CELL SIGNALING PATHWAYS IN CONTROL OF NEUROBLASTOMA AND HUMAN NEURAL CREST DEVELOPMENT

Receptor Tyrosine Kinases (RTKs) are a family of cell surface receptors that bind extracellular ligands and initiate cellular signaling pathways. These pathways ultimately influence cellular behaviors such as cell proliferation, migration, and differentiation. Consequently, mutations and amplifications of RTK genes contribute to the development of several cancers, including neuroblastoma. Neuroblastoma originates from neural crest cells, which are formed as the neural tube closes during vertebrate development. The neural crest differentiates and migrates to form cells of several lineages including the peripheral nervous system, melanocytes, and craniofacial cartilage. These processes are tightly controlled by RTK signaling, and failures in differentiation may lead to uncontrolled cell growth and tumor formation. While extensive research has characterized individual RTK signaling pathways in control of neural crest differentiation, it is unknown how signals from several different receptors are integrated to produce a cellular response. In Chapter 2, we investigate the regulation of RTK activity through endocytosis, which controls RTK degradation and receptor recycling to the plasma membrane. Here we show that the SRC family kinases (SFKs) are located in endosomes of neuroblastoma cells, and are activated by RTKs. Furthermore, SFK localization within different endosome populations changed in response to signaling by ALK and KIT. The scaffold protein, PAG1, forms signaling complexes containing SFKs as well as members of other pathways downstream of RTKs. To understand how PAG1 directs pSFK endocytosis and sequestration, we generated a PAG1 protein lacking the transmembrane domain. We define a model where PAG1 directs pSFK localization in responses tailored to different receptors and overall signaling. PAG1 mutation increased SFK activity in cells, but decreased SFK activity in endosomes and reduced cell differentiation. In Chapter 3, to better understand differences in signaling between human neural crest and neuroblastoma, we generated neural crest stem cells from human embryonic stem cells. During differentiation, neural crest cells unexpectedly formed cartilage, a cell lineage derived from neural crest. We investigated the differentiation of craniofacial cartilage and developed a protocol for production of cartilage organoids. Organoids are a promising model for neural crest development as well as a potential source of cartilage for facial reconstructions