아주대학교

목차

Chapter I. Introduction 1
1. Sensorineural hearing loss （SNHL） 1
2. In vitro model for cochlear hair cell generation: inner ear organoids 2
3. Limitations of current IEO models and the importance of the microenvironment 3
4. The pre-otic signaling environment: FGF, SHH, and retinoic acid in otic axis patterning 4
4-A. Roles of SHH （ventral axis signal） 5
4-B. Sequential differentiation of neurosensory domains 5
4-C. Retinoic acid as a pre-otic posterior signal: modulating the FGF–SHH environment 7
Chapter II. Study I: Early WNT-Mediated Posteriorization of the Organoid Microenvironment 9
1. Materials and methods 9
1-A. Inner ear organoid （IEO） generation and experimental design 9
1-B. Quantitative real-time PCR （qPCR） 10
1-C. Immunohistochemistry （IHC） 12
1-D. In vivo adult mouse cochlea immunohistochemistry 12
1-E. Statistical analysis 14
1-F. Notch inhibition and reaggregation cultures （supplementary） 15
2. Results and conclusions 16
2-A. Early WNT activation establishes a posterior microenvironment in IEOs during pre-otic development 16
2-B. Early WNT agonist delays otic fate but maintain otic progenitor competence 20
2-C. Early WNT activation induces spiral ganglion neurons but yields a hair-cell readout distinct from native cochlea 27
Chapter III. Study II: Combined Early WNT and RA Promote Posteriorization-Associated Ventralization and Cochlear-Leaning Specification 32
1. Materials and methods 32
1-A. Experimental design: combinatorial WNT and retinoic acid treatment 32
1-B. Drug treatment 33
1-C. Quantitative real-time PCR （qPCR） 33
1-D. Immunohistochemistry （IHC） 34
1-E. Flow cytometry （FACS） 34
1-F. Statistical analysis 34
2. Results and conclusions 39
2-A. Retinoic acid selectively induces posterior-hindbrain identity in IEOs during pre-otic development 39
2-B. WNT and RA together sustain otic induction and an Fgf-low, Shh-permissive window 43
2-C. Cochlear-leaning neurosensory output is associated with combined WNT priming and RA posteriorization 49
Chapter IV. Discussion 58
1. Study I: Early WNT reshapes the A–P character of the pre-otic microenvironment 59
2. Study II: RA selectively imposes posterior-hindbrain identity （RQ1） 61
3. FGF–SHH dynamics and the posteriorization-to-ventralization transition （RQ2） 63
4. Late otic development and cochlear-versus-neuronal output （RQ3） 65
5. Integrated interpretation: developmental and comparative context 69
6. Limitations 74
7. Future work 76
Chapter V. References 78
국문요약 93