Small molecule-induced ablation and subsequent regeneration of larval zebrafish melanocytes

Yang, Chao-Tsung; Johnson, Stephen L.

doi:10.1242/dev.02533

Cited by 90 publications

(135 citation statements)

References 54 publications

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“…Cells were initially highly motile and then became mostly stationary by 72 hpf. Melanocytes were continually added to the pattern on the head from ~25 hpf until 96 hpf, although the majority of melanocytes appeared established by 72 hpf, consistent with previous reports (Yang and Johnson, 2006). Crucially, we also noticed a previously undescribed population of melanocytes arising through division of pigmented cells (Fig.…”

Section: Time-lapse Imaging Of Direct-developing Melanocytessupporting

confidence: 91%

“…Skin melanocyte numbers also expand following ultraviolet radiation exposure, and in the mouse these cells appear to arise from proliferative undifferentiated melanoblasts (Kawaguchi et al, 2001;van Schanke et al, 2005;Walker et al, 2009). In zebrafish, most embryonic melanocytes are directly derived from the neural crest, and adult melanocytes are added to the embryonic melanocyte pattern by an expansive wave of development from undifferentiated melanocyte progenitors or MSCs (Hultman et al, 2009;Parichy, 2003;Yang and Johnson, 2006). Thus, in developing animals, melanocyte population numbers are achieved primarily by proliferation of unpigmented melanocyte precursor cells and pigmentation is associated with differentiation without evidence of cell division.…”

Section: Introductionmentioning

confidence: 99%

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Differentiated melanocyte cell division occurs in vivo and is promoted by mutations in Mitf

Taylor

Lister

Zeng³

et al. 2011

Development

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SUMMARYCoordination of cell proliferation and differentiation is crucial for tissue formation, repair and regeneration. Some tissues, such as skin and blood, depend on differentiation of a pluripotent stem cell population, whereas others depend on the division of differentiated cells. In development and in the hair follicle, pigmented melanocytes are derived from undifferentiated precursor cells or stem cells. However, differentiated melanocytes may also have proliferative capacity in animals, and the potential for differentiated melanocyte cell division in development and regeneration remains largely unexplored. Here, we use time-lapse imaging of the developing zebrafish to show that while most melanocytes arise from undifferentiated precursor cells, an unexpected subpopulation of differentiated melanocytes arises by cell division. Depletion of the overall melanocyte population triggers a regeneration phase in which differentiated melanocyte division is significantly enhanced, particularly in young differentiated melanocytes. Additionally, we find reduced levels of Mitf activity using an mitfa temperature-sensitive line results in a dramatic increase in differentiated melanocyte cell division. This supports models that in addition to promoting differentiation, Mitf also promotes withdrawal from the cell cycle. We suggest differentiated cell division is relevant to melanoma progression because the human melanoma mutation MITF 4T⌬2B promotes increased and serial differentiated melanocyte division in zebrafish. These results reveal a novel pathway of differentiated melanocyte division in vivo, and that Mitf activity is essential for maintaining cell cycle arrest in differentiated melanocytes.

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Section: Time-lapse Imaging Of Direct-developing Melanocytessupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Differentiated melanocyte cell division occurs in vivo and is promoted by mutations in Mitf

Taylor

Lister

Zeng³

et al. 2011

Development

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“…mitfa is a master transcription regulator required for both specification and differentiation of melanocytes (27). In the absence of mitfa function, melanocyte precursors are thought to die (27), but specific ablation of melanocytes can trigger regeneration of new melanocytes from surrounding unpigmented precursors (28,29). Therefore, one possible explanation for the lack of mitfa-deficient pigmentation phenotypes in the CRISPR/Cas9 system is that the few remaining mitfa-positive precursor cells surrounding the mitfa-null cells regenerate and mask the mitfa defects.…”

Section: Discussionmentioning

confidence: 99%

Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

Jao¹,

Wente²,

Chen

2013

Proc. Natl. Acad. Sci. U.S.A.

