Cranial neural crest cells (CNCCs) are a population of multipotent stem cells that give rise to craniofacial bone and cartilage during development. Bone morphogenetic protein (BMP) signaling and autophagy have been individually implicated in stem cell homeostasis. Mutations that cause constitutive activation of the BMP type I receptor ACVR1 cause the congenital disorder fibrodysplasia ossificans progressiva (FOP), which is characterized by ectopic cartilage and bone in connective tissues in the trunk and sometimes includes ectopic craniofacial bones. Here, we showed that enhanced BMP signaling through the constitutively activated ACVR1 (ca-ACVR1) in CNCCs in mice induced ectopic cartilage formation in the craniofacial region through an autophagy-dependent mechanism. Enhanced BMP signaling suppressed autophagy by activating mTORC1, thus blocking the autophagic degradation of β-catenin, which, in turn, caused CNCCs to adopt a chondrogenic identity. Transient blockade of mTORC1, reactivation of autophagy, or suppression of Wnt–β-catenin signaling reduced ectopic cartilages in ca-Acvr1 mutants. Our results suggest that BMP signaling and autophagy coordinately regulate β-catenin activity to direct the fate of CNCCs during craniofacial development. These findings may also explain why some patients with FOP develop ectopic bones through endochondral ossification in craniofacial regions.

BMP signaling plays pleiotropic roles in various tissues during embryogenesis and after birth. We have previously generated a constitutively activated Acvr1(ca-Acvr1) transgenic mouse line (line L35) through pronuclei injection to investigate impacts of enhanced BMP signaling in a tissue specific manner. However, line L35 shows a restricted expression pattern of the transgene. Here, we generated another ca-Acvr1 transgenic line, line A11, using embryonic stem (ES) transgenesis. The generated line A11 shows distinctive phenotypes from line L35, along with very limited expression levels of the transgene. When the transgene is activated in the neural crest cells in a Cre-dependent manner, line A11 exhibits cleft palate and shorter jaws, while line L35 develops ectopic cartilages and highly hypomorphic facial structures. When activated in limb buds, line A11 develops organized but smaller limb skeletal structures, while line L35 forms disorganized limbs with little mineralization. Additionally, no heterotopic ossification (HO) is identified in line A11 when bred with NFATc1-Cre mice even after induction of tissue injury, which is an established protocol for HO for line L35.Therefore, the newly generated conditional ca-Acvr1 mouse line A11 provides an additional resource to dissect highly context dependent functions of BMP signaling in development and disease.

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Summary Podoplanin, PDPN, is a mucin‐type transmembrane glycoprotein widely expressed in many tissues, including lung, kidney, lymph nodes, and mineralized tissues. Its function is critical for lymphatic formation, differentiation of type I alveolar epithelial lung cells, and for bone response to biomechanical loading. It has previously been shown that Pdpn null mice die at birth due to respiratory failure emphasizing the importance of Pdpn in alveolar lung development. During the course of generation of Pdpn mutant mice, we found that most Pdpn null mice in the 129S6 and C57BL6/J mixed genetic background die at the perinatal stage, similar to previously published studies with Pdpn null mice, while all Pdpn null mice bred with Swiss outbred mice survived. Surviving mutant mice in the 129S6 and C57BL6/J mixed genetic background showed alterations in the osteocyte lacunocanalicular network, especially reduced osteocyte canaliculi in the tibial cortex with increased tibial trabecular bone. However, adult Pdpn null mice in the Swiss outbred background showed no overt differences in their osteocyte lacunocnalicular network, bone density, and no overt differences when challenged with exercise. Together, these data suggest that genetic variations present in the Swiss outbred mice compensate for the loss of function of PDPN in lung, kidney, and bone.