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, 30 (1), 108-118

Brush Border Protocadherin CDHR2 Promotes the Elongation and Maximized Packing of Microvilli in Vivo

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Brush Border Protocadherin CDHR2 Promotes the Elongation and Maximized Packing of Microvilli in Vivo

Julia A Pinette et al. Mol Biol Cell.

Abstract

Transporting epithelial cells optimize their morphology for solute uptake by building an apical specialization: a dense array of microvilli that serves to increase membrane surface area. In the intestinal tract, individual cells build thousands of microvilli, which pack tightly to form the brush border. Recent studies implicate adhesion molecule CDHR2 in the regulation of microvillar packing via the formation of adhesion complexes between the tips of adjacent protrusions. To gain insight on how CDHR2 contributes to brush border morphogenesis and enterocyte function under native in vivo conditions, we generated mice lacking CDHR2 expression in the intestinal tract. Although CDHR2 knockout (KO) mice are viable, body weight trends lower and careful examination of tissue, cell, and brush border morphology revealed several perturbations that likely contribute to reduced functional capacity of KO intestine. In the absence of CDHR2, microvilli are significantly shorter, and exhibit disordered packing and a 30% decrease in packing density. These structural perturbations are linked to decreased levels of key solute processing and transporting factors in the brush border. Thus, CDHR2 functions to elongate microvilli and maximize their numbers on the apical surface, which together serve to increase the functional capacity of enterocyte.

