Three-dimensional imaging of embryonic mouse kidney by two-photon microscopy

Abstract

Developing mammalian embryonic kidney becomes progressively more elaborate as the ureteric bud branches into undifferentiated mesenchyme. Morphological perturbations of nephrogenesis, such as those seen in inherited renal diseases or induced in transgenic animals, require careful and often tedious documentation by multiple methodologies. We have applied a relatively quick and simple approach combining two-photon microscopy and advanced three-dimensional (3-D) imaging techniques to visualize and evaluate these complex events. As compared with laser confocal microscopy, two-photon microscopy offers superior optical sectioning deep into biological tissues, permitting analysis of large, heterogeneous, 3-D structures such as developing mouse kidney. Embryonic and newborn mouse kidneys were fluorescently labeled with lectins, phalloidin, or antibody. Three-dimensional image volumes were then collected. The resulting volume data sets were processed using a novel 3-D visualization technique. Reconstructed image volumes demonstrate the dichotomous branching of ureteric bud as it progresses from a simple, symmetrical structure into an elaborate, asymmetrical collecting system of multiple branches. Detailed morphology of in situ cysts was elucidated in a transgene-induced mouse model of polycystic kidney disease. We expect this integration of two-photon microscopy with advanced 3-D image analysis will provide a powerful tool for illuminating a variety of complex developmental processes in multiple dimensions.

Figure 1.

Stereopairs of developing embryonic kidneys imaged by two-photon microscopy. Whole kidneys were microdissected from wild-type mouse embryos, stained with fluorescently conjugated lectins, and imaged by two-photon microscopy. Three-dimensional image processing reveals weak staining of undifferentiated mesenchyme in an E11.5 metanephric rudiment stained with rhodamine-PNA (A). By day E13.5, the simple, dichotomously branching ureteric bud is noted to have a cobblestone surface when stained with rhodamine-PNA (B) and a spiculated internal structure when stained with fluorescein-SBA (C). Each image is 205 μm across. A shows projections collected over a depth of 68 μm, B a depth of 74 μm, and C a depth of 116 μm. These and all subsequent stereopairs are also reproduced as rotating projections located at http://renal.nephrology.iupui.edu/phillipsetal.

Figure 2.

Stereopairs of embryonic day 17.5 kidneys imaged by two-photon microscopy. E17.5 wild-type kidneys were microdissected and labeled with fluorescein-tagged PNA (A and B) or fluorescein-phalloidin (C) to illustrate branches of ureteric bud surrounded by differentiating mesenchyme. PNA shows intense staining of ureteral bud branches and ampullary tips but weakly stains mesenchyme undergoing conversion to epithelial structures (A and B). After phalloidin staining, this condensed mesenchyme can be seen adjacent to the circular cross sections of ureteric bud (C). Careful inspection of A and B reveals the emergence of faintly staining tubule segments that bridge between the developing nephrons and the ampullae. Each image is 205 microns across. A shows projections collected over a depth of 139 μm, B a depth of 137 μm, and C a depth of 16 μm.

Figure 3.

Stereopairs of newborn mouse kidneys imaged by two-photon microscopy. Vibratome sections of P5 kidneys incubated with rhodamine-conjugated lectins LCA (A) or PNA (B and C). A shows a normal arcuate artery giving off an interlobular artery which has two afferent arterioles with attached glomeruli. B shows normal proximal tubules and scattered collecting ducts with apical labeling of intercalated cells. C shows cystic proximal tubules from an inv/inv mouse. Compare the cystic collecting duct of C (lower left corner) to those in B. Each image is 205 microns across. A shows projections collected over a depth of 100 μm, B a depth of 77 μm, and C a depth of 130 μm.

Figure 4.

Renderings of the image volume of inv/inv kidneys shown in Figure 3C ▶ . Cysts arising from proximal tubules and a collecting duct are seen. The two perspectives shown here were produced using a voxel rendering program that performs real-time 3-D reprojection imaging (see Methods).

Figure 5.

Resolution and contrast in two-photon microscopy. A: A single optical section from the 3-D volume shown in Figures 3C and 4 ▶ ▶ collected at a depth of 50 μm into the tissue. B: A magnified portion of this image (A) demonstrates the high resolution and contrast of the 2-photon images. C: A high magnification optical section taken from the image volume shown in Figure 3B ▶ , collected at a depth of 17 μm into the tissue. D: A high magnification image of a single vertical section from the image volume shown in Figure 3C ▶ . E: A vertical section of the image volume shown in Figure 3B ▶ . Scale bar, 50 μm (A), 10 μm (B–D), and 25 μm (E).

Figure 6.

Stereopairs of dual-fluorochrome-labeled mouse renal tubules. Normal E17.5 (A) and P5 inv/inv (B) mouse kidneys were dual-labeled with rhodamine and fluorescein. In A, branches of ureteric bud (red) stained with PNA-rhodamine are separated by a network of mesenchyme stained with LCA-fluorescein (green). In B, cystic renal tubules labeled with DBA-rhodamine (red) are seen in close proximity to more normal caliber thick ascending loop of Henle labeled with anti-Tamm-Horsfall-fluorescein (green). Each image is 205 μm across. A shows projections collected over a depth of 68 μm and B a depth of 52 μm.