Project description:The epithelial-to-mesenchymal transition (EMT) and migration of cranial neural crest cells are critical processes that occur in the early embryo that permit proper craniofacial patterning. Disruptions in these processes not only impair development but also lead to various diseases, underscoring the need for their detailed understanding at the molecular level. The chick embryo has served historically as an excellent model for human embryonic development. While chick cranial neural crest cell EMT and migration have been characterized at the transcript level, studies at the protein level—to allow direct measurement of the active players—have not been undertaken to date. In this study, we applied mass spectrometry (MS)-based proteomics to establish a deep proteomics profile of the midbrain region during early embryonic development. We developed a proteomics method combining optimal lysis conditions and offline fractionation with nanoflow liquid chromatography coupled to high-resolution MS to analyze the tissue from this region, which identified >5,900 proteins involved in key pathways related to neural crest cell EMT and migration such as signaling, proteolysis/extracellular matrix (ECM), and transcriptional regulation. This study offers valuable insight into important developmental processes occurring in the midbrain region and demonstrates the utility of proteomics for characterization of various tissues during chick embryogenesis.
Project description:Neural crest cells are multipotent cells that delaminate from the neuroepithelium, migrating throughout the embryo. Aberrant migration causes developmental defects. Animal models are improving our understanding of neural crest anomalies, but in vivo migration behaviours are poorly understood. Here, we demonstrate that murine neural crest cells display actin-based lamellipodia and filopodia in vivo. Using neural crest-specific knockouts or inhibitors, we show that the serine-threonine kinase Glycogen Synthase Kinase-3 (GSK3), and the cytoskeletal regulator Lamellipodin (Lpd), are required for lamellipodia formation whilst preventing focal adhesion maturation. Lpd is a novel substrate of GSK3 and phosphorylation of Lpd favours interactions with the Scar/WAVE complex (lamellipodia formation) at the expense of VASP and Mena interactions (adhesion maturation and filopodia formation). This improved understanding of cytoskeletal regulation in mammalian neural crest migration has general implications for neural crest anomalies and cancer.
Project description:Neural crest cells are a transient embryonic population, hence are neither present after bith and nor are they readily accesible for analysis. Therefore, little is known about the genetic networks that regulate NC especification, delamitation and migration from the dorsal neural tube to their final destination along the embryo. Here we have developed a novel resource for lineage tracing and for the isolation of neural crest cells in the chick embryo, enabling us to perform a genome-wide expression screen in neural crest progenitors versus neuroepithelial cells.
Project description:Recent studies suggest that malignant melanoma heterogeneity includes subpopulations of cells with features of multipotent neural crest (NC) cells. Zebrafish and mouse models have shown that reactivation of neural crest-specific pathways determines the invasiveness of melanoma cells. In this study, we show that the neural crest-associated transcription factor FOXD1 plays a key role in invasion and migration capacity of metastatic melanomas both in vivo and in vitro. Gene expression profiling analysis identified on one hand, an upregulation of FOXD1 in NC and melanoma cells, and on the other hand, a downregulation of several genes related to cell invasion in FOXD1 knockdown cells, including MMP9 and RAC1B. Furthermore, we demonstrate that knockdown of RAC1B a tumor-specific isoform of RAC1, significantly impaired cell migration and invasion in melanoma and could abrogate enhanced invasiveness induced by FOXD1 overexpression. We conclude that FOXD1 may influence invasion and migration via indirect regulation of MMP9 and RAC1B alternative splicing in melanoma cells.