Defects in ErbB-Dependent Establishment of Adult Melanocyte Stem Cells Reveal Independent Origins for Embryonic and Regeneration Melanocytes

Abstract

Adult stem cells are responsible for maintaining and repairing tissues during the life of an organism. Tissue repair in humans, however, is limited compared to the regenerative capabilities of other vertebrates, such as the zebrafish (Danio rerio). An understanding of stem cell mechanisms, such as how they are established, their self-renewal properties, and their recruitment to produce new cells is therefore important for the application of regenerative medicine. We use larval melanocyte regeneration following treatment with the melanocytotoxic drug MoTP to investigate these mechanisms in Melanocyte Stem Cell (MSC) regulation. In this paper, we show that the receptor tyrosine kinase, erbb3b, is required for establishing the adult MSC responsible for regenerating the larval melanocyte population. Both the erbb3b mutant and wild-type fish treated with the ErbB inhibitor, AG1478, develop normal embryonic melanocytes but fail to regenerate melanocytes after MoTP-induced melanocyte ablation. By administering AG1478 at different time points, we show that ErbB signaling is only required for regeneration prior to MoTP treatment and before 48 hours of development, consistent with a role in establishing MSCs. We then show that overexpression of kitla, the Kit ligand, in transgenic larvae leads to recruitment of MSCs, resulting in overproliferation of melanocytes. Furthermore, kitla overexpression can rescue AG1478-blocked regeneration, suggesting that ErbB signaling is required to promote the progression and specification of the MSC from a pre–MSC state. This study provides evidence that ErbB signaling is required for the establishment of adult MSCs during embryonic development. That this requirement is not shared with the embryonic melanocytes suggests that embryonic melanocytes develop directly, without proceeding through the ErbB-dependent MSC. Moreover, the shared requirement of larval melanocyte regeneration and metamorphic melanocytes that develops at the larval-to-adult transition suggests that these post-embryonic melanocytes develop from the same adult MSC population. Lastly, that kitla overexpression can recruit the MSC to develop excess melanocytes raises the possibility that Kit signaling may be involved in MSC recruitment during regeneration. The promise of regenerative medicine lies in the ability to find or create stem cells that can be manipulated to replace damaged tissues and organs. Such ability requires an understanding of how adult stem cells are established and then later recruited to regenerate different tissues. Here, we study the zebrafish's ability to regenerate melanocytes, a pigment cell shared with humans, to understand these mechanisms. We show that the erbb3b gene is required to establish melanocyte stem cells in the embryo that are responsible for regenerating melanocytes after melanocytes are ablated in the larval zebrafish. Because this adult stem cell is not required for the development of embryonic melanocytes, we conclude that adult melanocyte stem cells develop in parallel to the embryonic tissues that they regulate. We also show that overexpressing kit ligand a will result in over-recruiting these adult stem cells to produce excess melanocytes. Further exploration into the mechanisms by which the zebrafish melanocyte stem cells are maintained and recruited will inform how adult stem cells might be manipulated for medical applications.