Department of Genetics, Washington University School of Medicine, 4566 Scott Avenue, St Louis, MO 63110, USA
*Author for correspondence (e-mail: sjohnson{at}sequencer.wustl.edu)
Accepted March 12, 2001
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SUMMARY |
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Key words: Melanocyte, Stem cell, Regeneration, kit, sparse, Conditional, Temperature sensitive, Zebrafish, Danio rerio
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INTRODUCTION |
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Following partial amputation, zebrafish fins rapidly regenerate to replace the missing tissue. Wound healing occurs in the first stage after amputation (1 stage is the equivalent of 1 day of regeneration at 25°C; Johnson and Weston, 1995), followed by recruitment of cells into the cell cycle between stages 1.5 and 2 (Johnson and Bennett, 1999), formation of visible blastemata by stage 3 and outgrowth of the regenerate through completion of regeneration (approximately stage 25-30). Precursors of regeneration melanocytes are observed by in situ hybridization proximal to the amputation plane as early as stage 1 to 1.5, and then distal to the amputation plane by stage 2 to 3. Pigmented de novo melanocytes are first observed throughout the regenerate at stage 4, and continue to differentiate in the distal-most, or developmentally youngest, part of the regenerate, until completion of regeneration. The observation that fish can regenerate their fins and re-establish their melanocyte stripes from undifferentiated cells in the stump through numerous cycles of amputation and regeneration, suggests the existence of melanocyte or pigment cell stem cells (Rawls and Johnson, 2000).
We have previously shown that zebrafish mutant for the kit receptor tyrosine kinase (formerly called sparse; Parichy et al., 1999) fail to develop regeneration melanocytes between stage 4 and stage 7. In the absence of kit function and absence of early regeneration melanocytes, a secondary regulatory class of melanocytes differentiates (starting around stage 8) and eventually re-establishes the fin stripes. Secondary regulatory melanocytes have little or no role in normal stripe regeneration (Rawls and Johnson, 2000), therefore the melanocytes that re-establish the fin stripes during normal regeneration are entirely or almost entirely kit dependent. However, to avoid complications arising from the possible minor contribution of secondary regulatory melanocytes to normal regeneration after stage 8, we now focus on melanocytes that develop prior to stage 7 in investigating the role of kit in regeneration melanocyte development.
In mammals, adult melanocyte pigment pattern is also maintained by stem cells (Kunisada et al., 1998), and loss of kit function similarly causes deficits in adult melanocytes (Silvers, 1979; Geissler et al., 1988; Nocka et al., 1990; Tan et al., 1990; Giebel and Spritz, 1991; Tsujimura et al., 1991; Besmer et al., 1993; Marklund et al., 1998). As kit function is also required for the migration and survival of embryonic neural crest-derived melanocytes and their precursors (Motro et al., 1991; Cable et al., 1995; Wehrle-Haller and Weston, 1995; MacKenzie et al., 1997; Parichy et al., 1999), it has been proposed that adult pigment pattern phenotypes in kit mutants might be due to requirements for kit during early stages of ontogeny to promote the development of adult melanocyte stem cells (Huszar et al., 1991; Wehrle-Haller and Weston, 1995; Yoshida et al., 1996; MacKenzie et al., 1997; Rawls and Johnson, 2000). For example, kit may be required during embryogenesis to establish melanocyte stem cells, or during larval development to maintain melanocyte stem cells. Adult roles for Kit in melanocyte development have been previously suggested by studies in mice, using conditional abrogation of gene function using Kit-directed antibodies (Nishikawa et al., 1991; Kunisada et al., 1998). In the absence of molecular markers for melanocyte stem cells, we sought to distinguish between possible roles for kit during early developmental stages, in establishing or maintaining adult melanocyte stem cells, or during regeneration, in promoting melanocyte differentiation, using a temperature-sensitive mutation of kit.
