published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Bone morphogenetic proteins (BMPs) are diffusible molecules involved in a variety of cellular interactions during development. In particular, Bmp4 expression accompanies the development of the ear sensory organs during patterning and specification of sensory cell fates, and it has been shown to play a role in inner ear development and morphogenesis. However, there is no understanding of the cellular effects of BMP4 in prosensory progenitors, and about its role in the process of sensory fate specification. The present thesis project was aimed at exploring the effects of BMP-signaling on the development of hair-cells, using the chick inner ear as a model.The specific aims proposed were:1- Analyze the cellular effects caused by addition of BMP4 in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.2- Analyze the cellular effects caused by inhibition of BMP4 signaling in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.3- Analyze the expression in the innear ear of downstream targets of BMP signalling, in particular, analyse the members of Id gene family.4- Analyze the regulation of Id genes by BMP signalling in the inner ear.5- Analyze the expression of genes involved in the process of terminal differentiation, in particular, Btg1 and Btg2 genes6- Analyze the regulation of Btg1 and Btg2 gene by BMP signalling in the inner ear

Inner ear hair cells have been identified as the sites of mechanoelectrical transduction from a mechanical event (e.g. hearing, motion) to an electrical event (e.g. neural response). Deflection of bundles of hair-like stereocilia extending from these cells has been associated with the transduction process. Stereocilia bundle structure and stiffness controls deflection and thus the fundamental sensitivity of the transduction process. The finite-element method was used along with analytical techniques to characterize individual stereocilium and stereocilia bundle stiffnesses. A three ‘stack’ bundle with a Young’s modulus of 3 GPa (F-actin protein) and Poisson’s ratio of 0.4 (nearly incompressible) resulted in a stiffness of K = 2.1 x 10⁻³ N/m. This value is within the range of experimentally determined stiffmesses. Tip-link and subapical band interconnecting structures each contribute significantly to bundle stiffness and each could act as the gating-spring in transduction models, which propose gating structures as a means of regulating ionic activity and therefore neural activity. Stiffness depends most strongly on individual stereocilium geometry and material description, tip-link orientation and material description, and stereocilia bundle width. Stiffness depends least on stereocilia height variations and subapical bands configuration. Linear analysis was reliable up to deflections of 3.5 um, the upper limit of physical response. Preliminary dynamic response indicates a natural frequency of 382 kHz for the vibration mode resembling physical deformation behavior. Future models should include hexagonal bundle arrangements, transversely isotropic stereocilia material descriptions, and viscoelastic tip-link behavior. / Master of Science

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex threedimensional structure. To achieve this complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tisúes surrounding the otic primordium, and more particularly the adjacent segmented hindbrain, have been implicated in specifying structures along the anteroposterior and dorsoventral axes of the inner ear. In this work we have first characterized the generation and axial specification of the otic neurogenic domain, and second, we have investigated the effects of the mutation of kreisler/MafB -a gene transiently expressed in the rhombomeres 5 and 6 of the developing hindbrain- in early otic patterning and cell specification. We show that kr/kr embryos display an expansion of the otic neurogenic domain, due to defects in otic patterning. Although many reports have pointed to the role of FGF3 in otic regionalization, we provide evidence that FGF3 is not sufficient to govern this process. Neither Krox20 nor Fgf3 null mutant embryos, in which Fgf3 is either downregulated or absent in r5 and r6, present ectopic otic neuroblasts in the otic primordium. However, Fgf3-/-Fgf10-/- double mutants show a phenotype very similar to kr/kr embryos: they present ectopic neuroblasts along the AP and DV otic axes. Finally, and remarkably, partial rescue of the kr/kr phenotype is obtained when Fgf3 or Fgf10 are ectopically expressed in the hindbrain of kr/kr embryos. These results highlight a compensatory mechanism between FGFs, and the importance of hindbrain-derived signals in instructing otic patterning and the establishment of the neurogenic domain.