Supplementary Materials

A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation

Charles K. Kaufman, Christian Mosimann, Zi Peng Fan, Song Yang, Andrew J. Thomas, Julien Ablain, Justin L. Tan, Rachel D. Fogley, Ellen van Rooijen, Elliott J. Hagedorn, Christie Ciarlo, Richard M. White, Dominick A. Matos, Ann-Christin Puller, Cristina Santoriello, Eric C. Liao, Richard A. Young, Leonard I. Zon

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Figs. S1 to S12
Table S1
Primers
Sequences of primers used for cloning and introduction of mutations in crestin_296bp.
Table S2
Microarray Data Annotated
Gene expression analysis was performed on single scales with crestin:EGFP (+) cell precursor patches versus those that were crestin:EGFP (-) from p53/BRAF/MCR/Na/crestin:EGFP zebrafish. Sheet 1 lists the top 48 annotated genes with ~ ≥2 fold enrichment in the crestin:EGFP (+) samples and annotation for those genes that are neural crest related (red) and/or melanoma related/expressed (green). Sheet 2 provides the complete probe list and expression with zebrafish Gene Symbol, Transcript, and Description.
Table S3
Super-enhancers in A375 Cells
Complete list of super-enhancer regions identified from H3K27Ac ChIP-seq performed on human A375 melanoma cell line. Columns H and I identify nearby genes and the closest gene, respectively, to the super-enhancer region listed in Column C and D. Super-enhancers are ranked for highest signal (highest to lowest, Column J).
Table S4
RNA-Seq Data crestin (+) cells
Complete set of RNA-Seq data from sorted 15 somite stage crestin:EGFP (+) cells compared to (-) cells. List is sorted by log2(fold_change) with genes having 0 reads in either condition placed at the end of the list (results in +/- infinity ratio). EGFP and crestin comparison also included (green box) as these genes were manually mapped to the HiSeq reads.
Table S5
RNA-Seq Data human melanoma lines
Complete set of RNA-Seq data for the human melanoma cell lines and normal human melanocytes (HeMA). Sheet 1 contains the data for all genes sorted alphabetically. Sheet 2 shows comparisons for selected genes (SOX10, DLX2, TFAP2 family members) with fold enrichment of melanoma expression compared to HeMA cells.
Table S6
GSEA Gene Lists
Contains individual sheets with the rnk and gmt file gene lists used for the GSEA analysis in Figure 3.

Images, Video, and Other Other Media

Movie S1
Lateral view of neural crest formation and migration using crestin reporter visualized by time-lapse imaging using Spinning Disk Confocal Z stack reconstructions of Tg(crestin_1kb:EGFP) embryo beginning at ~13 somites and followed at 7 minute intervals for 24 hours at 28°C embedded in 1% low melting point agarose in PTU (to inhibit pigment formation which blocks EGFP signal) and Tricaine (safely immobilizes embryos). Note the emergence of both cranial and thoracic crestin:EGFP (+) cells from the dorsum of the embryo and their rapid migration ventrally. The stellate appearance of migratory melanocyte precursors is apparent particularly over the midportion of the embryo. Also, note the rapid rostro-caudal (left to right) migration of the developing lateral line organ overlying the middle of the somites. The embryo tail does not extend normally due to the embedding in agarose, which prevents this movement.
Movie S2
Dorsal view of neural crest formation and migration using the crestin reporter from ~ 15 somites and followed at 6 minute intervals as above for 24 hours at 28°C. The head is down in this view with the developing eyes at the bottom. Again, note the bilateral emergence of crestin:EGFP (+) neural crest cells from the lateral borders of the dorsal neural tube as they then migrate downward with some cells moving over the round yolk and achieving a typical stellate appearance of melanocytes. Group migration of anterior lateral line and branchial arch primordia is visible in the center of the image. Cranial neural crest emergence and migration is again apparent in the lower portion of the embryo.
Movie S3
Adult Tumor Movie. Video of an adult p53/BRAF/crestin:EGFP fish with a melanoma tumor on the head visualized via ambient light and with a fluorescent flashlight and filter specific for EGFP (BlueStar by Nightsea). Note the specificity of the green fluorescence to the tumor. The second segment is a video of another adult p53/BRAF/crestin:EGFP fish with a very small 22 precursor/early tumor near the dorsal fin that is also visualized using epifluoresence both when the fish is briefly anesthetized and then when it is swimming freely. Video taken with iPhone 5S and edited using iMovie.