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

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

Cyclic AMP receptor protein-aequorin molecular switch for cyclic AMP

Molecular switches are designer molecules that combine the functionality of two individual proteins into one, capable of manifesting an "on/off" signal in response to a stimulus. These switches have unique properties and functionalities and thus, can be employed as nanosensors in a variety of applications. To that end, we have developed a bioluminescent molecular switch for cyclic AMP. Bioluminescence offers many advantages over fluorescence and other detection methods including the fact that there is essentially zero background signal in physiological fluids, allowing for more sensitive detection and monitoring. The switch was created by combining the properties of the cyclic AMP receptor protein (CRP), a transcriptional regulatory protein from E. Coli that binds selectively to cAMP with those of aequorin, a bioluminescent photoprotein native of the jellyfish Aequorea victoria . Genetic manipulation to split the genetic coding sequence of aequorin in two and genetically attach the fragments to the N and C termini of CRP resulted in a hybrid protein molecular switch. The conformational change experienced by CRP upon the binding of cyclic AMP is suspected to result in the observed loss of the bioluminescent signal from aequorin. The "on/off" bioluminescence can be modulated by cyclic AMP over a range of several orders of magnitude in a linear fashion in addition to the capacity to detect changes in cellular cyclic AMP of intact cells exposed to different external stimuli without the need to lyse the cells. We envision that the molecular switch could find applications in vitro as well as In Vivo cyclic AMP detection and/or imaging

Model for the parallel establishment of the zebrafish embryonic melanocyte lineage and the adult melanocyte stem cell lineage.

<p>Our results indicate that there are two distinct melanocyte lineages that develop in the zebrafish embryo: the embryonic or ontogenetic melanocyte lineage and the regenerative and metamorphic melanocyte lineage. In embryonic development, neural crest cells (squares) give rise to <i>dct+</i> melanoblasts (triangles), which later develop into embryonic melanocytes (black star). The regeneration and metamorphic lineage develops in parallel to embryonic melanocytes, prior to 48 hpf and presumably from the neural crest. The establishment of melanocyte stem cells (MSCs, dark circle) require ErbB signaling to progress from the pre-MSC state (light circle). The MSC can self-renew (circular arrow) and give rise to melanoblasts and melanocytes during metamorphosis or regeneration, when inhibition from embryonic melanocytes (block arrow) is relieved. The MSC–derived melanocytes are sensitive to developmental blockade by ICI-118,551 after the <i>dct</i>+ melanoblast stage. Ectopic expression of <i>kitla</i> can recruit the MSC lineage by either inducing MSCs or pre–MSCs resulting in overproliferation of melanocytes.</p

Regeneration requires ErbB signaling before 48 hpf.

<p>(A) Cartoon of drug treatment timeline. (B) Quantitation of average regenerated dorsal trunk melanocytes from somites 5–12 for each treatment in (A) for untreated (gray) and AG1478 treated (blue). Error bars represent standard deviation, * P<0.05, - P>0.05 (Student t-test, N = 10). Time shifts with AG1478 treatment reveal that it blocks regeneration when administered prior to 48 hpf. When AG1478 is added after 48 hpf regeneration is unaffected. The early effect is observed when MoTP treatment is withheld until after AG1478 washout at 48 hpf.</p

The regeneration specific drug, ICI-118,551, reveals that excess melanocytes in <i>PT2hsp70:kitla</i> larvae arise from the MSC lineage.

<p>(A) Cartoon of drug treatment timeline. (B) Quantitative data for ontogeny and regeneration represented as percentage of WT melanocyte numbers. Error bars show standard deviations, * P<0.05, - P>0.05 (Student's t-test, N>7). (C) WT larvae treated with ICI-118,551 develop faintly melanized ontogenetic melanocytes, in contrast to (D) failure to develop melanized melanocytes when challenged to regenerate in the presence of ICI-118,551. (E) <i>In situ</i> analysis reveals regeneration proceeds to the <i>dct</i>+ melanoblast stage (arrows) in the presence of ICI-118,551. (F) ICI-118,551 treated <i>PT2hsp70:kitla</i> embryos develop similar numbers of faintly melanized melanocytes as ICI-118,551 treated embryos shown in (C). Differences in iridophore appearance between (C) and (F) are results of slightly different illumination conditions.</p

ErbB signaling is not required for stem cell self-renewal.

<p>(A) Cartoon of drug treatment timeline for single and double regeneration assays. (B) Quantitation of average regenerated dorsal melanocytes for each treatment in (A). Error bars represent standard deviation, - P>0.05 (Student's t-test, N = 10 for single regeneration, >5 for double regeneration). As previously shown (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000544#pgen-1000544-g002" target="_blank">Figure 2</a>), when larvae are treated with AG1478 after 2 dpf, they regenerate normally following a single round of MoTP treatment. When animals are treated with two rounds of MoTP (from 2–4 dpf and from 5–7 dpf) they regenerate slightly less melanocytes than a single round by 9 dpf. To test whether AG1478 treatment would block stem cell self-renewal, we treated animals with MoTP and AG1478 from 2–4 dpf, and then with a second round of MoTP from 5–7 dpf. These animals regenerated melanocytes similar to non-AG1478 treated larvae for double regeneration (P = 0.9).</p

ErbB signaling is required for larval melanocyte regeneration.

<p>(A) Cartoon of drug treatment timeline for assaying melanocyte ontogeny and regeneration. Arrow indicates when embryos were collected for photos and melanocyte counts, in this case at 168 hpf. (B) Quantitation of average dorsal melanocytes from somites 1–26 for each treatment in (A) for melanocyte ontogeny (gray) and melanocyte regeneration (red). Error bars represent standard deviation, * represents P<0.05 (Student t-test, N = 10). Photos of representative larvae at 168 hpf for ontogeny (C, E, G) and regeneration (D, F, H). WT larvae regenerate nearly completely (compare D to C). WT treated with AG1478 (E, F) and <i>erbb3b</i> mutants (G, H) largely fail to regenerate but have normal ontogenetic number of melanocytes. A few regeneration melanocytes are observed in the head and sporadically in parts of the trunk (black bars in F and H) but are mostly absent throughout the trunk (red bars in F and H).</p