Lineage Relationship of Direct-Developing Melanocytes and Melanocyte Stem Cells in the Zebrafish

Abstract

Previous research in zebrafish has demonstrated that embryonic and larval regeneration melanocytes are derived from separate lineages. The embryonic melanocytes that establish the larval pigment pattern do not require regulative melanocyte stem cell (MSC) precursors, and are termed direct-developing melanocytes. In contrast, the larval regeneration melanocytes that restore the pigment pattern after ablation develop from MSC precursors. Here, we explore whether embryonic melanocytes and MSCs share bipotent progenitors. Furthermore, we explore when fate segregation of embryonic melanocytes and MSCs occurs in zebrafish development. In order to achieve this, we develop and apply a novel lineage tracing method. We first demonstrate that Tol2-mediated genomic integration of reporter constructs from plasmids injected at the 1–2 cell stage occurs most frequently after the midblastula transition but prior to shield stage, between 3 and 6 hours post-fertilization. This previously uncharacterized timing of Tol2-mediated genomic integration establishes Tol2-mediated transposition as a means for conducting lineage tracing in zebrafish. Combining the Tol2-mediated lineage tracing strategy with a melanocyte regeneration assay previously developed in our lab, we find that embryonic melanocytes and larval regeneration melanocytes are derived from progenitors that contribute to both lineages. We estimate 50–60 such bipotent melanogenic progenitors to be present in the shield-stage embryo. Furthermore, our examination of direct-developing and MSC-restricted lineages suggests that these are segregated from bipotent precursors after the shield stage, but prior to the end of convergence and extension. Following this early fate segregation, we estimate approximately 100 embryonic melanocyte and 90 MSC-restricted lineages are generated to establish or regenerate the zebrafish larval pigment pattern, respectively. Thus, the dual strategies of direct-development and MSC-derived development are established in the early gastrula, via fate segregation of the two lineages.

On the embryonic origin of adult melanophores: the role of ErbB and Kit signalling in establishing melanophore stem cells in zebrafish

“…Recovering melanoblasts emerge at the DRG and migrate along the spinal nerves Larval melanophores can regenerate if chemically or genetically ablated (Yang and Johnson, 2006;Hultman et al, 2009;Tryon et al, 2011). To investigate whether these newly formed melanophores originated from the putative DRG-associated stem cells, we depleted the directly differentiating melanophores with a mitfamorpholino.…”

Kit signaling is involved in melanocyte stem cell fate decisions in zebrafish embryos

“…This means that integration events can happen before fate segregation, generating both lineages (bipotential labelings), or after fate segregation, labeling only a single lineage. We had shown previously that ~60% of clones were bipotential, and the remainder generated only ontogenetic melanocytes or regeneration melanocytes (Tryon et al, 2011). Under Model 1 (Fig.…”

“…We have previously shown that all lineages that contain an MSC probably come from a neural crest precursor that will also produce an ontogenetic melanocyte lineage (Fig. 5A) (Tryon et al, 2011). Based on this model, we could imagine three possible ways to disrupt MSC establishment: (1) death of the neural crest precursor (Fig.…”

“…To study each process we can generate clones by injecting embryos at the one-to two-cell stage with a transposon plasmid containing GFP driven by the Tyrp1 promoter from fugu (fTyrp1>GFP), which is specifically activated in the melanocyte lineage. Using this protocol, we have shown that transposon integration occurs largely after the midblastula transition, centered on 6 hours post fertilization (Tryon et al, 2011). This generates melanocyte lineage clones, and we can infer the activity of the MSC and its daughters by analyzing the labeled melanocytes arising during regeneration.…”

“…Recently, these methodologies have been used to show the contributions of specific tissue types during complex regeneration events (Gargioli and Slack, 2004;Kragl et al, 2009;Rinkevich et al, 2011;Tu and Johnson, 2011). Additionally, they have been used to elucidate contributions of individual cells within a single tissue during growth, homeostasis and regeneration (Gupta and Poss, 2012;Snippert et al, 2010;Tryon et al, 2011).…”