Journal: bioRxiv

Article Title: Mitfa-Independent Melanocyte Progenitors are Highly Susceptible to GNAQ-induced Uveal Melanoma in Adult Zebrafish

doi: 10.1101/2025.05.05.652300

Figure Lengend Snippet: (A) TEAZ-Skin was used to induce GNAQ-positive melanoma using two strategies. Strategy A involves a single plasmid construct containing expression cassettes driven by the mitfa promoter for GNAQ Q209L -T2A-eGFP and Cas9, as well as a U6 promoter driving the expression of three separate gRNAs (targeting tp53 , ptena , and ptenb , or a non-targeting control). Strategy B uses a multi-plasmid approach with five plasmids for TEAZ-Skin. Expression cassettes include: (a) mitfa : GNAQ Q209L - mitfa :eGFP, (b) U6 :gRNA ( tp53 ), (c) U6 :gRNA ( ptena ), and (d) U6 :gRNA ( ptenb ). (B) Schematic of TEAZ-Skin using wild type (AB), nacre ( mitfa w2/w2 ) and casper ( mitfa w2/w2 : mpv17 a9/a9 ) zebrafish lines. (C) TEAZ-Skin using Strategy B plasmids in adult wild-type (AB) zebrafish. Representative brightfield and fluorescent images shown at 4, 8 and 12 weeks post electroporation. (D) Kaplan-Meier curves were used to estimate tumor-free survival in wild-type zebrafish defined as the time from electroporation until the first visible appearance of GFP+ nodular tumors. Experimental groups as labeled. (E) TEAZ-Skin using Strategy B plasmids in adult nacre zebrafish. Representative brightfield and fluorescent images shown at 2, 4 and 6 weeks post electroporation. (F) Kaplan-Meier curves were used to estimate tumor-free survival in nacre zebrafish defined as the time from electroporation until the first visible appearance of GFP+ nodular tumors. Experimental groups as labeled. (G) TEAZ-Skin using Strategy B plasmids in adult casper zebrafish. Representative brightfield and fluorescent images shown at 2, 4 and 6 weeks post electroporation. (H) Kaplan-Meier curves were used to estimate tumor-free survival in casper zebrafish defined as the time from electroporation until the first visible appearance of GFP+ nodular tumors. Experimental groups as labeled. ( I-J ) Kaplan-Meier curves comparing tumor-free survival among wild-type, nacre, and casper zebrafish following TEAZ-Skin induction using Strategy B plasmids with ( I ) non-targeting control gRNAs and ( J ) gRNAs targeting tp53, ptena and ptenb . Experimental groups as labeled.

Article Snippet: For immunofluorescence, slides were stained with primary antibodies against GFP (NB600-308 Rabbit anti-GFP polyclonal from Novus Biologicals at 1:250) and then fluorescent secondary antibodies against rabbit IgG (Alexa Fluor 488 goat anti-rabbit IgG at 1:500 from a 2 mg/mL stock).

Techniques: Plasmid Preparation, Construct, Expressing, Control, Electroporation, Labeling

Journal: bioRxiv

Article Title: Mitfa-Independent Melanocyte Progenitors are Highly Susceptible to GNAQ-induced Uveal Melanoma in Adult Zebrafish

doi: 10.1101/2025.05.05.652300

Figure Lengend Snippet: (A) Schematic of the zebrafish eye, dashed box indicates the retina and choroid. (B) H&E analysis of a wild type zebrafish eye (20x magnification). Eye structures as labeled with yellow asterisks marking choroidal melanocytes. ( C–D ) Immunohistochemical analysis of transgenic Tg(mitfa:GFP) zebrafish using an anti-GFP antibody. Sections were counterstained with DAPI to visualize nuclei. ( C ) Representative image showing GFP expression in mitfa -positive cells. ( D ) Secondary-only antibody was used as background control. ( E ) Schematic of TEAZ-Eye using choroidal targeted injection of Strategy B plasmids. ( F-K, F’-K’ ) Live imaging of adult wild-type zebrafish over 42 days post-electroporation (dpe) using the TEAZ-Eye method with Strategy B plasmids to induce uveal melanoma. (F-K) brightfield imaging and (F’-K’) the corresponding GFP fluorescence images highlighting tumor progression. ( L-N, L’-N’ ) Live imaging of adult casper zebrafish over 21 days post-electroporation (dpe) using the TEAZ-Eye method with Strategy B plasmids to induce uveal melanoma. (L-N) brightfield imaging and (L’-N’) the corresponding GFP fluorescence images highlighting tumor progression. ( O ) H&E analysis of uveal melanoma in casper zebrafish induced by TEAZ-Eye. Images were acquired at 20× magnification and stitched from 85 fields using Thunder imaging software. Normal eye tissue and uveal melanoma as labeled. ( P-Q ) anti-GFP immunohistochemical analysis of TEAZ-Eye induced uveal melanoma. Sections were counterstained with DAPI to visualize nuclei. (P) Images were acquired at 20x magnification and stitched from 85 fields using Thunder imaging software. (Q) Representative 40x magnification image showing GFP expression in tumors cells.

