www.sciencemag.org/cgi/content/full/science.1254837/DC1

Supplementary Materials for

Local reorganization of xanthophores fine-tunes and colors the striped

pattern of zebrafish

Prateek Mahalwar, Brigitte Walderich, Ajeet Pratap Singh,* Christiane Nüsslein-

Volhard*

*Corresponding author. E-mail: ajeet.singh@tuebingen.mpg.de (A.P.S.); christiane.nuesslein-

volhard@tuebingen.mpg.de (C.N.-V.)

Published 28 August 2014 on Science Express

DOI: 10.1126/science.1254837

This PDF file includes:

Materials and Methods

Figs. S1 to S11

Caption for Movie S1

Full Reference List

Other Supplementary Material for this manuscript includes the following:

(available at www.sciencemag.org/cgi/content/full/science.1254837/DC1)

Movies S1

2

Materials and Methods

Zebrafish lines: The following zebrafish lines were used: Wild-type (WT, Tübingen strain from

the Tübingen zebrafish stock center), albino (21), nacre (22), pfeffer (23), Tg(fms:GAL4) (13),

Tg(UAS:E1b:nfsB.mCherry) (13), Tg(UAS:Cre) (transgenic line from Alessandro Mongera, C.N-

V. lab), Tg(UBI:loxp-EGFP-loxp-mCherry) (24), Tg (pax7:GFP) (transgenic line from Sören

Alsheimer, C.N-V. lab), Tg(UAS:EGFP-CAAX) (25). Tg(sox10:ERT2

-Cre) (14), Tg(sox10:Cre)

(16), Tg(βactin2:loxP-STOP-loxP-DsRed-express) (26). Zebrafish were raised as described

previously (27). Staging of metamorphic zebrafish was done as described by Frohnhöfer et al (3).

Different methods of labelling and lineage-tracing xanthophores: Xanthophores were

labelled using the following transgenic lines: Tg(fms:Gal4.VP16),Tg(sox10:Cre), Tg (pax7:gfp)

and Tg(sox10:ERT2

-Cre) in combination with appropriate UAS and loxp reporter lines described

below.

Tg(fms:Gal4.VP16) fish were crossed with the following reporter lines to drive fluorophore

expression in xanthophores: Tg(UAS:E1b:nfsB.mCherry), Tg(UAS:EGFP-CAAX), and

Tg(UAS:Cre); Tg(βactin:loxp-STOP-loxp-DsRed).

Tg(sox10: Cre) was used in combination with Tg(UBI:loxp-EGFP-loxp-mCherry) for the

analysis of xanthophore clusters. sox10 promoter labels a large set of neural crest-cell derivatives

including xanthophores (fig S5). Animals with patchy labelling of xanthophore clusters were

selected for long-term imaging and analysis.

To get small clusters of labelled xanthophores, chimeric animals were generated by

transplantation of Tg(pax7:GFP) positive cells into albino host embryos at blastula stage (28).

The number of transplanted cells was estimated to be in the range between 1 – 10 cells. On an

average, three xanthophore clusters per fish were obtained. Fish were raised and analysed at

different timepoints throughout metamorphosis until adulthood.

3

Cre induction: 4-5 dpf old zebrafish larvae carrying a single copy of Tg(sox10:ERT2

-Cre) and

Tg(βactin2:loxP-STOP-loxP-DsRed-express) were treated with 5µM 4-hydroxytamoxifen (4-

OHT; Sigma, H7904) for 1-2 hours. Cre induction between 4-5 dpf predominantly labels

iridophore progenitors (5), but in rare cases leads to xanthophore clones, which were followed

until postmetamorphic stages.

Throughout the text, we use the term ‘cluster’ for xanthophores labeled in transplantation

experiments and the Tg(sox10: Cre) experiments. These xanthophores are derived from a small,

but unknown number of progenitor cells; we may also refer to them as polyclones. The

xanthophores labeled using Tg(sox10:ERT2

-Cre), onthe other hand, are referred to as clones as all

the labeled xanthophores must be related by lineage in these experiments.

