The ophthalmic trigeminal placode (opV) exclusively gives rise to sensory neurons. A number of signaling pathways including Wnt, PDGF, FGF, and Notch are all involved in the progression of an undifferentiated cell in the opV placode to a proneural cell in the condensing opV ganglion. However, the regulatory relationships between these signal transduction pathways are still unknown. To determine if FGF activation acts to modulate Notch signaling in the sensory neurogenesis pathway, a novel multifactorial approach was employed: FGF signaling was inhibited in individual cells and globally with simultaneous inactivation of Notch signaling in chick embryos to investigate if FGF activation downregulates Notch thereby driving neurogenesis. These experiments resulted in few differentiating opV cells in the mesenchymal region of future ganglion formation suggesting an alternate regulatory relationship between FGF and Notch where either reduced Notch activity allows for FGFR4 expression (leading to FGF signaling and neurogenesis), or a parallel relationship where FGF and Notch act independently of one another to induce neurogenesis. To distinguish between these two possibilities Notch signaling was inhibited with DAPT, a gamma-secretase inhibitor, and assayed for FGFR4 mRNA expression. These results indicated FGFR4 is not upregulated by reduced Notch activity, suggesting that FGF and Notch act in parallel to promote neurogenesis. During these experiments it was observed that Notch inhibition resulted in an undefined ectoderm in the opV placode region. To investigate this, FGF and Notch were inhibited by SU5402, an FGF antagonist, and DAPT, and later sectioned and stained for Laminin. In DAPT treated embryos the basement membrane became highly fragmented, a remarkable observation not yet reported. From these data a proposed mechanism was established where activation of FGF with parallel downregulation of Notch leads to disruption of extracellular matrix proteins in the basement membrane resulting in fragmentation and subsequent delamination of differentiating opV placode cells.

This dissertation is divided into three chapters, each discussing the study of different regulatory molecules involved in sensory neurogenesis occurring in the trigeminal placode. Chapter one is a spatiotemporal description of Notch pathway genes in chick opV placode by stage-specific expression analysis, showing expression of many Notch pathway genes and effectors in the opV placode. Notch pathway gene expression is primarily confined to the ectoderm with highest expression of these genes at the beginning stages of peak neuronal differentiation. This information preceded studies of the functional roles that Notch signaling has in the opV placode and how it may affect the transcription factor, Pax3. Chapter two is a study of the transcription factor Pax3 and its role in opV placode development and sensory neuron differentiation. Pax3 is known to activate or repress gene transcription, and its activity may be dependent on the splice variant or isoform present. We show through RT-PCR that alternative splice forms of Pax3 are present at stages of chick development corresponding to cellular competence, cellular differentiation and ingression, and cellular aggregation. We have named these splice forms, Pax3V1 and Pax3V2. Using quantitative RT-PCR we show that Pax3V2 is consistently expressed at lower levels compared to Pax3 during cellular competence and differentiation. In order to determine the function of the three splice forms, we misexpressed them in the opV placode and analyzed the effect on neurogenesis. We looked at markers for neuronal differentiation of targeted cells after in ovo electroporation of Pax3, Pax3V1, and Pax3V2, which showed a significant difference between the control and each construct, but not between the groups of constructs. To enhance the process of neurogenesis we exposed the electroporated embryos to DAPT, a Notch signaling inhibitor that enhances sensory neurogenesis. Using this method we found that misexpression of Pax3 and Pax3V1 resulted in cells failing to differentiate, while Pax3V2 misexpression more closely resembles the neuronal differentiation seen in controls. These results show that the Pax3V2 isoform allows for neuronal differentiation of opV placodal cells after misexpression, while the Pax3 isoform and the Pax3V1 isoform block neuronal differentiation. Chapter three is a study of the necessity of Wnt signaling originating from the neural tube to induce Pax3 expression in the opV placode. A double knockout of Wnt1 and Wnt3a was produced to determine the necessity of these genes in opV placode development. Pax3 expression in the opV placode at E8.5 and E9.5 was markedly reduced in the double mutants when compared to wild type mice. This study shows that Wnt1 and Wnt3a genes are necessary for normal Pax3 expression, but that other signals may contribute to its induction.