The utility of temperature-sensitive mutations to remove or restore gene function has been useful in dissecting a variety of biological processes in yeast (Hartwell et al., 1974), worms (Vowels and Thomas, 1992) and flies (Suzuki et al., 1976). As poikilothermic vertebrates such as zebrafish can grow in a wide range of temperatures (Schirone and Gross, 1968), temperature-sensitive mutations in fish can also be identified (Abdelilah et al., 1994; Johnson and Weston, 1995; Winkler et al., 2000). We therefore generated a temperature-sensitive allele of zebrafish kit in order to assess the temporal requirements of kit. We show that kit is required after fin amputation to promote the population of the regenerate by melanoblasts, rather than during earlier developmental stages to establish melanocyte stem cells. As early regeneration melanocytes do not form in the absence of kit function, we also used the temperature-sensitive kit mutant to determine the role that kit plays in these cells following differentiation. These studies revealed a transient role for kit in promoting the survival of differentiated regeneration melanocytes.
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MATERIALS AND METHODS |
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Non-complementation screen for temperature-sensitive kit alleles
Pre-meiotic ENU-mutagenesis (Solnica-Krezel et al., 1994) was performed on wild-type SJD males. Mutant clones produced after such treatment typically account for less than 0.1% of sperm from individual males (S. L. J., unpublished). Pooled sperm from subsets of 45 mutagenized males was used to fertilize eggs from homozygous kitb5 females in a C32 background, and progeny were initially reared at 33°C. Non-complementing embryos displaying the kit mutant embryonic melanocyte phenotype (Parichy et al., 1999) were shifted to 25°C and reared to maturity for further analysis. Over 83,000 embryonic progeny were screened in this manner, yielding 247 non-complementing mutants. Temperature sensitivity of new mutants was assessed by backcrossing founders to homozygous kitb5 testers, splitting the clutch in half between the permissive temperature (25°C) and the restrictive temperature (33°C). Clutches with approximately 50% kit mutant phenotype embryos and 50% wild-type embryos at 25°C, and 100% kit mutant phenotype at 33°C were considered to be temperature sensitive. Sixty different founders, most of them presumably identifying independent alleles, were screened in this manner. From these, six temperature-sensitive kit alleles were identified and five of these alleles were recovered. Although it remains possible that these temperature-sensitive kit alleles are not independent, the lack of duplicated lesions in the 10 kit alleles so far sequenced from this screen (Parichy et al., 1999; this study; J. F. R. and S. L. J., unpublished) suggests that this possibility is remote.
Quantitative assessment of regeneration melanocyte survival
Fins regenerated at 25°C until upshift to 33°C at stage 7, 11, 15 or 20. At the time of upshift, animals were treated with 1 mg/ml epinephrine for 5 minutes to contract melanosomes and facilitate cell counting (Johnson et al., 1995; Rawls and Johnson, 2000; Sugimoto et al., 2000) and photographed. Following upshift to 33°C, fish were maintained in 0.2 mM phenylthiourea (PTU) to block melanin synthesis and therefore inhibit pigmentation of any new melanocytes (Milos and Dingle, 1978; Rawls and Johnson, 2000). After six stages at 33°C, fish were again treated with epinephrine and photographed. The rate of melanocyte survival during these treatments was assessed by dividing the number of melanocytes in the presumptive central stripe in an individual regenerate after six stages at 33°C, by the number of melanocytes in the presumptive central stripe at the time of upshift (7-10 individuals per timepoint). We define the presumptive central stripe as the region of the regenerate distal of the central stripe in the stump. As the pigmentation of further de novo melanocytes is inhibited with PTU and migration of differentiated regeneration melanocytes is minimal (C. Beckett and S. L. J., unpublished), no further melanocytes become located in the presumptive central stripe following upshift for the duration of the experiment.
