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. 2019 Oct 17;8:e48474. doi: 10.7554/eLife.48474

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© 2019, Thomson et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 2. — (A) Confocal microscopy of whole mount eyes revealed reduced CD31+ SC area in adult Tek^+/- haploinsufficient mice. This phenotype was blunted in Tek^+/-;Ptprb^NLS-LacZ/WT double heterozygous animals, confirming the importance of TEK activation in canal development. Ptprb^NLS-LacZ/WT heterozygous controls had normal SC area (n = 8 WT, 12 Ptprb^NLS-LacZ/WT, 11 Tek^+/- and 11 Tek^+/-;Ptprb^NLS-LacZ/WT mice). 20x fields shown represent an area of 65,536 μm². Images were captured as 10-frame Z stacks with a step size of 1.67 μm and a pinhole of 1.2 Airy units, and are shown as maximum intensity projections. (B, quantified in C) At postnatal day 5 (P5), confocal microscopy of the developing SC in eye whole mounts revealed reduced numbers of proliferating Ki-67-positive SC ECs (Ki67-PROX1 double positive cells) in Tek haploinsufficient animals compared to littermate WT or Ptprb^NLS-LacZ/WT controls. Normal proliferation was observed in Tek^+/-;Ptprb^NLS-LacZ/WT animals. (D) Compared to control and Ptprb^NLS-LacZ/WT mice, PROX1 expression was reduced in Tek^+/- littermate eyes. Expression was normal in Tek^+/-;Ptprb^NLS-LacZ/WT double heterozygotes. n = 8 (WT), 4 (Ptprb^NLS-LacZ/WT), 8 (Tek^+/-) and 5 (Tek^+/-;Ptprb^NLS-LacZ/WT) Shown are maximum intensity projections from 8-frame confocal Z stacks captured using a 20x objective, step size of 1 μm and pinhole of 1.2 Airy units. Norm. AFU: Normalized, background subtracted arbitrary fluorescence units. (E) Compared to control and Ptprb^NLS-LacZ/WT littermates, confocal analysis of adult SC revealed a marked increase in the number of focal convolutions and narrowings in the eyes of Tek^+/- and Tek^+/-;Ptprb^NLS-LacZ/WT mice. N = 4 (WT), 7 (Ptprb^NLS-LacZ/WT), 5 (Tek^+/-) and 6 (Tek^+/-;Ptprb^NLS-LacZ/WT). Horizontal lines indicate population means. *p<0.05, **p<0.01, ***p<0.001 as determined by 1-way ANOVA followed by Bonferroni’s correction.