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) is the birth place of one cell type of sensory neurons contributing to the trigeminal ganglion. Signals from the neural tube induce placodal identity within the surface ectoderm. Specified opV placode cells then up-regulate neuron differentiation markers and migrate to the ganglion. Several molecular pathways have been shown to act in opV placode cell development. Despite this, signals that specify individual neurons from within the opV placode remain unknown. However, it is known that components of the Notch signaling pathway are expressed in the opV placode. I tested the role of Notch signaling in opV placode development by separately inhibiting and over-activating the pathway. Using DAPT, an inhibitor of gamma-secretase, I inhibited Notch signaling in 13-15 somite stage chick embryo heads. Attenuated Notch signaling caused increased neuronal differentiation of opV cells at 13-15 somites. I also observed an increase in migratory opV placode (Pax3+) cells in the mesenchyme and expression of neuronal marker Islet1 in the ectoderm. Further, I activated Notch signaling by misexpressing the Notch intracellular domain (NICD) by in ovo electroporation of 10-12 somite stage chick embryos. This resulted in Pax3+ targeted cells failing to differentiate and remain instead in the ectoderm. Thus, Notch/Delta signaling plays an important role in selecting ophthalmic trigeminal cells to differentiate and migrate to the trigeminal ganglion.

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.

Chez les vertébrés, une première ébauche du système nerveux central à partir du neurectoderme est obtenue par la neurulation. Ce processus mène à la formation du tube neural (TN) à partir de la plaque neurale. La neurulation est coordonnée avec l’induction d’une population de cellules multipotentes aux bordures latérales de la plaque neurale: les cellules de la crête neurale (CCNs). Le gène Pax3 encode un facteur de transcription qui est essentiel pour la formation du TN et des CCNs. Une petite région régulatrice d’environ ~250pb dans le promoteur proximal de Pax3, appelée NCE2, est suffisante pour récapituler l’induction de Pax3 ainsi que sa restriction aux bordures latérales de la plaque neurale. Le NCE2 de Pax3 est connu pour intégrer des signaux instructifs antéropostérieur (AP) provenant de la voie Wnt, via les protéines CDX (CDX 1, 2, 4), pouvant induire l'expression de Pax3 dans la plaque neurale postérieure (PNP). Nous avons démontré ici que, en plus des signaux AP, le NCE2 de Pax3 intègre des signaux instructifs dorsoventraux (DV) provenant de la voie BMP, via ses effecteurs SMAD1/5. Nos résultats indiquent que les protéines SMAD1/5 pourraient être le cofacteur manquant dans le contrôle CDX-dépendant de l’expression de Pax3 et que ce serait ces protéines qui permettraient de conférer le patron d’expression restreint de Pax3 aux bordures latérales de la PNP. Pour étayer cette affirmation, nous fournissons de nouvelles preuves que l’activité de BMP-SMAD1/5 sur l’expression de Pax3 est médiée par les CDX. Comme des défauts affectant la formation du TN et des CCNs sont à la base de plusieurs syndromes génétiques et malformations congénitales chez l’humain, nos résultats offrent ainsi une meilleure compréhension des mécanismes moléculaires sous-tendant ces pathologies. / In vertebrates, a first draft of the central nervous system from the neurectoderm is obtained by neurulation. This process leads to the formation of the neural tube (NT) from the neural plate. Neurulation is coordinated with the induction of a population of multipotent cells at the neural plate border: neural crest cells (NCCs). The Pax3 gene encodes a transcription factor that is essential for the formation of the NT and NCCs. A small regulatory region of ~250bp in the proximal promoter of Pax3, called NCE2, is sufficient to recapitulate the induction of Pax3 and its restriction to the lateral borders of the neural plate. The Pax3NCE2 is known to incorporate anterior-posterior (AP) instructive cues from the Wnt pathway, via CDX proteins (CDX1, 2, 4), which can induce the expression of Pax3 in the posterior neural plate (PNP). We have demonstrated that, in addition to the AP cues, Pax3NCE2 integrates instructive dorsal-ventral (DV) cues from the BMP pathway, via SMAD1/5 proteins. Our results indicate that SMAD1/5 proteins could be the missing co-factor in the CDX-dependent expression of Pax3 that restrict Pax3 expression to the lateral borders of the PNP. To support this assertion, we provide further evidence that the activity of BMP-SMAD1/5 on the expression of Pax3 is mediated by CDX proteins. As defects affecting the formation of the NT and NCCs are the basis of many genetic syndromes and birth defects in humans, our results provide a better understanding of the molecular mechanisms underlying these pathologies.

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