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RESULTS |
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To determine if kit is required during regeneration to recruit melanocyte precursors to form differentiated melanocytes, we amputated fins from kitj1e99 animals reared at the permissive temperature, and then shifted them to the restrictive temperature. These regenerates failed to develop de novo melanocytes by stage 7 (Fig. 1F), indicating that kit is required after amputation to promote development of regeneration melanocytes.
kit is required after stage 2 to promote population of the regenerate by melanoblasts
To determine the role of kit during regeneration in promoting the development of de novo melanocytes at stage 4, we first observed the location of melanoblasts in wild-type and mutant regenerating fins using in situ hybridization. During wild-type fin regeneration, kit-expressing presumptive melanoblasts are located in the stump and at the amputation plane as early as stage 1.5 (Rawls and Johnson, 2000). By stage 4 in wild-type regenerates, kit-expressing melanoblasts have typically migrated into the regenerate and have begun expressing melanin (Fig. 2A). In contrast, we detected kit-expressing melanoblasts in the stump and at the amputation plane but not in the regenerate in stage 4 kitj1e99 mutants held at the restrictive temperature (Fig. 2B). This shows that kit is required to promote population of the regenerate by kit-expressing melanoblasts, and is consistent with possible roles for kit in migration of melanoblasts into the regenerate as well as the subsequent survival of melanoblasts in the regenerate prior to differentiation. As previously described (Rawls and Johnson, 2000), kit transcript was undetectable in kitb5 homozygotes (Fig. 2C). Presumably this failure to detect kitb5 transcript was due to degradation of the mutant transcript via nonsense-mediated mRNA decay (Culbertson, 1999) caused by the premature stop codon encoded in the kitb5 lesion (Parichy et al., 1999).
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Requirement for kit extends until late stages of melanocyte differentiation
In a reciprocal series of temperature shifts, kitj1e99 regenerates shifted from the permissive temperature to the restrictive temperature as late as stage 3.5 failed to develop regeneration melanocytes (Fig. 2E). In shifts to the restrictive temperature at stage 4, when some regeneration melanocytes had already developed at the permissive temperature prior to the upshift, few or no additional melanocytes developed following the shift to the restrictive temperature (through stage 7; not shown). These results suggest that a temporal requirement for kit extends until late stages of melanoblast differentiation, perhaps as late as onset of pigmentation. That this requirement persists beyond stage 4 in fin regeneration presumably reflects the continuous differentiation of new melanocytes as the fin regenerate grows.
Consistent with a role for kit in the continuous formation of new melanocytes through later stages of regeneration, we find that regeneration melanocyte precursors in kitj1e99 regenerates at the restrictive temperature remain competent to respond to restoration of kit function. In kitj1e99 animals reared at the restrictive temperature and then shifted to the permissive temperature during later stages of regeneration (stages 7-11), de novo melanocytes subsequently appeared in distal positions in the regenerate (not shown). Notably, these new melanocytes consistently appeared within two stages of downshift to the permissive temperature, but never earlier than stage 4, which is when regeneration melanocytes normally form. This shows that kit is required continuously for new melanocyte differentiation, and suggests that melanoblasts or their precursors remain competent to respond to kit function beyond stage 4, which is when they would normally complete pigmentation.
kit is required for survival of differentiated regeneration melanocytes
Because regeneration melanocytes fail to form in the absence of kit function, the role of kit in regeneration melanocytes following differentiation was unknown. As larval melanocytes in kit mutants undergo programmed cell death and extrusion from the animal within 6 days of differentiation (Parichy et al., 1999), we hypothesized that differentiated regeneration melanocytes would also require kit function for survival. To test this model, we permitted kitj1e99 fish to develop regeneration melanocytes normally at the permissive temperature until specific stages of regeneration, when they were shifted to the restrictive temperature to remove kit function. To monitor regeneration melanocyte survival, fish were maintained in the presence of the melanin synthesis inhibitor, phenylthiourea (PTU; Milos and Dingle, 1978; Rawls and Johnson, 2000), after the shift to the restrictive temperature. This enabled us to follow selectively those melanocytes that were differentiated prior to PTU treatment and upshift. kitj1e99 fins regenerating at the permissive temperature formed typical dendritic melanocytes throughout the dermis of the regenerate by stage 7 (Fig. 3A). However, shifting those fish to the restrictive temperature caused regeneration melanocytes to undergo typical teleost melanocyte cell death (Parichy et al., 1999; Sugimoto et al., 2000), as evidenced by contraction of their dendritic processes and displacement from the dermis into the epidermis by stage 10 (Fig. 3C), and their disappearance from the fin by stage 13 (Fig. 3B). This shows that kit is required for the survival of differentiated regeneration melanocytes, in addition to the aforementioned requirement for their differentiation.
