Molecular basis and cellular events of brachydactyly type A1 pathogenesis

Abstract

The synovial joints connect articulating elements and provide mobility, while proper embryonic joint development is crucial to ensure postnatal function. Interzones form as joint progenitors appear, differentiate and cavitate to form joint components. Amongst, cavitation is poorly understood but a defective process likely contributes to joint ablating condition. Here, programmed cell death (PCD) is detected from early interzone till end of cavitation in phalangeal joint and corresponds to specific joint developmental stage. Inhibiting caspases result in poor cavitation and inability to maintain interzone molecular signature, resulting in anlage fusion. Therefore, the controversial PCD is essential to initiate or progress joint cavitation, forming the basis of dominant Indian hedgehog (Ihh)E95K mutation-causing brachydactyly type A1 (BDA1) pathogenesis. In human and homozygous mouse model, BDA1 is characterized as short middle phalanges in digit II to V, with an absent distal phalangeal joint in digit V. Despite extended IhhE95K range and attenuated signalling capacity are responsible for short anlage, the disease mechanism for ablated joint remains elusive. Here, TUNEL quantification revealed BDA1 joints have significantly less PCD, with a more severe reduction in distal joints. Indeed, the ablated joint developed a delayed interzone-like structure but no PCD event, suggesting inability to cavitate. Single cell transcriptomics revealed a decrease in pro-apoptotic pathway in BDA1 joint where B cell lymphoma (Bcl-2) family of intrinsic apoptosis regulators is tipped towards anti-apoptosis. Henceforth, when apoptosis is introduced specifically to developing interzone of ex vivo BDA1 paw culture, the absent joint is partially rescued with signs of cavitation. The extended IhhE95K signalling field is likely the driving mechanism for the cellular changes. Recent discoveries on growth arrest specific-1 (Gas1) and cell adhesion associated/oncogene regulated (Cdo) Hh co-receptors demonstrated their pro-apoptotic and Hh potentiating property. They express in developing interzone, muscle and periinterzone cells. In Gas1 genetically overexpressed joint, anlage has extended Ihh range but lower Ihh expression, leading to BDA1-like deranged growth plate with reduction in hypertrophy and ossification. This molecular and phenotypic pattern is opposite of Cdo-/- mutants. These suggest Gas1 and Cdo function in developing limb as long range Ihh promoter to inhibit growth plate differentiation. Therefore, the 2-fold increase in Cdoexpressing cells and strong Gas1 and Cdo staining in BDA1 joints likely further joint chondrogenicity and enhance BDA1 growth plate phenotype. Cdo-/- mutants demonstrated Cdo pro-apoptotic property in IhhE95K/+ interzones but opposite in WT interzones. Meanwhile, Gas1 overexpression promotes PCD in developing joint. The reciprocal expression nature between joint Gas1 and Cdo indicates the final Cdo/Gas1/Ihh output in regulating PCD occurrence. The absence of Gas1 and Cdo expression in BDA1 ablated joint in conjunction with increased pro-survival Ihh contributed to insufficient PCD, resulting in cavitation failure and an absent joint.