ZFIN ID: ZDB-FIG-180716-24
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:
Fish:
Conditions:
Knockdown Reagents:
Anatomical Terms:
Stage: Adult
PHENOTYPE:
Fish:
Conditions:
Knockdown Reagents:
Observed In:
Stage: Adult

Fig. 2

Shh-Signaling-Dependent ascl1a Regulation in the Injured Retina

(A and B) RT-PCR (A) and qPCR (B) analysis of ascl1a in the post-injured retina; n = 6 biological replicates.

(C) Fluorescence ISH (FISH) and IF microscopy images of a 0.5-μm-thick optical section of retina showing co-localization of ascl1a with ptch1 in BrdU+ MGPCs at 4 dpi. Arrowheads mark co-expression of genes in BrdU+ cells.

(D–F) BF microscopy images of ascl1a mRNA ISH in retina at 4 dpi with cyclopamine treatment, shha or gli1 knockdowns (D), and gli3 or sufu knockdowns (E). The number of ascl1a+ cells from (E) is quantified in (F).

(G) qPCR analysis of ascl1a mRNA with cyclopamine treatment and shha or sufu knockdown in 2 dpi retina.

(H) Schematic of the ascl1a promoter with a putative Gli-binding site (Gli-BS) cluster. Arrows mark ChIP primers, N.S marks the negative control, and capital letters mark putative Gli-BSs.

(I) Retinal ChIP assay at 4 dpi showing both Gli1 and Gli3 bound to the ascl1a promoter.

(J) Luciferase assay in 24 hpf embryos co-injected with ascl1a:GFP-luciferase vector and sufu or shha MOs.

(K) Luciferase assay was done with mutated Gli-BS of ascl1a promoter in an experiment similar to (J).

Scale bars represent 10 μm in (C) and 20 μm in (D) and (E). Asterisk indicates the injury site (C–E). Error bars represent SD. p < 0.0001 (F); p < 0.01 (G); p < 0.01 (J). n = 6 biological replicates (F and G); n = 3 (J). See also Figures S2, S6, and S7.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
ascl1a 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 RTPCR
Adult regenerating tissue retina ISH
WT + MO2-gli1 perforation: retina Adult regenerating tissue retina ISH
WT + MO2-sufu perforation: retina Adult regenerating tissue retina RTPCR
Adult regenerating tissue retina ISH
WT + MO3-gli3 perforation: retina Adult regenerating tissue retina ISH
WT + MO5-shha + MO6-shha perforation: retina Adult regenerating tissue retina ISH
Adult regenerating tissue retina RTPCR
ptch1 WT perforation: retina Adult regenerating tissue Muller cell ISH
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
WT perforation: retina, chemical treatment: Cyclopamine Adult regenerating tissue retina ascl1a expression decreased amount, abnormal
Adult regenerating tissue retina ascl1a expression decreased amount, abnormal
WT + MO2-gli1 perforation: retina Adult regenerating tissue retina ascl1a expression decreased amount, abnormal
WT + MO2-sufu perforation: retina Adult regenerating tissue retina ascl1a expression increased amount, abnormal
Adult regenerating tissue retina ascl1a expression increased amount, abnormal
WT + MO3-gli3 perforation: retina Adult regenerating tissue retina ascl1a expression increased amount, abnormal
WT + MO5-shha + MO6-shha perforation: retina Adult regenerating tissue retina ascl1a expression decreased amount, abnormal
Adult regenerating tissue retina ascl1a 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.