Date

1-2014

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Cell and Developmental Biology

Institution

Oregon Health & Science University

Abstract

In the developing vertebrate head, essential structures that constitute the paired sensory organs arise from discrete regions of sensory epithelium, known as cranial placodes. These regions reside outside the axial ectoderm of the forming central nervous system (CNS). Cranial placodes are morphologically defined as transient ectodermal thickenings, with columnar or pseudostratified epithelial cell morphology; on a molecular level, placodal cells exhibit a unique expression profile of transcription factors. Through physical interactions with neighboring tissues and in response to extrinsic signals, cells of the ectodermal placodes delaminate and/or invaginate to form structures as diverse as the optic lens, the otic vesicle, and neurons of the cranial ganglia. My studies have focused on the more posterior placodes, the otic and epibranchial (EB) placodes. The otic placode gives rise to all inner ear structures, including the sensory epithelia and its associated neurons. The EB placodes form the sensory neurons of the EB ganglia, including the facial, glossopharyngeal, and vagal ganglia. EB neurons act as a relay for information from the sensory organs (e.g. taste buds of the gustatory system, baroreceptors of the heart, and sensory enteric nerves of the gut) to the CNS. Initially, the EB placode precursors are intermingled with other pan-placodal cell populations in a single domain called pan-placodal ectoderm (PPE). Subsequently, cells within this domain undergo multiple sorting steps in order to segregate into distinct placodes. The molecular and cellular mechanisms that govern the segregation and the subsequent migration of precursors are largely unknown. In zebrafish, precursors for the EB and otic xi placodes segregate from the PPE and form a posterior domain called the posterior placodal area (PPA) shortly after gastrulation. This domain is marked by expression of the transcription factors Pax2a, Pax8, and Sox3. Specification of both the otic and EB placodes requires extrinsic signaling from Fibroblast growth factors (Fgf), which in turn controls Pax2a, Pax8 and Sox3 expression. In this work, we used lineage tracing and live imaging analyses to show that cells of the PPA become biased towards certain fates as early as 12 hours post fertilization (hpf). Ablation studies verified the requirement of the PPA to form the otic and EB placodes, and support a spatial correlation bias with final structure contribution. We also discovered that levels of Pax2a expression correlate to cell fate: high Pax2a+ cells segregate into the otic placode and low Pax2a+ cells segregate into the EB placodes. Pax2a overexpression and suboptimal morpholino knockdown of Pax2a together with Pax8 confirm that high levels of Pax expression bias cells towards an otic fate, while low levels of Pax expression bias cells toward an EB fate. Final, we found the Wnt signaling from the neural tube is responsible for inducing high levels of Pax2a in a subset of cells of the PPA to enforce an otic identity. Once EB placode precursors segregate from the nascent otic placode, their subsequent differentiation requires continuous Fgf signaling. When Fgf receptor signaling is globally inactivated between 12 and 24 hpf the EB placodes are lost, while other placode derived structures (like the otic vesicle) remain relatively intact. Notably, activation of local Fgf signaling through Fgf ligand soaked beads implanted near forming EB placodes expands placodal Pax2a xii expression. Analysis of various Fgf ligands during this critical period revealed that Fgf3 and Fgf10a are responsible for maturation of the EB placodes. Injection of fgf10a morpholino in fgf3-/- embryos resulted in loss of the EB placodes as marked by Pax2a and sox3, a reduction in the glossopharyngeal and vagal placodes at 24 hpf, and a loss of the respective ganglia at 72 hpf. Loss of Fgf3/10a did not result in an otic deficit, indicating a process specific to EB placode development. We find that the endoderm is the tissue source of Fgf3 responsible for EB placode maturation, and provide evidence that the lateral line system and possibly the anterior otic vesicle is the signaling center of Fgf10a. Our studies contribute to a better understanding of sensory organ development and early neural progenitor cell induction and specification in a unique setting outside of the CNS.

Identifier

doi:10.6083/M4NV9GK5

School

School of Medicine

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