Figures

FIGURE 1:
FIGURE 1:
CDHR2 KO mice exhibit growth defects. (A) Cartoon of the strategy used to generate the tm1d CDHR2 KO allele from tm1a mice. Binding sites of forward and reverse primers used to genotype these animals are shown with black arrows. Expected PCR band sizes for the tm1c and tm1d alleles are also given. (B) PCR genotyping confirmation of the allele conversion to CDHR2tm1d upon crossing CDHR2tm1c animals with mice harboring villin-CRE. (C) Western blotting validation of loss of CDHR2 expression in the isolated intestinal epithelium of CDHR2tm1d/CDHR2tm1d mice. (D) Photograph of WT and CDHR2 KO littermates at P30. (E) Comparison of male WT and CDHR2 KO littermate body weights at P15 (WT, n = 9, 6.6 ± 0.7 g; KO, n = 9, 6.4 ± 1.7 g), P30 (WT, n = 20, 14.0 ± 2.6 g; KO, n = 20, 13.2 ± 2.9 g), P60 (WT, n = 13, 24.0 ± 2.3 g; KO, n = 9, 22.4 ± 3.5 g), and P90 (WT, n = 14, 27.2 ± 1.3 g; KO, n = 28, 25.2 ± 3.3 g). Values are mean ± SD, P90, *p < 0.05. Whiskers on box plots indicate min/max values. (F) H&E-stained Swiss roll sections of paraffin-embedded small intestine from WT and CDHR2 KO mice. Scale bars are 2 mm for whole Swiss roll and 500 μm for zoom panels. (G) Quantification of villus length in WT and CDHR2 KO Swiss roll sections. WT, n = 75 measurements (25/Swiss roll, 1 Swiss roll/mouse, three mice), 431 ± 65 μm; KO, n = 7; (25/Swiss roll, 1 Swiss roll/mouse, three mice), 357 ± 65 μm. Values indicate mean ± SD; ****, p < 0.0001. (H) Quantification of crypt depth in WT and CDHR2 KO Swiss roll sections. WT, n = 75 measurements (25/Swiss roll, 1 Swiss roll/mouse, three mice), 107 ± 29 μm; KO, n = 75 measurements (25/Swiss roll, 1 Swiss roll/mouse, three mice), 82 ± 11 μm. Values indicate mean ± SD; ****, p < 0.0001.
FIGURE 2:
FIGURE 2:
CDHR2 KO mice exhibit perturbations in the intestinal epithelium. (A) SEM images for intestinal tissue samples from WT and CDHR2 KO mice. Scale bars are 500 μm for Ai, 200 μm for Aii, 100 μm for Aiii; yellow asterisks in KO Aii indicate a large region of the epithelium lacking villi. (B) Single image planes of Alexa488-phalloidin–stained frozen tissue sections from WT and CDHR2 KO mice, acquired with a laser-scanning confocal microscope. Signal is inverted to facilitate visualization. Scale bars are 25 μm. (C) Three-dimensional reconstructions (8 μm thick) of deconvolved laser-scanning confocal volumes from the WT and CDHR2 KO samples imaged in B. Signal is inverted to facilitate visualization. Scale bars are 10 μm. (D) Single confocal image planes of WT and CDHR2 KO tissue sections stained with anti-villin (green) to highlight the brush border and anti–E-cadherin (magenta) to label cell margins. Zoom 1 shows the region in the main panel highlighted by the small white box; zoom 2 shows the region in zoom 1 highlighted by the small white box. Scale bars are 100 μm for main panels, 25 μm for zoom 1, and 10 μm for zoom 2.
FIGURE 3:
FIGURE 3:
IMAC components are mislocalized in CDHR2 KO brush borders. Single confocal image planes of WT and CDHR2 KO paraffin-embedded tissue sections stained with anti-villin (magenta) to highlight the brush border and (A) anti-MYO7B (green), (B) anti-USH1C (green), or (C) anti-CDHR5 (green). Scale bars are 100 μm for main panels and 25 μm for zoom panels. Zoom panels show the region in the main merge panel highlighted by the small white box.
FIGURE 4:
FIGURE 4:
Microvillar ultrastructure is perturbed in CDHR2 KO brush borders. (A) TEM images of WT and KO brush borders in a plane parallel to the microvillar axis; white dashed boxes show regions highlighted in zoom panels to the right. Black arrows in WT zoom panel highlight intermicrovillar adhesion links at the distal tips of microvilli. (B) Quantification of microvillar length in WT and KO brush borders. WT, n = 85 measurements, 1084 ± 110 nm; KO, n = 82 measurements, 682 ± 180 nm. Values indicate mean ± SD; ****, p < 0.0001. Length measurements were highly selective such that only protrusions with actin cores that are clearly visible along their full length were scored. (C) TEM images of WT and KO brush borders in a plane perpendicular to the microvillar axis; white boxes show region highlighted in zoom panels below. Image montage to the right of the KO panel shows several examples of distorted microvillus cross-sections. White outlined arrows in the KO zoom panel highlight a subset of the poorly consolidated and misshapen core actin bundles. Yellow outlined arrows in the KO zoom panel highlight remnant intermicrovillar link-like structures. (D) Frequency distributions of circularity measurements on microvillar cross-sections, where perfect circles exhibit a circularity of 1, less circular objects <1 (n = 203 cross -sections for WT and n = 321 for KO). (E) Quantification of microvillar cross–sectional area. WT, n = 98 cross-sections, 11,895 ± 1227 nm2; KO, n = 98, 15,872 ± 5394 nm2. Values indicate mean ± SD; ****, p < 0.0001. All scale bars are 500 nm.
FIGURE 5:
FIGURE 5:
Microvillar packing density is decreased in CDHR2 KO brush borders. (A) SEM images show en face views of microvilli in WT and KO brush borders at intermediate magnification. Insets in both panels show FFT frequency domain images calculated from the regions highlighted by the white boxes. (B) High-magnification SEM images of microvilli in WT and KO brush borders reveal packing defects in KO samples. (C) Black spots represent the positions of microvillar tips in the images shown in B. The number of microvilli counted in each field is shown in the upper right-hand corner. (D) Frequency distributions of the nearest-neighbor distance calculated for WT and CDHR2 KO microvilli. WT, n = 24,036 measurements, 93.5 ± 14.1 nm; KO, n = 18,268 measurements, 111.4 ± 13.9 nm. Values indicate mean ± SD; ****, p < 0.0001. Vertical dashed blue line on the KO plot indicates mean nearest-neighbor distance for WT. All scale bars are 1 μm.
FIGURE 6:
FIGURE 6:
Levels of key apical markers are reduced in CDHR KO tissues. (A) Single confocal image planes of WT and CDHR2 KO paraffin tissue sections stained with primary antibodies targeting the apical marker indicated at the top of each image column. Top row shows native 40× magnification; bottom row shows tissue sections at threefold higher magnification. LUTs were inverted to facilitate visualization of intensity differences between WT and CDHR2 KO samples. All scale bars are 50 μm. (B) Quantification 12-bit fluorescence intensities from the brush border of WT and CDHR2 KO samples. Each plot consists of 20 measurements from sections representing multiple animals of each genotype. IAP, WT: 1325 ± 420 vs. KO 435 ± 119; SI, WT: 321 ± 92 vs. KO 123 ± 25; DPPIV, WT: 1558 ± 997 vs. KO 304 ± 124; NHE3, WT: 630 ± 249 vs. KO 94 ± 35; p-ERM, WT: 1605 ± 638 vs. KO 803 ± 173; ezrin, WT: 1884 ± 639 vs. 570 ± 176. Units = 12-bit fluorescence intensity; all values indicate mean ± SD; ****, p < 0.0001.

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