Article Snippet: For immunofluorescence, slides were stained with primary antibodies against GFP (NB600-308 Rabbit anti-GFP polyclonal from Novus Biologicals at 1:250) and then fluorescent secondary antibodies against rabbit IgG (Alexa Fluor 488 goat anti-rabbit IgG at 1:500 from a 2 mg/mL stock).

Techniques: Labeling, Immunohistochemical staining, Transgenic Assay, Expressing, Control, Injection, Imaging, Electroporation, Fluorescence, Software

Journal: bioRxiv

Article Title: Mitfa-Independent Melanocyte Progenitors are Highly Susceptible to GNAQ-induced Uveal Melanoma in Adult Zebrafish

doi: 10.1101/2025.05.05.652300

Figure Lengend Snippet: Immunohistochemistry of the choroid using an anti-GFP antibody in ( A ) Tg ( mitfa :GFP) with 10x magnification and ( B ) TEAZ-Eye–injected zebrafish expressing an eGFP construct driven by the mitfa promoter ( mitfa:GFP ) with 40x magnification. Sections were counterstained with DAPI to visualize nuclei.

Article Snippet: For immunofluorescence, slides were stained with primary antibodies against GFP (NB600-308 Rabbit anti-GFP polyclonal from Novus Biologicals at 1:250) and then fluorescent secondary antibodies against rabbit IgG (Alexa Fluor 488 goat anti-rabbit IgG at 1:500 from a 2 mg/mL stock).

Techniques: Immunohistochemistry, Injection, Expressing, Construct

Journal: bioRxiv

Article Title: Mitfa-Independent Melanocyte Progenitors are Highly Susceptible to GNAQ-induced Uveal Melanoma in Adult Zebrafish

doi: 10.1101/2025.05.05.652300

Figure Lengend Snippet: (A) Kaplan-Meier curves comparing tumor-free survival among wild-type, nacre, and casper zebrafish following TEAZ-Eye induction using Strategy B plasmids. (B) H&E analysis showing the retina and choroid of wild-type (left) and nacre (right) zebrafish eyes (20x magnification). Note the loss of melanocytes in the choroid of nacre eyes. (C) Immunohistochemical analysis of transgenic Tg(mitfa:GFP); mitfa w2/w2 (nacre) zebrafish using an anti-GFP antibody. Sections were counterstained with DAPI to visualize nuclei. Representative images showing GFP positive cells in the ciliary body and choroid are shown by yellow arrow heads. Secondary-only antibody was used as background control. (D) UMAP representation of single-cell RNA sequencing (scRNA-Seq) data from three dissociated control-injected eyes ( mitfa :Cas9 with non-targeting gRNAs) and from eyes harboring uveal melanoma (UM) tumors induced using Strategy B via TEAZ-Eye. Annotated cell clusters are labeled. Progenitor cells and UM tumor clusters are outlined with dashed red lines. Representative brightfield images of a normal eye and a UM tumor in casper zebrafish are shown in the top left corner of the UMAP. (E) Re-clustered UMAP representing progenitor cells from control eyes and uveal melanoma (UM) tumor cells from TEAZ-Eye–injected casper zebrafish. Heterogeneous cell populations as labeled. (F) Re-clustered UMAP from (E) showing melanocyte progenitor cells from control eyes in red and UM tumor cells from TEAZ-Eye in blue. Expression plots for pax3a, foxd3 and sox10 are shown, dashed line marks the progenitor cell population. (G) Dot plot illustrating differentiational expressed genes (log2FC > 0.5; Adj p-value < 0.05) between heterogeneous cell populations in (E). Dot size indicates the percentage of cells expressing each gene; color intensity (blue to red) reflects normalized average expression levels (low to high). (H) UMAP obtained after clustering GFP-positive cells sorted from Tg(mitfa:GFP) and Tg(mitfa:GFP); mitfa w2/w2 (nacre) zebrafish embryos at 28 hours post fertilization (hpf). Annotated cell clusters as labelled. (I) Comparison of relative cluster size for the two genotypes; Tg(mitfa:GFP) and Tg(mitfa:GFP); mitfa w2/w2 (nacre) in (H). (J) Dot plot illustrating differentiational expressed genes (log2FC > 0.5; p-value < 0.05) in melanophores and progenitor cells in mitfa w2/w2 and sibling embryos at 28hpf. Dot size indicates the percentage of cells expressing each gene; color intensity (grey to red) reflects normalized average expression levels (low to high). ( K-K’ ) Kaplan–Meier curves comparing tumor-free survival in wild-type (n=12) and casper (n=6) zebrafish following TEAZ-Eye injection using Strategy B plasmids. Wild-type zebrafish were injected with either Strategy B plasmids plus non-targeting gRNAs (n=6) or Strategy B plasmids plus mitfa -targeting gRNAs (n=6). Casper zebrafish were injected with Strategy B plasmids and mitfa -targeting gRNAs (n=6). ( K’ ) Representative brightfield and GFP-overlay images of tumors that formed in casper (6 of 6) and wild-type zebrafish injected with mitfa -targeting gRNAs (1 of 6). Metastatic cells are indicated by red arrows.