1,194

1,081

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A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.genome engineering | RNA-guided mutagenesis | pigmentation R ecent methodological innovations that exploit zinc finger nucleases (ZFNs) and transcription-activator-like effector nucleases (TALENs) have made it possible to introduce sitespecific genomic modifications in cell culture systems and in a wide range of species (1, 2). These designer nucleases target and cleave specific genomic sequences. Error-prone nonhomologous end joining (NHEJ) DNA repair then generates mutations. The engineering of efficient ZFNs requires extensive technical expertise and empirical testing to find efficient enzymes. The TALEN technology provides an attractive alternative to ZFNs, but like ZFNs, it requires the assembly of two relatively large DNA-binding proteins for each target. Most recently, a new class of genome editing tool based on the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been developed (3).CRISPR systems in eubacteria and archaea use small RNAs and CRISPR-associated proteins (Cas) proteins to target and cleave invading foreign DNAs (4-6). One of the simplest CRISPR systems is the type II CRISPR system from Streptococcus pyogenes: an endonuclease Cas9 and two small RNAs, CRISPR RNA (crRNA) and a transacting RNA (tracrRNA), are sufficient for RNA-guided cleavage of foreign DNAs (7). Recent studies show that a single guide RNA (gRNA) chimera that mimics the crRNA:tracrRNA complex can guide Cas9 to introduce sitespecific DNA double-stranded breaks in vitro (3) and in mammalian cell lines (8-11), bacteria (12, 13), yeast (14), zebrafish (15, 16), and mice (17) with ...

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“…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.…”

Section: Stationary Mps Are Associated With Drgsmentioning

confidence: 99%

“…As the NC is no longer present, these cells must arise from hypothetical NC-derived stem cells that are set aside in the embryo (Rawls et al, 2001;Parichy et al, 2003;Yang and Johnson, 2006;Budi et al, 2011). These cells are quiescent during embryonic development but start to proliferate during juvenile development (Quigley et al, 2004;Mellgren and Johnson, 2004;Hultman et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2013

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SUMMARYPigment cells in vertebrates are derived from the neural crest (NC), a pluripotent and migratory embryonic cell population. In fishes, larval melanophores develop during embryogenesis directly from NC cells migrating along dorsolateral and ventromedial paths. The embryonic origin of the melanophores that emerge during juvenile development in the skin to contribute to the striking colour patterns of adult fishes remains elusive. We have identified a small set of melanophore progenitor cells (MPs) in the zebrafish (Danio rerio, Cyprinidae) that is established within the first 2 days of embryonic development in close association with the segmentally reiterated dorsal root ganglia (DRGs). Lineage analysis and 4D in vivo imaging indicate that progeny of these embryonic MPs spread segmentally, giving rise to the melanophores that create the adult melanophore stripes. Upon depletion of larval melanophores by morpholino knockdown of Mitfa, the embryonic MPs are prematurely activated; their progeny migrate along the spinal nerves restoring the larval pattern and giving rise to postembryonic MPs associated with the spinal nerves. Mutational or chemical inhibition of ErbB receptors blocks all early NC migration along the ventromedial path, causing a loss of DRGs and embryonic MPs. We show that the sparse like (slk) mutant lacks larval and metamorphic melanophores and identify kit ligand a (kitlga) as the underlying gene. Our data suggest that kitlga is required for the establishment or survival of embryonic MPs. We propose a model in which DRGs provide a niche for the stem cells of adult melanophores. KEY WORDS: Kit ligand a, ErbB, Dorsal root ganglia, Melanophore stem cells

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Small molecule-induced ablation and subsequent regeneration of larval zebrafish melanocytes

Cited by 90 publications

References 54 publications

Differentiated melanocyte cell division occurs in vivo and is promoted by mutations in Mitf

Differentiated melanocyte cell division occurs in vivo and is promoted by mutations in Mitf

Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

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

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