For wild-type bone analysis, C57BL/6J male mice were used unless stated otherwise. All EC-specific gene targeting experiments were performed with Cdh5(PAC)-CreERT2 transgenic mice24 (link). For Rbpj deletion in the postnatal endothelium, mice carrying loxP-flanked Rbpj (Rbpjlox/lox) alleles25 (link) and Cdh5(PAC)-CreERT2 transgenics were interbred. To induce Cre activity and gene deletion, offspring was injected with 500µg tamoxifen (Sigma, T5648) intraperitoneally every day from P10 to P14. The resulting RbpjiΔEC (CreERT2T/+Rbpjlox/lox) mutants and Cre- littermate controls were sacrificed at P28, and femurs and tibiae were collected for analysis. Identical breeding and tamoxifen administration strategies were used to generate EC-specific mutants with Fbxw7lox/lox (Ref.10 (link)), Dll1lox/lox (Ref. 13 (link)), Dll4lox/lox (Ref. 14 (link)), or Jag1lox/lox (Ref. 15 (link)) mice and inducible osteoblast-specific Rbpj knockouts (RbpjiOB). The latter were generated with Tg(Col1a1-creERT2)6.1.ICS transgenic mice (Institut Clinique de la Souris, France).
For Notch gain-of-function experiments, Gt(ROSA)26Sortm1(Notch1)Dam/J mice carrying a Cre-inducible transgene for Notch1 intracellular domain overexpression26 (link) and Cdh5(PAC)-CreERT2 transgenics were interbred. Tamoxifen administration (see above for injection schedule) was used to generate CreERT2-positive (NICDiOE-EC) mutants overexpressing NICD in ECs and controls. For experiments with EC-specific Dll4iΔEC/NICDiOE-EC double mutants, interbreeding of Dll4lox/lox conditional mice14 (link) with Gt(ROSA)26Sortm1(Notch1)Dam/J and Cdh5(PAC)-CreERT2 transgenics generated triple heterozygote males, which were bred to Dll4lox/+Gt(ROSA)26Sortm1(Notch1)Dam/J double heterozygous females. This produced CreERT2-negative controls together with Dll4lox/lox NICD+/+CreERT2T/+ (Dll4iΔEC) and Dll4+/+ NICD+/OECreERT2T/+ (NICDiOE-EC) single mutants, and Dll4iΔEC/NICDiOE-EC double mutants, which received tamoxifen and were analysed as described above. For mutant analysis, both male and female mice were used.
For Noggin administration experiments, mice were injected intraperitoneally once daily with 500µg/kg recombinant Noggin (R&D Systems) from P15 to P27, after completion of tamoxifen injections (P10-P14) and before analysis at P28.
All animal experiments were performed in compliance with the relevant laws and institutional guidelines and were approved by local animal ethics committees.
For Notch gain-of-function experiments, Gt(ROSA)26Sortm1(Notch1)Dam/J mice carrying a Cre-inducible transgene for Notch1 intracellular domain overexpression26 (link) and Cdh5(PAC)-CreERT2 transgenics were interbred. Tamoxifen administration (see above for injection schedule) was used to generate CreERT2-positive (NICDiOE-EC) mutants overexpressing NICD in ECs and controls. For experiments with EC-specific Dll4iΔEC/NICDiOE-EC double mutants, interbreeding of Dll4lox/lox conditional mice14 (link) with Gt(ROSA)26Sortm1(Notch1)Dam/J and Cdh5(PAC)-CreERT2 transgenics generated triple heterozygote males, which were bred to Dll4lox/+Gt(ROSA)26Sortm1(Notch1)Dam/J double heterozygous females. This produced CreERT2-negative controls together with Dll4lox/lox NICD+/+CreERT2T/+ (Dll4iΔEC) and Dll4+/+ NICD+/OECreERT2T/+ (NICDiOE-EC) single mutants, and Dll4iΔEC/NICDiOE-EC double mutants, which received tamoxifen and were analysed as described above. For mutant analysis, both male and female mice were used.
For Noggin administration experiments, mice were injected intraperitoneally once daily with 500µg/kg recombinant Noggin (R&D Systems) from P15 to P27, after completion of tamoxifen injections (P10-P14) and before analysis at P28.
All animal experiments were performed in compliance with the relevant laws and institutional guidelines and were approved by local animal ethics committees.
