FIGURE SUMMARY
Title

Biotagging of Specific Cell Populations in Zebrafish Reveals Gene Regulatory Logic Encoded in the Nuclear Transcriptome

Authors
Trinh, L.A., Chong-Morrison, V., Gavriouchkina, D., Hochgreb-Hägele, T., Senanayake, U., Fraser, S.E., Sauka-Spengler, T.
Source
Full text @ Cell Rep.

Genetically Encoded Biotagging Toolkit in Zebrafish

(A) Schematic of the binary transgenic system for cell-type-specific in vivo biotinylation. BirA drivers are in red and Avi effector lines in blue. POI, protein of interest.

(B–E) Widefield fluorescent image of biotagging drivers expressing BirA under the sox10 (B), myl7(C), and zic (D), or ubiquitous (E, βactin) promoters; schematics of the transgenic constructs are shown above the images. (B′–E′) Corresponding confocal images of BirA-equivalent membCherry expression in the pharyngeal arches and hindbrain (white arrow, B), myocardium (white arrow, C), and hindbrain (white arrow, D).

(F) Widefield image of Avi-RanGap(nucAvi) effector with schematic of transgenic construct above image. (F′) Confocal image of Avi-RanGap expression in the somite.

(G and H) Widefield fluorescent (G) and projection of confocal (H) microscope images of ncBirA(BAC) driver with schematic of recombineering BAC cassette and tol2 containing BAC. Arrow points to otic vesicle, and arrowheads point to midbrain expression.

Scale bars represent 50 μm (B′–D′) and 20 μm (F′ and H).

Biotagging Avi-Tagged Effectors

(A and B) Schematic of Avi-tagged constructs for generating nuclear effector (nucAvi) (A) and ribosome effector (riboAvi) (B).

(C–F) Confocal 3D projection of nucAvi (C and D) and riboAvi (E and F) expression in the developing inner ear (C and E) and somite (D and F) at 32 hpf. Arrow points to nucleoli.

(G–I) Confocal 3D projection of BirA driver (G and I in NC; H in myocardium, red) and Avi effector (G,H, nucAvi, and I, riboAvi, blue). Scale bars, 20μm.

Specific In Vivo Biotinylation of Avi-Tag Proteins and Purification of Subcellular Compartments

(A and B) Antibody staining for Avi-RanGap (green) and HA-BirA (red), with anti-GFP and anti-hemagglutinin (anti-HA) antibodies, respectively. In fixed samples, Avi-RanGap localize more discretely to the nuclear envelope. Anti-HA staining shows BirA (red) expressed in both nuclei and cytoplasm of cells. Scale bars, 20 μm.

(C and D) Streptavidin, anti-GFP, and anti-HA western blot of nuclear (C) and ribosome (D) extracts from BirA drivers (ncBirA or ncBirA(BAC)) and Avi-tagged (nucAvi, C) or (riboAvi, D) effector embryos. (C) Arrow points to biotinylated Avi-RanGap (C, lane 3), shifted to larger size after biotinylation when detected with anti-GFP (compare lanes 2 and 3).

(E) Bright-field image of harvested nucleus from BirA;nucAvi embryos after incubation with streptavidin Dynabeads and isolated by magnetic capture. (E′) DAPI stained of nucleus in (E). (E″) Merge of images in (E) and (E′).

(F) Quantification of total RNA yield from biotagged nuclei or ribosomes or FACS isolation protocols using ncBirA and respective Avi-tagged effectors. RNA from cellular compartments calculated per 100 embryos. Error bars represent SDs from two sequenced replicates. Significance calculated using Student’s t-test (one-tailed, two-sample equal variance).

(G–K) Representative Bioanalyzer profile of total RNA extracted from Streptavidin-bound biotagged nuclei (G), ribosomes (J), flow-through (unbound) (H and K), and whole embryo (I).

Biotagging sox10 BirA drivers and Avi-tagged RanGap effectors. Related to Figures 1 and 2.

A-C Differential expression between sox10 biotagging transgenic and BAC drivers. Wide-field image of TgBAC(Sox10:BirA-mCherry)ox104a (ncBirA(BAC)) (A). Tg(Sox10:BirA-membCherry)ct706a (ncBirA) and Tg(Sox10:BirA-membCherry)ct706bsox10 Biotagging transgenic exhibit expression in different neural crest derivatives that are included in the overall sox10 expression pattern, by sox10 Biotagging BAC (A). Arrow points to lack of expression in the otic vesicle, while arrowheads point to lack of expression in the midbrain of sox10 transgenes.

D-L Biotagging Avi-tagged nuclear localized effectors. Schematic of two variant Avi-tagged RanGap constructs for generating Avi effector transgenes. N-terminal Avi-tag construct (Avi-Cerulean-Rangap) contains the beta-actin2 (β actin) promoter upstream of Avi-tag (steelblue), the Tobacco Etch Virus protease cleave site (TeV, green), Cerulean (turquoise), the C-terminal domain of RanGap (purple), and a polyA signal (D). C-terminal Avi-tag construct (RanGap-Cerulean-Avi) contains the beta-actin2 ( βactin) promoter upstream of the C-terminal domain of RanGap (purple), Cerulean (turquoise), the Tobacco Etch Virus protease cleave site (TeV, green), Avi-tag (steelblue), and a polyA signal (E). Both constructs are flanked by tol2 elements (yellow) for transgenesis by Tol2 transposition. 3-D projection of confocal Z-stack of Avi-RanGap (F-G) and RanGap-Avi (H-I) of the developing inner ear (F, H) and somite (G, I), imaged at 32hpf. Both Avi-Cerulean-RanGap and Rangap-Cerulean-Avi proteins localize similarly to the nucleus of all cells in the embryo. Schematic of Avi-RanGap effector construct with ubiquitin promoter (ubiq) upstream of N-terminal Avi tagged RanGap elements (J). Confocal image of hindbrain (K) and eye (L) of Tg(ubiq:Avi-RanGap) embryo (nucAvi(ubiq)). Scale bars: 20 μm, except 50 μm in (K, L).

Acknowledgments
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