Analysis of Abnormal Craniofacial and Ear Development of a Transgenic Mutant with Ectopic Hoxb3 Expression

This dissertation, "Analysis of Abnormal Craniofacial and Ear Development of a Transgenic Mutant With Ectopic Hoxb3 Expression" by Yee-man, Elaine, Wong, 王怡雯, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Analysis of abnormal craniofacial and ear development of a transgenic mutant with ectopic Hoxb3 expression Submitted by Wong Yee Man Elaine for the degree of Doctor of Philosophy at The University of Hong Kong in August 2006 An evolutionarily conserved function of Hox genes is to assign positional information to body segments along the anteroposterior axis. Hoxb3 is a homeobox gene encoding a transcription factor that regulates other genes. As Hoxb3 knockout mice are largely normal due to functional redundancy with its paralogous group 3 members, the role of Hoxb3 in early embryogenesis is still unknown. To determine the potential roles of Hoxb3 and to identify genes involved in Hoxb3 regulatory pathways, a gain-of-function mutation approach was employed in this study. Using a Hoxb2 rhombomere 4 (r4) and second branchial arch specific enhancer element, ectopic Hoxb3 was directed to express in r4 and second branchial arch of the transgenic mutant named SL2. The SL2 mutants exhibited a spectrum of craniofacial, outer, middle and inner ear defects including pointed face, facial paralysis, reduced pinna size, impaired reaching response, abnormal circling behaviour and deafness. To understand the molecular bases of these abnormalities, in situ hybridization was performed with marker genes expressed in the rhombomere, branchial arch and otic vesicle using SL2 embryos. The ectopic expression of Hoxb3 in r4 resulted in down-regulating the expression of Hoxb1 and its downstream targets, Hoxb2 and Gata3, in r4; but maintained a molecularly distinct r4-, r5-, r6-like territory. A Hoxb3 binding site was identified in the Hoxb1 gene, a direct binding mechanism between Hoxb3 protein and Hoxb1 gene was demonstrated by electrophoresis mobility shift assay. Therefore Hoxb3 could normally suppress Hoxb1 expression in hindbrain development. het hom The reduction in size of the second branchial arch in the SL2 and SL2 embryos at 10.5 dpc was shown by scanning electron microscopy and quantitative measurement. As shown by histological sections and skeletal staining, first arch neural-crest derived structures including malleus, incus, tympanic ring, mandible and squamous bone; and second arch neural-crest derived structures including styloid process, stapes, hyoid bone and facial nerve were affected. ET1 and its downstream targets, dHAND, Gsc, Dlx5, Prx2 and Twist, were found to be specifically affected in the first and second branchial arches in the SL2 mutants. It is possible that Hoxb3 may be an upstream factor of the ET1 regulatory pathway involved in the epithelial-mesenchymal interaction in the branchial arch. The dorsal and ventral otic vesicle derived inner ear structures, including endolymphatic duct, common crus, lateral semi-circular canal and ampulla, utricle, saccule, cochlea and vestibuloacoustic ganglion were affected in the SL2 mutants. In the developing otic vesicle, the expression domains of dorsal genes, Hmx3 and Gata3, were expanded ventrally while the expression domains of the ventral genes, Otx1, het Lfng, Bmp4, Fgf3, Ngn1 and NeuroD, were reduced in the SL2 embryos. Enhanced het apoptosis in the ventral otic vesicle was also observed in the SL2 embryos. These phenotypes were similar to that of Six1 knockout mutants. However, Six1 expression het patterns in the SL2 embryos remained the same as wild-type embryos, indicating th