Article Snippet: For immunofluorescence, slides were stained with primary antibodies against GFP (NB600-308 Rabbit anti-GFP polyclonal from Novus Biologicals at 1:250) and then fluorescent secondary antibodies against rabbit IgG (Alexa Fluor 488 goat anti-rabbit IgG at 1:500 from a 2 mg/mL stock).

Techniques: Immunohistochemical staining, Transgenic Assay, Control, RNA Sequencing, Injection, Labeling, Expressing, Comparison, Eye Injection

Journal: bioRxiv

Article Title: Mitfa-Independent Melanocyte Progenitors are Highly Susceptible to GNAQ-induced Uveal Melanoma in Adult Zebrafish

doi: 10.1101/2025.05.05.652300

Figure Lengend Snippet: Melanocyte rescue experiments in nacre zebrafish by TEAZ-Eye using plasmids containing mitfa : mitfa ; mitfa :GFP (miniCoopR ). ( A-A’ ) H&E images (10x and 40x magnification) of control wild-type (AB) eyes following TEAZ-Eye injection with ( A ) control mitfa -GFP or ( A’ ) miniCoopR plasmids. ( B-B’ ) H&E images (10x and 40x magnification) of control mitfa w2/w2 ( nacre ) eyes following TEAZ-Eye injection with ( B ) control mitfa -GFP or ( B’ ) miniCoopR plasmids, rescued melanocytes in the choroid are indicated by a red asterisk. ( C ) Representative dorsal image of a transgenic Tg(mitfa:GFP); mitfa w2/w2 injected with mitfa-GNAQ Q209L following TEAZ-Eye injection.

Article Snippet: For immunofluorescence, slides were stained with primary antibodies against GFP (NB600-308 Rabbit anti-GFP polyclonal from Novus Biologicals at 1:250) and then fluorescent secondary antibodies against rabbit IgG (Alexa Fluor 488 goat anti-rabbit IgG at 1:500 from a 2 mg/mL stock).

Techniques: Control, Eye Injection, Transgenic Assay, Injection

Journal: Immunity

Article Title: Indoleamine 2,3-dioxygenase 1 activation in mature cDC1 promotes tolerogenic education of inflammatory cDC2 via metabolic communication

doi: 10.1016/j.immuni.2022.05.013

Figure Lengend Snippet:

Article Snippet: Next, slides were washed and stained with fluorescein isothiocyanate (FITC)-labelled phalloidin (1:250) for actin labeling and counterstained with 4’, 6’-diamidino-2phenylindole DAPI (#P36931, Thermo Fisher Scientific) for nuclei.

Techniques: Produced, Virus, Subcloning, Recombinant, Electron Microscopy, Protease Inhibitor, Purification, Adjuvant, Modification, Selection, Western Blot, In Situ, Enzyme-linked Immunosorbent Assay, Reverse Transcription, SYBR Green Assay, Bradford Protein Assay, Control, Retroviral, Mouse Assay, Sequencing, Real-time Polymerase Chain Reaction, Cloning, Software