Clonal Association between the Three Pigment Cell Types

(A) Adult zebrafish and (A′) close-up of the striped pattern on zebrafish trunk.

(B-H) Types of pigment cell clones in young adult zebrafish carrying Tg(sox10:ER^T2-Cre) and Tg(βactin2:loxP-STOP-loxP-DsRed-express): (B) a single melanophore (M), (C) iridophores (I), (D) xanthophores (X), (E) melanophore and iridophore (MI), (F) melanophore and xanthophore (MX), (G) iridophore and xanthophore (IX; note that this is the only IX that we obtained), and (H) melanophore, iridophore, and xanthophore (MIX) clones. Representative images in (B)-(G) (DsRed/bright field) are from clones obtained from Cre activation at 15 dpf.

(I) Quantification of the clonal association between pigment cell types obtained by Cre activation at 16 hpf (n = 48 clones from 43 animals, median standard length [SL] at the time of image acquisition = 20.8 ± 1.84 mm); 5 dpf (n = 54 clones from 36 animals, median SL at the time of image acquisition = 24 ± 1.77 mm); 15 dpf (n = 112 clones from 74 animals, median SL at the time of image acquisition = 21 ± 1.77 mm); and 21 dpf (n = 38 clones from 23 animals, median SL at the time of image acquisition = 20 ± 2.07 mm).

(J) Schematic representation of the segregation of pigment cell fates.

N, neuron; M, melanophore; I, iridophore; X, xanthophore.

Insets in (B)-(D) are blow-ups. Scale bars represent 5 mm (A) and 250 µm (B-H). See also Figures S1 and S2 and Tables S1 and S2.

Shared Multipotent Progenitors for Pigment Cells and Neural Cells

(A-A5) Example of a clone with labeled neurons and pigment cells in the trunk skin, scales, and dorsum. Dashed curves in (A) show scale pigmentation. Dashed squares in (A2) show regions enlarged in (A3)-(A5). Scale bar, 500 µm.

(B) Quantification of the clonal association between pigment cells and neural cells.

(C and D) A clone in the skin of 20-dpf zebrafish with DRG (asterisk) and neurons. Scale bar, 100 µm.

(E) Xanthophores, iridophores, and melanophores appear in this clone at 25 dpf. Asterisk indicates the DRG. Scale bar, 100 µm.

N, neuron; M, melanophore; I, iridophore; X, xanthophore.

Long-Term Imaging of Progenitors that Make Neurons and Three Pigment Cell Types

(A-L′) Repeated imaging of a clone induced at 5 dpf in zebrafish carrying Tg(sox10:ER^T2-Cre) and Tg(βactin2:loxP-STOP-loxP-DsRed-express). Grayscale in (A)-(L): clone; Red/gray scale in (A′)-(L′): clone/bright field. Asterisks indicate the DRG. Yellow arrows indicate nerve routes, unless indicated otherwise; blue arrows and arrowheads show undifferentiated progenitors; yellow arrowheads in (E)-(H′) show undifferentiated progenitors that make melanophores; dashed curves indicate pigment cells of a scale. Scale bars, 100 µm. See also Figure S3.

Regional Distribution of Clonally Derived Pigment Cells

(A-D) Clones in young adult animals obtained from Cre activation at 15 dpf (genotype Tg(sox10:ER^T2-Cre)/+; Tg(βactin2:loxP-STOP-loxP-DsRed-express)/+) show variability in clone span along the dorsoventral body axis: the clonally derived pigment cells may predominantly contribute to pigment cells of the (A) dorsal, (B) lateral, or (C) ventral regions. Scale bars represent 250 µm. (D) Quantification of the extent of the clones along the dorsoventral body axis; each line represents the dorsoventral span of a single clone. Color code indicates clone type according to pigment cell composition. MIX clones tend to be larger than clones containing only a single pigment cell type. D, dorsal; V, ventral; X0, first light stripe.

(E) Quantification of the number of labeled melanophores in each clone; each line represents the number of melanophores in an individual clone. Color code indicates the dark stripe: blue, 1D; brown, 1V; green, 2V.

Neural Crest-Derived Progenitors are Multipotent but Do Not Have Stereotypic Outcome

(A-F) Clones in young adult animals obtained from Cre activation at (A, A′, D, G) 16 hpf, (B, B′, E, H) 5 dpf, and (C, C′, F, I) 21 dpf (genotype Tg(sox10:ER^T2-Cre)/+; Tg(βactin2:loxP-STOP-loxP-DsRed-express)/+) reveal a progressive reduction in the size and span of the clonally derived pigment cells, albeit several clones remain MIX, suggesting existence of multipotent progenitors at all time points. (D-F) Quantification of the extent of the clones along the dorsoventral body axis; each line represents dorsoventral span of a single clone. Color code shows clone type according to pigment cell composition.

(G-I) Quantification of the number of labeled melanophores in each clone; each line represents the number of melanophores in an individual clone. Color code indicates the dark stripe: blue, 1D; brown, 1V; green, 2V.

