Arabidopsis WRINKLED1 (AtWRI1) (At3g54320) is a pivotal transcription factor in the regulation of plant oil biosynthesis. Our previous study identified a WRI1 homolog highly expressed in oil palm mesocarp (EgWRI1). EgWRI1 protein is 93 amino acids shorter than AtWRI1 with low sequence conservation and all deletions occurring in the Cterminal region. We asked whether this divergent EgWRI1 protein, from a non-seed tissue with very high oil content, and from a phylogenetically distant species is able to complement the Arabidopsis loss-of-function mutant wri1-1. We show that expression of EgWRI1 is able to restore several phenotypes of wri1-1, including reduced seed fatty acid content, the “wrinkled" appearance of the seed coat, reduced seed germination, and impaired seedling establishment. Comparison of the protein sequence of WRI1 orthologs across many diverse plant species revealed the conservation of a 9 bp exon encoding “VYL". However, this small exon is missing in one of three predicted AtWRI1 splice forms. To understand expression of WRI1 splice forms we performed RNASeq analysis of Arabidopsis developing seeds and queried other Arabidopsis EST and RNASeq databases derived from a number of tissues and from a range of plant species. In all cases, only splice form 3 is expressed in Arabidopsis and VYL is observed in the cDNA of all WRI1 orthologs investigated. Site-directed mutagenesis showed that amino acid substitutions within the ‘VYL' exon of AtWRI1 results in failure to restore reduced oil content of wri1-1 seeds, providing direct evidence for the crucial role of this small exon in AtWRI1 function.

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.