GSE183253
Suppression of apoptosis impairs phalangeal joint formation in the pathogenesis of brachydactyly type A1
Brachydactyly type A1 (BDA1), due to mutations in IHH, is characterized with shortened and/or missing middle phalangeal bones. These mutations alter the capacity and range of IHH signaling, reducing mesenchymal cells recruiting into the cartilage anlagen of a growing digit, result in a shorter middle phalange. However, reason for a missing middle phalange is unclear. Here, in IhhE95K BDA1 mouse, we showed a link to impaired apoptosis normally needed for interzone cavitation, thus a joint is not formed and the middle phalange is deemed absence. Hedgehog interacting partners (PTCH, CDON, GAS1and HHIP) are expressed in the interzone. Our data support a model for interzone cavitation, where CDON/GAS1 function as dependency receptors. Normally, IHH level is low at the center of the interzone, and the “ligand-free” CDON/GAS1 activate cell death for cavitation. In BDA1 joint, this is suppressed due to excess IHH. Our findings provided new insights into the role of IHH and CDON in joint formation, with relevance to hedgehog signaling in developmental biology. Overall design: Digit III M/P1 interzones (GFP-labelled) at E14.5 were dissected with minimum surrounding tissues (GFP negative) were harvested with the aid of Lgr5-GFP in WT (Ihh+/+; Lgr5GFP/+) and Lgr5-DTR-GFP in BDA1 (IhhE95K/E95K/Lgr5DTR-GFP/+). Two litters of the same father were used for each genotype and 14 joints were harvested per genotype. Dissected tissues were dissociated with 0.02% collagenase P (Roche 11213865001) for 45mins at 37oC, followed by 40?M cell strainer. Single cells were encapsulated using 10X Genomics Chromium Single Cell Chip (10x genomics) for encapsulations with Chromium Single Cell 3' Reagent Kits v2 according to manufacturer's instructions, performed by Centre of PanorOmic Sciences, HKU. Libraries were sequenced by HiSeq1500 (Illumina) and NovaSeq6000 (Illumina).