(J1-J5) Skin of F₀ adults from embryos injected with albino knockout CRISPR.

Scale bars in (A)-(C′) represent 500 µm. See also Figure S4.

Progenitors Increase in Number between Embryogenesis and Metamorphosis; Proliferation Ability Diminishes with Fate Restriction

(A-D) Tg(sox10mG) expression in zebrafish trunk at (A) 16 hpf, (B) 5 dpf, (C) 15 dpf, and (D) 21 dpf. Asterisk indicates DRG; arrows point to sox10-positive cells along the nerve tracts. Gray, membrane-tagged GFP; red, nuclear RFP.

(E) Schematic representation of the association between pigment cell progenitors and peripheral neurons.

(F-I) Committed melanoblasts generate a small number of melanophores. Melanophore distribution in (F) MIX, (G) MI, and (H) M clones. (I) Quantification of the number of labeled melanophores; each dot represents the number of labeled melanophores in an individual clone.

Scale bars represent 100 µm in (A)-(D) and 250 µm in (F)-(H). See also Figures S5 and S6.

Dorsoventral Organization of Clones in Pigmentation Mutants

(A-I) Clones in the background of (A-C) sparse/kita lacking a subset of melanophores (n = 18 clones), (D-F) shady/ltk lacking iridophores (n = 20 clones), and (G-I) pfeffer/csf1ra lacking xanthophores (n = 20 clones). Scale bars, 250 µm. See also Figure S7.

Cre-activation leads to induction of small clones.

(A-H) examples of clones obtained on 16 dpfupon ere-activation on 15 dpfin fish carrying Tg(so10:ER^T2-Cre) and Tg(βactin2:loxP-STOP-loxP-DsRed-express). 8 clones were obtained after screening 101 fish. Scale bars = 100 µm.

Pigment cells can be identified by their shape, color and location in the skin.

(A) Schematic representation of the layered arrangement of the three pigment cell types that results in appearance of distinct light and dark stripe regions. (B) Pigment cells of the light stripe region - (B1-B1′′) xanthophores; dashed square indicates xanthophore that is enlarged in B2-B2′′, (B3-B3′′) dense and silvery s-iridophores. (C) Pigment cells of the dark stripe region - (C1-C1′′) xanthophores, (C2-C2′′) loose and blue s-iridophores, (C3-C3′′) melanophores - note the black melanin pigment, (C4-C4′′) elongated L-iridophores. In Band C: leftmost panel- fluorescence; middle panel - bright field; rightmost panel - merge. (D-D1′) Iridophores (I) and xanthophores (X) of the light stripe are distinguished by their characteristic shape. Genotype - Tg(soxlO:ER^T2-Cre)/+; Tg(βactin2:loxP-STOP-loxP-DsRed-express)/+. Scale bars = 100 µm.

Nerve-associated progenitors make scale pigmentation.

(A-O′) Repeated imaging of a clone induced on 5 dpf in zebrafish carrying Tg(sox10:ER^T2-Cre) and Tg(βactin2:loxP-STOP-loxP-DsRed-express) reveal production of scale pigmentation from nerve-associated progenitors that produce pigment cells of the body. Blue arrows: neuronal branches; yellow arrows: scale melanophores and their progenitors; asterisks: undifferentiated progenitors that produce scale xanthophores; arrowheads: iridophores; dpf; days post-fertilization. Dashed lines indicate pigment cells of a scale. Panels (A-D′) are reproduced from Singh et ai, 2014. Scale bars = 100 µm.

Melanophore quantification in wild-type.

(A) Skin of a wild-type young adult zebrafish (22 mmSL, 81 dpf). (B) Graph showing number of melanophores per segment from five different animals. Color code indicates the dark stripe: blue - 1D; brown - 1V; green - 2V Melanophores were counted in the region between the pectoral fin and the anal fin.

Clones with labeled melanophores obtained from Cre-activation.

(A1-A3′) Cre-activation at 16 hpf. (A1-A1′) MIX clone, (A2-A2′) MX clones, (A3-A3′) MI clone, and (A4-A4′) M clone. (B1-B3′) Cre-activation at 15 dpf. (B1-B1′) MIX clone, (B2-B2′) MI clone, (B3-B3′) M clone. MX clones are not shown. (C1-C3′) Cre-activation at 21 dpf. (C1-C1′) MIX clone, (C2-C2′) MI clone, (C3-C3′) M clone. N: nerves; M: melanophore; I: iridophore, X: xanthophore. Scale bars = 250 µm.

Committed iridophore and xanthophore clones.

Iridophore clones obtained from Cre-activation at (A1-A2) 5dpf, (B1-B2) 15 dpf, and (C1-C2) 21 dpf. Xanthophore clones obtained from Cre-activation at (D) 16 hpf, (E) 5dpf, (F) 15 dpf, and (G) 21 dpf. (H) Quantification of the number of labelled xanthophores - each dot represents the number of labelled xanthophores in an individual clone. Scale bars = 250 µm.

Long-term imaging of clones in shady (ltk) and pfeffer(csf1ra).