The highly conserved Notch signaling pathway regulates cell-cell communication pathways, cell fate, cell determination, cell death, embryonic development, and adult tissue pathways in metazoans. The Notch receptors and ligands that bind to Notch are single pass, transmembrane proteins that communicate cell to cell via juxtacrine signaling. There are reports of the divergent function and localization of the Deltalike 3 (Dll3) ligand. In Mus musculus (an eutherin mammal) the DLL3 protein inhibits the Notch signaling pathway and is localized in the Golgi apparatus. In contrast, the DLL3 protein from zebrafish, Danio rerio (a teleost) activates Notch and is located on the cell surface. This study will focus on examining the evolutionary pathway/evolutionary similarities, localization, and function of the A. carolinensis dll3 gene in comparison to other vertebrate species. This is important because there is not much known about the evolutionary divergence of the DLL3 A. carolinensis protein, its function in Notch signaling, and its subcellular localization.
Evolutionary analysis of vertebrate DLL3 protein sequences using phylogenetic trees showed that D. rerio and A. carolinensis are more evolutionarily similar in comparison to M. musculus suggesting that they may have similar intracellular localization. However, immunofluorescence staining experiments showed that the A. carolinensis DLL3 protein co-localized significantly with an endoplasmic reticulum (ER) specific primary antibody. Since this protein is localized in the secretory system, similar to that of M. musculus DLL3, it suggests that its function is to inhibit the Notch signaling pathway. Protein sequence alignments were created that suggested that there is a region in the protein sequences where the lizard and mouse sequence are conserved, while the zebrafish sequence simultaneously varies. This region of the amino acid sequence could be responsible for the difference in localization and function of the protein in these two species.