“…We elected to use optical projection tomography (OPT) (Sharpe et al, 2002) to examine branching morphogenesis, principally because it is suited to the visualisation of mouse embryos (Alanentalo et al, 2008;Short et al, 2010;Walls et al, 2008). However, the approach is equally applicable to other imaging modalities that can produce isotropic 3D datasets of labelled branching epithelia.…”

“…A 'root' for the skeleton is manually selected as the basis for subsequent analysis. To the best of our knowledge, no commercially available software can accomplish the refinement of these structures across different organ types, including our previously published, kidney-specific attempt at such analysis (Short et al, 2010). Our approach can be fine-tuned for different tissues by modulating minimum feature size, the strength of the Gaussian field and the weighting placed on the vectors used for skeletonisation.…”

“…However, these thin, avascular organs develop outside of the foetal milieu, significantly disrupting normal spatial tissue organisation. Consequently, they often poorly reflect in vivo organ development (Al-Awqati and Goldberg, 1998;Short et al, 2010). As a result, our understanding of the spatial development and patterning of organs, even during normal development, is often poor.…”

A spatially-averaged mathematical model of kidney branching morphogenesis

“…More recently, initiation of kidney branching has been simulated using a ligand-receptor based Turing model that represents the known Gdnf-RetWnt11 feed forward loop and this has been refined into a laboratory based model based on organ culture of kidneys (Adivarahan et al (2013)). Organ culture, while useful for simple studies of branching, does not recapitulate normal branching mechanics (Short et al, 2010). None of these studies have used data from in vivo kidneys, across development and in three dimensions.…”