Pax3 has been used as a valuable marker in research aimed at understanding tissue interactions involved in trigeminal ophthalmic (opV) placode development. While Pax3 expression coincides with opV neuron specification, the function of Pax3 in these cells has not previously been investigated. Splotch mutant mice (which lack Pax3) have a reduced trigeminal ganglion; however it is not clear whether this reduction is due to neural crest or placode cells. We have used electroporation in the chick model system to block or ectopically express Pax3 at key times in opV placode development. Using several markers of placode cell differentiation, we have determined the experimental effects manipulating Pax3. Blocking placodal Pax3 with gene specific morpholinos resulted in a loss of migratory placode cells, and a downregulation of all opV placode markers in targeted cells. Ectopic expression of Pax3, either within the placode domain or in adjacent cranial ectoderm, resulted in the upregulation of some but not all placode markers. We conclude that opV placodal Pax3 expression is required for normal placode cell development, and hypothesize that its expression must be tightly regulated in order for placode cells to fully differentiate. The precise role of Pax3 and Pax7 in the restriction and differentiation of dorsal interneuron progenitors has been difficult to assess due to the many additional factors involved in specification and patterning of the neural tube. We have used electroporation in the chick model system to ectopically express Pax3 and Pax7 unilaterally in the neural tube. Using several markers for differentiation of ventral and dorsal neuronal progenitors, we have experimentally determined the effects of Pax3 and Pax7 ventrally and dorsally. Ectopic expression of these transcription factors in the ventral neural tube resulted in the loss of motorneurons. Though mis-expression did not qualitatively affect commissural neurons as assayed by neurofilament staining, ectopic expression of Pax3 and Pax7 in the dorsal neural tube stopped dorsal interneuron progenitors from differentiating. We conclude that Pax3 and Pax7 expression is sufficient to restrict ventral neuron identity. We also hypothesize that downregulation of these transcription factors in the dorsal neural tube is required for normal dorsal interneuron differentiation.

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.