Browsing by Subject "sox2"
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Item Inner Ear Sensory Epithelia Development and Regulation in Zebrafish(2011-10-21) Sweet, Elly MaeThe inner ear is a complex sensory organ of interconnected chambers, each with a sensory epithelium comprised of hair cells and support cells for detection of sound and motion. This dissertation focuses on the development and regulation of sensory epithelia in zebrafish and utilizes loss of function, gain of function and laser ablation techniques. Hair cells and support cells develop from an equivalence group specified by proneural genes encoding bHLH transcription factors. The vertebrate Atoh1 bHLH transciption factor is a potential candidate for this role. However, data in mouse has led some researchers to conclude it does not have a proneural activity, but, rather, is involved in later stages of hair cell differentiation. In addition, the factors regulating Atoh1 are mostly unknown. We address these issues in zebrafish and show that the zebrafish homologs atoh1a and atoh1b are required during two developmental phases, first in the preotic placode and later in the otic vesicle. They interact with the Notch pathway and are necessary and sufficient for specification of sensory epithelia. Our data confirm atoh1 genes have proneural function. We also go on to show Atoh1 works in a complex network of factors, Pax2/5/8, Sox2, Fgf and Notch. Misexpression of atoh1 alters axial patterning and leads to expanded sensory epithelia, which is enhanced by misexpression of either fgf8 or sox2. Lastly, we examine the role of sox2 in sensory epithelia development and regeneration. Sox2 has been implicated in maintainence of pluripotent stem cells as well as cell differentiation. In the inner ear, Sox2 is initially expressed in the prosensory domain and is required for its formation. Eventually, Sox2 is downregulated in hair cells and maintained in support cells; however, its later role has not been determined. We show that in the zebrafish inner ear, sox2 is expressed after sensory epithelium development has begun and, like in mouse, expression is down regulated in hair cells and maintained in support cells. Our data demonstrate a role for sox2 in maintenance of hair cells and in transdifferentation of support cells into hair cells after laser ablation. Additionally, sox2 is regulated by Aoth1a/1b, Fgf, and Notch.Item Neurosensory Development in the Zebrafish Inner Ear(2012-02-14) Vemaraju, ShrutiThe vertebrate inner ear is a complex structure responsible for hearing and balance. The inner ear houses sensory epithelia composed of mechanosensory hair cells and non-sensory support cells. Hair cells synapse with neurons of the VIIIth cranial ganglion, the statoacoustic ganglion (SAG), and transmit sensory information to the hindbrain. This dissertation focuses on the development and regulation of both sensory and neuronal cell populations. The sensory epithelium is established by the basic helixloop- helix transcription factor Atoh1. Misexpression of atoh1a in zebrafish results in induction of ectopic sensory epithelia albeit in limited regions of the inner ear. We show that sensory competence of the inner ear can be enhanced by co-activation of fgf8/3 or sox2, genes that normally act in concert with atoh1a. The developing sensory epithelia express several factors that regulate differentiation and maintenance of hair cells. We show that pax5 is differentially expressed in the anterior utricular macula (sensory epithelium). Knockdown of pax5 function results in utricular hair cell death and subsequent loss of vestibular (balance) but not auditory (hearing) defects. SAG neurons are formed normally in these embryos but show disorganized dendrites in the utricle following loss of hair cells. Lastly, we examine the development of SAG. SAG precursors (neuroblasts) are formed in the floor of the ear by another basic helix-loophelix transcription factor neurogenin1 (neurog1). We show that Fgf emanating from the utricular macula specifies neuroblasts, that later delaminate from the otic floor and undergo a phase of proliferation. Neuroblasts then differentiate into bipolar neurons that extend processes to hair cells and targets in the hindbrain. We show evidence that differentiating neurons express fgf5 and regulate further development of the SAG. As more differentiated neurons accumulate, increasing level of Fgf terminates the phase of neuroblast specification. Later on, elevated Fgf stabilizes the transit-amplifying phase and inhibits terminal differentiation. Thus, Fgf signaling regulates SAG development at various stages to ensure that proper number of neurons is generated.Item The Function and Genetic Interactions of Zebrafish atoh1 and sox2: Genes Involved in Hair Cell Development and Regeneration(2010-10-12) Millimaki, Bonny ButlerThe sensory cells of the inner ear, hair cells, provide vertebrates with the ability to detect auditory stimuli and balance. In mammals, cochlear hair cells, those responsible for hearing, do not regenerate. Zebrafish hair cells do regenerate. Gaining an understanding of the role and regulation of the genes involved in the formation and regeneration of these cells may provide information important for the development of genetic therapies. We show that zebrafish atoh1 acts as the proneural gene responsible for defining the equivalence group from which hair cells form. Expression of atoh1 is dependent upon Fgf and Pax. Atoh1 induces expression of delta, resulting in activation of Notch and subsequent lateral inhibition. Another factor known to be important for hair cell formation in mice is Sox2. In zebrafish, sox2 expression is downstream of Atoh1, Notch and Fgf. Zebrafish Sox2 is not required for hair cell formation, but rather Sox2 is important for hair cell maintenance. In zebrafish, otic hair cell regeneration has not yet been characterized. We show that, following laser ablation, hair cells regenerate by way of transdifferentiation. We further show that this regeneration requires Sox2 activity. These data suggest that Sox2 acts to maintain support cell plasticity. This role is likely conserved because Sox2 is also important for stem cell plasticity in mammals. This new understanding of the role and regulation of both Atoh1 and Sox2 provides essential information that can be used to further efforts to provide genetic therapies for hair cell regeneration in mammals.