Premigratory and Migratory Neural Crest Cells are Multipotent In Vivo and The Roles of Sall4 in Melanoma

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The neural crest (NC) is a transient embryonic stem/progenitor cell population and a hallmark of vertebrate development. The NC is induced during neurulation at the border between the neuronal and non-neuronal ectoderm. Upon induction NC cells undergo an epithelial to mesenchymal transition (EMT), delaminate from the neural folds and migrate extensively through the developing embryo. NC cells give rise to a broad variety of cell types including the sensory and autonomic neurons of the peripheral nervous system, the myelinating Schwann cells and the melanocytes, among others. Despite the broad differentiation potential of NC cells it has been highly debated whether the NC consisted of multipotent cells or whether it was rather a heterogeneous population of restricted progenitors. In fact, although several earlier studies have described multipotency of NC cells, the existence of multipotent NC cells has been questioned both by cell culture experiments and in recent in vivo studies performed in chick embryos. In this study, we solved a longstanding controversy regarding a pivotal question in the field of stem cells. Using genetic lineage tracing in the mouse, we revealed for the first time the broad developmental potential of NC cells in a mammalian system and we demonstrated that the majority of premigratory and migratory NC cells are multipotent in vivo. Embryogenesis and tumorigenesis share several mechanisms in common and it has become more and more evident that knowledge in developmental biology can provide further insights into tumor biology. For instance, melanoma, a malignancy of NC-derived melanocytes, can exploit various NC developmental programs for disease progression. Malignant melanoma cells can indeed aberrantly activate EMT master regulators, such as members of the Snail, Twist and Zeb families, which are normally activated by NC cells during migration in the embryo. In the course of melanoma progression malignant cells brake through the basament membrane, invade the underlying mesenchyme, reach blood and lymphatic vessels and metastasize to distant organs. Moreover, similar to NC cells, melanoma cells can possess multipotency features and be able to express different lineage markers. We discovered that the zinc-finger transcription factor Sall4 is expressed in NC cells and it is downregulated upon differentiation. Sall4 is a crucial factor for the maintenance of self-renewal and pluripotency of embryonic stem cells. Moreover, SALL4 has been associated with tumorigenesis and to worse patient outcome in various cancer types. However, whether SALL4 may also play a role in melanoma 2 formation and progression has not been addressed so far. We observed that SALL4 was mostly expressed in human proliferative melanoma cell lines, while it was absent in more invasive melanoma cell lines or upon EMT induction. Interestingly, SALL4 downregulation induced, in turn, an EMT signature in a proliferative melanoma cell line, suggesting that there may be a regulatory feedback loop. In vivo we induced Sall4 loss in the Tyr::NRasQ61K Ink4a-/- Tyr::CreERT2 Sall4lox/lox R26R::GFP melanoma mouse model and could observed that primary tumor formation was impaired. However, Sall4 loss was linked to a reduced survival of the knock out animals and recombined cells were detected in lymph nodes and in some lungs. Further investigations now urge to be performed to prove whether Sall4 loss is necessary and sufficient for EMT induction.