Image acquisition, immuno-staining and processing: Repeated imaging of zebrafish and BrdU

staining were performed as described (5). Images were acquired on Zeiss LSM 780 NLO

confocal and Leica M205 FA stereo-microscopes. ImageJ (29), Fiji (30), Adobe Photoshop,

Adobe Illustrator and Imaris were used for image processing and analysis. Maximum intensity

projections of confocal scans of the fluorescent samples were uniformly adjusted for brightness

and contrast. Scans of the brightfield were stacked using ‘stack focuser’ plugin and tile scans

were stitched in Fiji (31). For the final imaging, animals were treated with (±) Epinephrine

hydrochloride (Sigma E4642) to induce melanosome aggregation to better visualize the clones.

Xanthophore Count: Individual xanthophores were identified by their morphology and pigment

content. Metamorphic xanthophores typically display a single and prominent pigment-rich spot

inside the cell. The xanthophores were manually counted using cell counter plugin in ImageJ.

4

Fig. S1. Xanthophores tinge the stripe pattern of zebrafish. Adult zebrafish stripe colour

pattern of (A,D) wild-type, (B,E) pfeffer and (C,F) nacre mutants lacking xanthophores and

melanophores respectively, showing residual stripe formation. Stripe nomenclature is depicted

on the right in (D); name of the stripes starts with a numeral whereas name of the interstripes

starts with X. Bright-field picture of wild-type (G) and (H) pfe at 8 mm standard length (31 dpf).

Absence of xanthophores does not affect the basic metamorphic pattern and melanophores and

iridophores appear in correct positions.

5

Fig. S2 – (A) Bright-field image of wild-type zebrafish after treatment with epinephrine

hydrochloride showing yellow pigmented xanthophores in stripe (arrow) and interstripe region

(arrowhead). (B) DsRed positive xanthophores labelled with Tg(fms:Gal4.VP16); Tg(UAS:Cre);

Tg(actin2:loxP-STOP-loxP-DsRed-express) in stripe region (arrow in B’) appear above

melanophores. Arrowhead: xanthophores in the interstripe region. (C) adult zebrafish trunk skin

showing blue stripes and golden interstripes. (D) Numbers of interstripe xanthophores (XI),

stripe xanthophores (XS) and melanophores (M) per segment in adult zebrafish trunk skin (n=5,

bars represent average±SD). Scale bars in all figures: 100 m.

6

Fig. S3 - Xanthophores cover the dorsolateral trunk skin before arrival of other two

pigment cell types. (1-1’’) Xanthophores are present in the skin prior to arrival of (2-2’’)

metamorphic iridophores and (3-3’’) melanophores. Genotype: Tg(sox10:Cre); Tg(UBI:loxp-

EGFP-loxp-mCherry). Scale bars: 100 µm.

7

Fig. S4 Change in xanthophore shape with the appearance of metamorphic iridophores.

(A1-a10’) During metamorphosis iridophores appear along the horizontal myoseptum,

xanthophores above these iridophores change their shape indicating an interaction between

iridophores and xanthophores during the formation of first interstripe. Genotype: Tg(sox10:Cre);

Tg(UBI:loxp-EGFP-loxp-mCherry). Dashed circle in A1’-10’ indicates area zoomed in a1-10’.

Arrows indicate a xanthophore. White arrowheads indicate iridophore platelets. Red arrowheads

8

indicate a melanophore that appears in the stripe region and expands in size; in the meanwhile,

neighbouring xanthophores retract. (B) Graph showing reduction in the length of xanthophore

filopodia (in µm) upon arrival of metamorphic iridophores. The points on X axis of graph -

Before and after arrival of iridophores indicate the situation shown in a1 and a10 respectively

(n=20, bars represent average±SD). Scale bars: 100 µm.

9

Fig. S5 – Tg(sox10:Cre) allows labelling of neural crest-derived pigment cells. Labelled

pigment cells (red) in the postmetamorphic and juvenile skin of Tg(sox10:Cre); Tg (UBI:loxp

gfploxpmcherry), (A-A’) a clone where all the pigment cells are labelled, (B-D’) patchy clones

with labelling of a fraction of pigment cells in the skin. Different types of pigment cells can be

identified by their distinct shape, location, and pigment content. (E-F’) a labelled melanophore

(asterisk) in the stripe of a juvenile shows characteristic morphology and black pigment. (F-H’)

arrows indicate xanthophores, (H-H’) arrowhead indicates iridophores. Scale bar: 100 m.

