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. 2020 Jul 10;217(10):e20200140. doi: 10.1084/jem.20200140

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© 2020 Hong et al.

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Figure 3. — Characterization of tetO-Adamts5 mice. (A) Schematic representation of the genetic strategy used to generate ADAMTS5^BEC-GOF mice in which doxycycline drives inducible expression of V5-tagged ADAMTS5 specifically in BECs. (B) Co-immunostaining of V5 and PECAM (EC marker) in P10 cerebellums reveals expression of endothelial ADAMTS5-V5 in ADAMTS5^BEC-GOF mice compared with littermate controls. Results are representative of n > 3 litters. (C) Immunostaining of versican (C, left) and DPEAAE (ADAMTS-cleaved versican; C, right) in serial cerebellar sections of P10 mice. Boxed regions are shown at higher magnification to the right. Scale bars, 150 µm. (D) Quantitation of versican (left) and DPEEAE (right) IntDen along the white matter tracts in ADAMTS5^BEC-GOF mice relative to littermate controls (versican: n = 10–12 from eight litters; DPEEAE: n = 9–12 from seven litters, Student’s t test). (E and F) Versican immunostaining is reduced in ADAMTS5^BEC-GOF mice to an extent comparable to Krit1^BECKO animals at P4 (E). Scale bars, 100 µm. Quantitation of versican IntDen along cerebellar white matter tracts relative to Slco1c1-Cre;Krit1^fl/+ mice (F; n = 5 from at least four litters, ANOVA + Tukey test). (G) Immunostaining for KLF4 in the cerebellar vessels of the white matter at P10. Scale bars: 50 µm. Results are representative of n > 3 litters. (H) qPCR analysis of mRNA levels of Klf2 and Klf4 in ECs isolated from control and ADAMTS5^BEC-GOF P10 cerebellums (n = 4–6 from two litters, Student’s t test). Controls (B–D, G, and H) were littermate animals carrying one or two of the following alleles: the Cre transgene, the rtTA transgene, or the tetO-Adamts5-V5 allele. Error bars represent ± SEM. Not significant (n.s.), P > 0.05; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.