The human ARF tumor suppressor senses blastema activity and suppresses epimorphic tissue regeneration
- University of California, San Francisco, United States
Figures
Figure 1 with 3 supplements
ARF, not normally present in highly regenerative vertebrates, is specifically activated in blastemas of ARF transgenic zebrafish.
(A) Comparison of amino acid sequences of proteins produced by the Cdkn2a/b loci of zebrafish (Danio rerio), amphibians including the axolotl (Ambystoma mexicanum) and western clawed frog (Xenopus …
Figure 1—figure supplement 1
In vitro analysis of ARF promoter constructs in zebrafish and human cells.
Representative luciferase reporter data of three replicates: relative luminescence generated by ARF promoter-firefly luciferase reporter constructs transfected into zebrafish (ZKS, ZF4) and human …
Figure 1—figure supplement 2
GFP reporter activity in WT or ARF:GFP zebrafish at multiple developmental and regenerative time points.
(A) Immunostaining (sagittal confocal images) for GFP at 24 hpf, 48 hpf, and 72 hpf in WT and ARF:GFP embryos. Scale bars: 200 μm. GFP expression is restricted to the hearts of transgenic fish due …
Figure 1—figure supplement 3
ARF is not activated during wound healing in the absence of a blastema.
(A) At day 0, dorsal fin lobes were wounded (interray laceration, 0 dpi), while ventral fin lobes were amputated (0 dpa). At day 1, GFP expression was assayed in the healing (dorsal) and …
Figure 2 with 1 supplement
Rb1 hyperphosphorylation and E2f1 binding of the human ARF promoter in the blastema during regeneration.
(A) Representative Western blot of three experimental replicates of Rb pathway components, E2f1, Rb1, and hyperphosphorylated Rb1 (p-Rb1), before injury (uninj.), at 2 dpa, and during embryogenesis …
Figure 2—figure supplement 1
Promoter sequences evaluated for E2f1 enrichment using an E2f1 antibody to perform a ChIP assay.
Both ARF and tk1 promoters contain E2f binding sites (bold; del Arroyo et al., 2007, Tfsitescan), but the tk1- promoter does not.
Figure 3 with 1 supplement
Expression of the mammalian tumor suppressor ARF in zebrafish driven by heat shock promoter.
In vivo analysis of transgenic zebrafish expressing human ARF under the control of an inducible heat shock promoter, Tg (hsp70l:ARF) (hs:ARF). Schematic of the hs:ARF transgene (top left). The ARF …
Figure 3—figure supplement 1
Analysis of ARF expression in zebrafish cells.
(A) Immunofluorescence for Mdm2 and ARF (top) and Tp53 (bottom) in zebrafish cells (ZKS) transfected with pcDNA-ARF. ARF and Mdm2 co-localize in the nucleolus (arrow) when ARF is expressed; in cells …
Figure 4 with 1 supplement
ARF suppresses fin regeneration.
(A) Schematic of heat shock regimen. An initial hour long, 37°C heat shock is delivered 3 hr prior to amputation (0 dpa) and then every 6 hrs thereafter for 6 days. Regenerates are then assessed …
Figure 4—figure supplement 1
ARF expression during regeneration promotes Tp53, tp53, and cdkn1a upregulation and regeneration inhibition is reversible.
(A) Representative (sagittal confocal) images of WT and hs:ARF fins at 4 dpa. Scale bars: 50 μm. Dashed lines represent amputation planes. ARF localizes to the nucleus (inset). Scale bars: 10 μm. (B)…
Figure 5
Human ARF functions through the Tp53 pathway in fish to suppress regeneration.
(A) Quantification of regenerate length and area at 6 dpa in tp53M214K/M214K, hs:ARF, and hs:ARF; tp53M214K/M214K fins exposed to the heat shock regimen as in Figure 4 (left; N = 30 fins). …
Figure 6
ARF suppresses fin regeneration by inducing apoptosis and cell-cycle arrest.
(A) Quantification of EdU staining at 2, 4, and 6 dpa in wild type (WT) and hs:ARF fins exposed to heat shock (left). At 2 dpa, 6.0% ± 1.1% of cells in WT regenerates were EdU + compared with …
Figure 7 with 3 supplements
ARF senses regenerative signals and suppresses fin regeneration.
