Serine Lipid Modification Of Wnt1 Is Required For Cell Proliferation And Specification In The Chick Spinal Cord

Spinal cord development and patterning is heavily regulated by Wnts. Wnts are a family of lipid modified signaling proteins that play crucial roles in embryonic development and adult homeostasis, in processes such as cell migration, cell proliferation, and cell fate. In the developing spinal cord, Wnts form a dorsal to ventral concentration gradient, controlling cell proliferation and inducing different neuronal types. Lipid modification of Wnts by the enzyme Porcupine (Porcn) is essential for their secretion and signaling activity during development. Wnt1 is lipidated on two conserved amino acids, C97 and S224. However, the substrate specificity of Porcn has not been established nor has the in vivo relevance of the serine residue in amniotes. We hypothesize that overexpression of Wnt1 bearing mutations of the serine residue (S224A, S224C) will yield reduced cell proliferation and altered specification within the neural tube, as compared to wild-type Wnt1. To test this hypothesis, expression constructs encoding for mutated and wild-type Wnt1 were electroporated into the developing chick spinal cord. Successfully electroporated embryos were assayed for changes in cell proliferation and neuron specification by immunostaining transverse sections with antibodies specific for proliferative and specification markers. Data in confocal images were quantified using software to evaluate cell proliferation and differentiation. Current analysis of the S224A mutation reveals depleted Wnt signaling in comparison to wild-type Wnt1, suggesting that lipid modification of S224 is indeed required for the biological activity of Wnt1 in amniotes. Experiments with S224C are now underway to test the enzyme specificity of Porcn.