10

Fig. S6 – Xanthophores do not undergo global reorganization but de-cluster locally upon

arrival of melanophores in the stripe region. (A1-4’) Xanthophore clusters can be recognized

during stripe morphogenesis suggesting that these cells do not undergo large-scale reorganization

(these figures are part of the time-series displayed in Figure 2C). (B1-6’) Upon arrival of

metamorphic melanophores, xanthophores begin to loosen up leading to de-clustering of

xanthophore clusters. Dashed lines indicate a xanthophore cluster that seems to break into two

upon arrival of melanophores in the stripe region. Scale bar: 100 µm.

11

Fig. S7 – Xanthophore undergo local proliferation in the skin. (A1-4’) Time-lapse imaging of

xanthophores (red; arrows and arrowheads) shows local increase in xanthophore numbers. (B)

xanthophores (green) take up BrdU (red) suggesting that these cells have potential to undergo

cell-division. Scale bar: 100 m.

12

Fig. S8 - Arrival of metamorphic melanophores leads to netlike xanthophore organization

in the stripe region. (A1-5’) With the appearance of melanophores in the stripe region,

xanthophores acquire net-like organization over the melanophores. Dashed square in A1’-A5’

indicate the region that is magnified in a1-a5’. Genotype: Tg(sox10:Cre); Tg(UBI:loxp-EGFP-

loxp-mCherry). Scale bars: 100 m

13

Fig. S9 : Blastomere transplants confirm different morphological states of xanthophores in

the stripe and the interstripe region. (A1-B’) transplantation of Tg(pax7:GFP) into wild type

14

hosts at the blastula stage leads to labelling of small clusters of xanthophores (green). (A1-A3’)

During metamorphosis xanthophores in the presumptive stripe region change their morphology

and extend long filopodia (arrow in A3’). Xanthophores of the interstripe region become densely

packed (arrowhead in A2’, A3’, B’, C’) during these stages. (C1-D2’) transplantation of Tg

(pax7:GFP) into Tg(fms:Gal4.VP16); Tg(UAS:E1b:nfsB.mCherry) hosts at the blastula stage

leads to labelling of small clusters of green xanthophores surrounded by red xanthophores.

Xanthophores maintain the cluster identity; grow in numbers by local proliferation and do not

exhibit extensive mixing between donor-derived green xanthophores and host-derived red

xanthophores. Scale bars: 100 m.

15

Fig. S10 - Xanthophores exhibit short scale movements towards the interstripe region. (A1-

A18’) Larval xanthophores maintain their continuity to contibute to the adult stripe pattern and

exhibit local proliferation, rearrangement and short local movements. Time-lapse imaging of a

DsRed labelled xanthophore clone induced at 4-5 dpf in Tg(sox10:ERT2

-Cre); Tg(βactin:loxp-

STOP-loxp-DsRed). Arrow indicates xanthophore movement from the stripe area to the

interstripe. Scale bars: 100 m.

16

Fig. S 11. Xanthophore reorganization during stripe pattern formation. (A1-B5) schematics

showing reorganization of xanthophores (yellow-orange) during stripe pattern formation; (A1-5)

lateral view, (B1-5) skin cross-section, outside to the right. A single xanthophore and its progeny

are shown in red to depict a xanthophore clone and local events. (A1,B1) premetamorphic stages.

(A2,B2) Xanthophores begin to increase in number at the onset of metamorphosis. Subsequently,

17

iridophores organize the first interstripe. (A3,B3) Xanthophores above the dense iridophores

forming the first interstripe become compact and acquire yellow-orange pigment. Melanophores

appear in the stripe region. Iridophores disperse along the dorsoventral axis to organize new

interstripes. (A4,B4) Iridophores aggregate to form new interstripe at a distance from the first

interstripe. (A5,B5) Xanthophores become stellate in the stripe region. Reiteration of these

cellular behaviors leads to addition of new stripes and interstripes in the growing fish.

18

Movie S1 - Dorso-lateral migration of larval xanthophores. Time-lapse imaging of

Tg(fms:Gal4.VP16); Tg(UAS:E1b:nfsB.mCherry) with a time interval of every 10 minutes for 14

hours. mCherry positive cells appear at the dorsal side of the embryo at ~ 24-27 hpf. They

migrate dorso-laterally and mature into xanthophores in the skin. Two mCherry labelled cells are

digitally marked with blue and green dot to follow them over time. After reaching the skin larval

xanthophores show limited movement but exhibit extensively dynamic filopodial extensions.

Neighbouring xanthophores frequently contact each other with their dynamic filopodia

suggesting that the xanthophores communicate with each other even after covering the larval

skin.

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