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Requirements for the kit receptor tyrosine kinase during regeneration of zebrafish fin melanocytes

Department of Genetics, Washington University School of Medicine, 4566 Scott Avenue, St Louis, MO 63110, USA

View larger version (142K): [in a new window]   Fig. 1. Requirement for kit function in regeneration melanocyte development in regenerating fins. Shown are stage 7 regenerates from (A) wild type, (B) temperature-sensitive kit mutant (kitj1e99) reared and regenerated constitutively at 25°C, (C) kitj1e99 fish reared at 33°C and shifted to 25°C upon amputation, (D) kitb5-null mutant, (E) kitj1e99 mutant reared and regenerated constitutively at 33°C and (F) kitj1e99 mutant reared at 25°C and shifted to 33°C upon amputation. Regeneration melanocytes are indicated by black arrows. Amputation planes are indicated by black arrowheads, with distal to the right of figures. In Figures 1-3, red lines and arrows represent growth at 33°C, blue lines and arrows represent growth at 25°C, and associated small black arrowheads represent time of amputation. Scale bars: 200 µm.

View larger version (153K): [in a new window]   Fig. 2. Temporal requirement for kit function in regeneration melanocyte development. In situ hybridization detects kit-expressing melanoblasts (white arrowheads) in stage 4 regenerates in (A) wild type and (B) kitj1e99 mutants regenerated at 33°C, but not in (C) kitb5-null mutants (Parichy et al., 1999). (D) Histogram shows average number of pigmented melanocytes in stage 4 regenerates of kitj1e99 mutants held constitutively at 25°C (blue), at 33°C for stages 0-2 and then shifted to 25°C for remainder of experiment (black), at 33°C for stages 0-3 and then shifted to 25°C (white), and held constitutively at 33°C (red). Melanocytes indicated by white and red bars consist largely of cells that were pigmented prior to amputation and subsequently migrated into the proximal regenerate (see Rawls and Johnson, 2000). Error bars show 95% confidence intervals. (E) Stage 6 kitj1e99 regenerate shifted from 25°C to 33°C at stage 3.5. Amputation planes are indicated by black arrowheads, with distal to the right of figures. Scale bars: 20µm (A,B,C), 200µm (E).

View larger version (70K): [in a new window]   Fig. 3. Transient requirement on kit for differentiated melanocyte survival. kitj1e99 mutant regenerating fin photographed at stage 7 following regeneration at 25°C (A), and immediately shifted to 33°C and later photographed at stage 13 (B). Note most regeneration melanocytes are lost by stage 13, with persistent melanocytes (black arrows in (B) and (E)) located only in the most proximal region of the regenerate (indicated by green bars in A,B,D,E). (C) Longitudinal section of a stage 10 kitj1e99 regenerate shifted to 33°C at stage 7 shows punctate distal melanocytes in the epidermis (white arrowheads), and proximal dermal dendritic melanocytes (black arrows). Basement membrane is indicated by an asterisk. (D,E) kitj1e99 mutant regenerating fin photographed at stage 11 following regeneration at 25°C (D), immediately shifted to 33°C at stage 11 and later photographed at stage 17 (E). Note that region of persistent melanocytes (green bars) has expanded distally. Amputation planes are indicated by black arrowheads, with distal to the right of figures. Scale bars: 200 µm (A,B,D,E), 20 µm (C).

View larger version (26K): [in a new window]   Fig. 4. Regeneration melanocytes acquire kit-independence as they mature during late stages of regeneration. Melanocyte survival rates (y-axis) from kitj1e99 (black) and wild-type regenerates (red) shifted from 25°C to 33°C at the stages of regeneration indicated (x-axis), or kitj1e99 held constitutively at 25°C (blue) are shown. To document the acquisition of kit-independence by regeneration melanocytes, melanocytes in the presumptive central stripe of caudal fins regenerating at 25°C were counted at the stages of regeneration indicated. The fish were then moved into PTU to inhibit the appearance of new melanocytes, and either shifted to 33°C or maintained at 25°C for six subsequent stages. The final number of melanocytes in the presumptive stripe from an individual regenerate after six stages was assessed and then divided by the initial number of melanocytes to determine the percent survival rate. The persistent melanocytes in kitj1e99 regenerates following upshift at stage 7 (black; 38%) consist largely of cells which were pigmented prior to amputation and subsequently migrated into the proximal regenerate (not shown). Error bars show 95% confidence intervals. (See Materials and Methods for further details.)

View larger version (11K): [in a new window]   Fig. 5. Temporal requirement for kit in regeneration melanocyte development. Requirement for kit in development of melanocytes that develop first at stage 4 in regenerating wild-type fins. Melanocytes stem cells (MSC) are presumably established during embryonic development and persist through adult stages. After fin amputation (amp.), melanocyte stem cells are recruited to form melanoblasts (MB) by regeneration stage 2. Melanoblasts differentiate into melanocytes (MC) by stage 4, as evidenced by their expression of melanin (indicated by shaded area). Minimal stages of kit requirement are spanned by a black bar, with maximal possible stages of kit requirement spanned by error bars. Requirement for kit in the regeneration melanocyte lineage begins between regeneration stages 2 and 3 and extends beyond the onset of melanin synthesis at stage 4. kit is required for survival of stage 4 regeneration melanocytes until they acquire kit-independence between stages 7 and 11. Regeneration melanocytes which pigment later than stage 4 require kit for a similar duration of time before and after their differentiation (not shown). Note that kit is not required during embryonic or adult stages to promote the establishment or maintenance of melanocyte stem cells.