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DISCUSSION |
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Conditional mutations facilitate the dissection of known mutant phenotypes, as well as uncover later roles for genes in cell types that are missing in constitutively mutant animals. Since kit-dependent regeneration melanocytes fail to develop in kit null mutants, we were able to assess late roles for kit in the temperature-sensitive mutant by allowing regeneration melanocytes to develop first at the permissive temperature before removing kit function by shifting to the restrictive temperature. Temperature shift experiments revealed a transient requirement for kit in promoting the survival of differentiated regeneration melanocytes. Because de novo melanocytes first appear at stage 4, and the first regeneration melanocytes to acquire kit independence between stages 7 and 11 are the developmentally oldest, we infer that the first regeneration melanocytes to pigment at stage 4 subsequently acquire kit independence between stages 7 and 11 (Fig. 5). While acquisition of both growth factor and growth factor receptor independence in neuronal development has been well documented in vivo by disrupting gene function with neutralizing antibodies (Johnson et al., 1980; Schwartz et al., 1982), the mechanisms that underlie this phenomenon remain largely unknown. Although dependence on kit and its growth factor ligand, steel, for melanocyte precursor survival and subsequent acquisition of independence has been described (Nishikawa et al., 1991; Morrison-Graham and Weston, 1993; Yoshida et al., 1996), our results now demonstrate transition to kit-independence in differentiated melanocytes, as revealed by genetic manipulation.
Our finding that kit is required only during post-stem cell stages in the zebrafish melanocyte lineage is in contrast to the temporal roles for murine Kit during germ cell development. In the germ cell lineage, Kit and its ligand have been shown to be required early in ontogeny to establish primordial germ cells in the gonad, as well as during later stages of germ cell maintenance and differentiation (see Sette et al., 2000). Interestingly, the temporal requirements for zebrafish kit in the melanocyte lineage are similar to the requirements for Kit during murine hematopoietic development. In the hematopoietic lineage, kit and its ligand are not required to establish totipotent hematopoietic stem cells, but are required later in development to promote their maintenance and recruitment into specific developmental pathways (see Broudy, 1997). As zebrafish kit is not required in the germ cell or hematopoietic lineages and kit-null mutants are therefore fertile and viable (Parichy et al., 1999), genetic analysis of kit-dependent pathways in the zebrafish melanocyte lineage may inform future studies of Kit-dependent processes in mammals.
Transgenic techniques for generating conditional mutations have been effective for analysis of temporal gene requirements in mice and flies (for example, see Shin et al., 1999), yet such tools have not yet been developed for the zebrafish. However, the ability of this poikilothermic vertebrate to live at a fairly wide range of temperatures (Schirone and Gross, 1968) permits the conditional disruption of gene function using temperature-sensitive mutations (Abdelilah et al., 1994; Johnson and Weston, 1995). The frequency of temperature-sensitive alleles found at the kit locus described in this study (6/60 or 10%) is consistent with frequencies of temperature-sensitive mutations in other model systems (Suzuki et al., 1967; Bartel and Varshavsky, 1988). Therefore, the frequency of temperature-sensitive mutations in zebrafish is sufficiently large to prompt screening for temperature-sensitive mutations in forward genetic screens (Johnson and Weston, 1995; J. F. R. and M. R. F., unpublished).
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ACKNOWLEDGMENTS |
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