(A) Schematic of transgene expressing human ARF under the control of the human ARF promoter (left). Representative images of age- and sex-matched ARF:ARF and WT zebrafish (right; 5 months …
Figure 7—figure supplement 1
The ARF:ARF transgene does not interfere with development, whereas forced ARF expression causes embryonic lethality.
(A) Quantification of embryonic mortality at 48 hpf and 72 hpf in wild type (WT) and ARF:ARF embryos (left; N = 90, p>0.05). Representative sagittal confocal images of ARF expression at 24 hpf in WT …
Figure 7—figure supplement 2
ARF:ARF fins do not completely regenerate even after 30 days.
Representative images of fin regeneration at 15 dpa and 30 dpa in wild type (WT) and ARF:ARF fins. Scale bars: 1 mm.
Figure 7—figure supplement 3
ARF:ARF expression and p53-dependent functions during regeneration.
(A) Representative images of wild type (WT) (left) and ARF:ARF (right) fins at 6 dpa. Scale bars: 1 mm. Representative images of ARF expression at 6 dpa in WT (left) and ARF:ARF (right) fins. Scale …
Figure 8
Model of ARF function in the context of Rb pathway activity during zebrafish development and fin regeneration.
ARF is not active during development during which a moderate level of mitogenic signaling causes modest phosphorylation of Rb1 (top); however, during regeneration, high mitogenic signaling induces …
Tables
Table 1
Primary antibodies.
| Host species | Antigen | Company | Cat. No. | Dilution | Application |
|---|---|---|---|---|---|
| Mouse | Tp53 | Abcam | ab77813 | 1:50 | IHC |
| Rabbit | Mdm2 | Santa Cruz | C-18 | 1:50 | IHC |
| Rabbit | GFP | Torrey Pines | TP401 | 1:3000 | IHC |
| Chicken | GFP | Abcam | ab13970 | 1:3000 | IHC |
| Mouse | Msxb | DSHC | 4G1-c | 1:50 | IHC |
| Rabbit | PCNA | Abcam | ab2426 | 1:500 | IHC |
| Mouse | p14ARF | Cell Signaling | 2407 | 1:100/1:500 | IHC/WB |
| Rabbit | Rb1 | AnaSpec | 55432 | 1:500 | WB |
| Rabbit | p-Rb1 (S780) | Abcam | ab47763 | 1:500 | WB |
| Rabbit | Beta-actin | Millipore | EP1123Y | 1:1000 | WB |
| Rabbit | E2f1 | Abcam | ab14769 | 1:1000 | WB |
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IHC: Immunohistochemistry; PCNA: Proliferating cell nuclear antigen; WB: Western blot.
Table 2
Chromatin immunoprecipitation primers.
| Gene | Ensembl ID | Target site | Forward primer | Reverse primer |
|---|---|---|---|---|
| CDKN2A | ENSG00000147889 | TSS | 5′-GCTGAGGGTGGGAAGATG-3′ | 5′-CCTTAACTGC AGACTGGGA-3′ |
| tk1 | ENSDARG00000086561 | TSS | 5′-AGTCACTGTGCCGGTTTATT-3′ | 5′-GTCGTCTGCTTG TTGTCTTTATTT-3′ |
| tk1- | ENSDARG00000086561 | 2 kbp 5′ of TSS | 5′-CAGGCTTACGGAGACAGCAA-3′ | 5′-AGTGTTTGCTG CTGGATCAC-3′ |
-
TSS: Transcriptional state site.
Table 3
Quantitative polymerase chain reactionprimers.
| Gene | RefSeq ID | Forward primer | Reverse primer |
|---|---|---|---|
| CDKN2A | NM_058195.3 | 5′-ATGGTGCGCAGGTTCTTGGTGA-3′ | 5′-CACCACCAGCGTGT CCAGGAAG-3′ |
| actb2 | NM_181601.4 | 5′-CGAGCAGGAGATGGGAACC-3′ | 5′-CAACGGAAACGCT CATTGC-3′ |
| tp53 | NM_131327.2 | 5′-CTGAAGTGGTCCGCAGATG-3′ | 5′-CGTTTGGTCCCAG TGGTGG-3′ |
| cdkn1a | NM_001128420.1 | 5′-AGCTGCATTCGTCTCGTAGC-3′ | 5′-TGAGAACTTACT GGCAGCTTCA-3′ |
