ZFIN ID: ZDB-FIG-180716-25
Kaur et al., 2018 - let-7 MicroRNA-Mediated Regulation of Shh Signaling and the Gene Regulatory Network Is Essential for Retina Regeneration. Cell Reports   23:1409-1423 Full text @ Cell Rep.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibody:
Fish:
Conditions:
Knockdown Reagents:
Anatomical Terms:
Stage: Adult
PHENOTYPE:
Fish:
Conditions:
Knockdown Reagents:
Observed In:
Stage: Adult

Fig. 3

Lin28a-let-7 Axis Regulates Shh Signaling Component Genes in the Injured Retina

(A) FISH and IF microscopy images of a 0.5-μm-thick optical section of retina showed co-localization of lin28a with ptch1 in BrdU+ MGPCs at 4 dpi. Arrowheads mark co-expression of genes in BrdU+ cells.

(B and C) BF microscopy images of lin28a mRNA ISH in the retina at 4 dpi with cyclopamine treatment and shha or sufu knockdown (B), which was quantified by qPCR (C). Arrowheads mark co-expression of genes in BrdU+ cells in (B).

(D and E) Schematic of the lin28a promoter with a potential Gli-BS cluster, where arrows mark ChIP primers and capital letters mark consensus sequence of Gli-BS (D). A 4 dpi retinal ChIP assay showed both Gli1 and Gli3 bound to one of the two Gli-BS clusters (E).

(F) Luciferase assay in 24 hpf embryos co-injected with lin28a:GFP-luciferase vector and sufu or shha MOs.

(G) Luciferase assay with mutated Gli-BSs of the lin28a promoter in an experiment similar to (F).

(H and I) ISH and IF microscopy of retina showing co-exclusion of let-7a microRNA (H) and co-localization of Shha protein (I) in BrdU+ MGPCs in the retina at 4 dpi. Arrowheads mark expression of let-7a in BrdU cells and arrows mark co-exclusion of let-7a from BrdU+ cells in (H). Arrowheads mark co-expression of Shha in BrdU+ cells in (I).

(J) let-7 microRNA downregulated the translation of GFP fused with the indicated gene constructs harboring microRNA-binding regions in a dose-dependent manner in HEK293T cells.

(K) Western blot of Shha in lin28a-MO electroporated retina at 4 dpi.

Scale bars represent 10 μm (A, H, and I) and 20 μm (B). Asterisk indicates the injury site (A, B, H, and I). Error bars represent SD.p < 0.001 (C); p < 0.001 (F). n = 6 biological replicates (C, F, and G). GS, glutamine synthetase. See also Figures S3, S6, and S7.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
lin28a WT perforation: retina Adult regenerating tissue Muller cell ISH
Adult regenerating tissue retina ISH
Adult regenerating tissue retina RTPCR
WT perforation: retina, chemical treatment: Cyclopamine Adult regenerating tissue retina ISH
Adult regenerating tissue retina RTPCR
WT + MO2-sufu perforation: retina Adult regenerating tissue retina ISH
Adult regenerating tissue retina RTPCR
WT + MO5-shha + MO6-shha perforation: retina Adult regenerating tissue retina ISH
ptch1 WT perforation: retina Adult regenerating tissue Muller cell ISH
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-shha WB WT perforation: retina Adult regenerating tissue retina
WB WT + MO1-lin28a perforation: retina Adult regenerating tissue retina
Phenotype Details
Fish Conditions Stage Phenotype
WT perforation: retina, chemical treatment: Cyclopamine Adult regenerating tissue retina lin28a expression decreased amount, abnormal
Adult regenerating tissue retina lin28a expression decreased amount, abnormal
WT + MO1-lin28a perforation: retina Adult regenerating tissue retina Ab1-shha labeling decreased amount, abnormal
WT + MO2-sufu perforation: retina Adult regenerating tissue retina lin28a expression increased amount, abnormal
Adult regenerating tissue retina lin28a expression increased amount, abnormal
WT + MO5-shha + MO6-shha perforation: retina Adult regenerating tissue retina lin28a expression decreased amount, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Cell Reports for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Cell Rep.