Roles of hedgehog, bone morphogenetic protein 4 and CXCL12 signaling on middle ear development

Abstract

Mammalian hearing system consists of three types of ears: outer, middle and inner ear. Middle ear consists of a chain of ossicles namely malleus, incus and stapes. These middle ear ossicles transmit the sound waves from outer ear to the inner ear. Any developmental defects in the middle ear ossicle can result in conductive hearing loss. These middle ear ossicles are derived from the neural crest cells (NCCs). The NCCs from rhombomere (r) 1 and r2 and few NC cells from r3 migrate into branchial arch (BA) 1 and develop into Meckel’s cartilage, malleus and incus. The NCCs from r4 and few cells from r3 and r5 will migrate into the BA2 to form stapes. It is still unclear that how the NCCs were able to find their final destinations and differentiate into the final target tissue. In order to answer this question, we approached cre-loxp technology to delete our genes of interest either in the NCCs or pharyngeal endoderm and analyzed the gene expression patterns in controls and their conditional knockout (cko) littermates. When we delete Smoothened (Smo) gene in the NCCs using Wnt1Cre or Sonic hedgehog (Shh), malleus-incus initial condensation was greatly reduced in the BA1 at embryonic day (E) 10.5 and the stapedial condensation is slightly reduced in the BA2 region. At E11.5, the stapedial condensation was disappeared in the Smocko embryos. This loss of malleus-incus condensation or the stapedial condensation was due to the failure of NC survival in the absence of hedgehog (HH) signaling confirming the HH signaling is required for the NC survival during the middle ear condensation stage. When HH signaling was constitutively activated in the NCCs, there was the increase in the condensation of both malleus-incus and stapes of SmoM2 mutants. At later stages, we observed an ectopic cartilage which appeared to be the fused and dislocated middle ear ossicles in the SmoM2 mutants. Similarly, when we deleted Smad4 gene in the NCCs or bone morphogenetic protein 4 (Bmp4) in the pharyngeal endoderm, we observed no change in malleus-incus condensation of both Smad4cko and Bmp4cko at E10.5 and also observed that stapedial condensation was absent. This loss of stapedial condensation was due to lack of the NCCs in the prospective stapes region. BMP4 signaling is not essential for the NC proliferation or survival. Based on previous studies, we hypothesized that CXCL12, a chemokine signaling can act as NCC chemoattractant and endodermal Bmp4 is required to induce the Cxcl12 expression in the prospective stapes region. We observed that Cxcl12 was expressed in the prospective stapes region although Bmp4 was deleted in the pharyngeal endoderm. This result suggests us that endodermal Bmp4 is not required to induce Cxcl12 expression in the prospective stapes region. When Cxcl12 was deleted in the whole body (Cxcl12+/-), we observed normal malleus and incus cartilages at E14.5; but, stapes was greatly malformed. This result confirms that Cxcl12 is required normal stapes development, but not required to initiate the stapedial condensation. These all results together confirm that the HH and Bmp4 signaling from the endoderm are required for the NCCs to initiate the malleus-incus and stapedial condensations, respectively.