ZFIN ID: ZDB-FIG-180716-23
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:
Antibodies:
Fish:
Conditions:
Anatomical Terms:
Stage: Adult
PHENOTYPE:
Fish:
Conditions:
Knockdown Reagents:
Observed In:
Stage Range: Day 4 to Adult

Fig. 1

Shh Signaling Is Necessary for MG Dedifferentiation in the Injured Retina

(A and B) RT-PCR (A) and qPCR (B) analysis of Shh signaling component genes in the retina at indicated time points post-injury; n = 6 biological replicates. p < 0.001; ∗∗p < 0.003.

(C and D) Immunofluorescence (IF) microscopy images of Shh signaling components in wild-type BrdU+ MGPCs (C), and Shh expression in 1016 tuba1a:GFP transgenic fish at 4 dpi (D). Arrowheads mark protein expression in cells in (C) and (D).

(E) RT-PCR assay of Shh signaling component genes in GFP-positive MGPCs and the rest of the cells from 1016 tuba1a:GFP transgenic retina at 4 dpi.

(F and G) Bright-field (BF) images of 4-days post-fertilized embryos treated with 5% (v/v) DMSO and 30 μM cyclopamine (F), and quantification of the number of cyclopia embryos (G).

(H–J) IF microscopy images showing a dose-dependent decline in GFP+ and BrdU+ MGPCs in 1016 tuba1a:GFP transgenic (H) and wild-type (I) retinae, respectively, at 4 dpi upon cyclopamine treatment, which is quantified in (J).

(K) BF microscopy images of blastema during caudal fin regeneration in cyclopamine-treated wild-type zebrafish at 6 days post-amputation.

(L and M) IF microscopy images of retinal sections with shha or sufu knockdowns (L), and quantification of the number of BrdU+ cells at the injury site (M). p < 0.0001; n = 4 biological replicates. Lissamine tag on MO shows red fluorescence in (L).

(N–P) RT-PCR analysis of ascl1a, lin28a, her4.1, and insm1a in uninjured control, 2.5 dpi DMSO-treated, and 2.5 dpi cyclopamine-treated retina (N); qPCR analysis of mRNA levels of insm1a and her4.1 with cyclopamine treatment (O); and BF images of corresponding mRNA in situ hybridization (ISH) of these genes in the retina at 4 dpi (P).

(Q) Single-cell-stage embryos were injected with insm1a:luciferase or her4.1:luciferase vectors along with Renilla luciferase mRNA for normalization and then treated with cyclopamine for 24 hr before lysing for quantification of insm1a and her4.1 promoter activity using a dual luciferase assay.

Scale bars represent 10 μm in (C), (D), (H), (I), (L), and (P) and 500 μm in (F) and (K). Asterisk indicates the injury site (C, H, I, L, and P). Error bars represent SD. p < 0.0001 (J); p < 0.001 (M). n = 6 biological replicates. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; UC, uninjured control. See also Figures S1, S2, S6, and S7.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
ascl1a WT perforation: retina Adult regenerating tissue retina RTPCR
WT perforation: retina, chemical treatment: Cyclopamine Adult regenerating tissue retina RTPCR
disp1 WT perforation: retina Adult regenerating tissue retina RTPCR
disp2 WT perforation: retina Adult regenerating tissue retina RTPCR
GFP mi4Tg perforation: retina Adult regenerating tissue Muller cell IHC
gli1 WT perforation: retina Adult regenerating tissue retina RTPCR
gli2a WT perforation: retina Adult regenerating tissue retina RTPCR
gli3 WT perforation: retina Adult regenerating tissue retina RTPCR
mi4Tg perforation: retina Adult regenerating tissue Muller cell RTPCR
her4.1 WT perforation: retina 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
insm1a WT perforation: retina Adult regenerating tissue retina RTPCR
Adult regenerating tissue retina ISH
WT perforation: retina, chemical treatment: Cyclopamine Adult regenerating tissue retina RTPCR
Adult regenerating tissue retina ISH
lin28a WT perforation: retina Adult regenerating tissue retina RTPCR
WT perforation: retina, chemical treatment: Cyclopamine Adult regenerating tissue retina RTPCR
ptch1 WT perforation: retina Adult regenerating tissue retina RTPCR
mi4Tg perforation: retina Adult regenerating tissue Muller cell RTPCR
ptch2 WT perforation: retina Adult regenerating tissue retina RTPCR
shha WT perforation: retina Adult regenerating tissue retina RTPCR
mi4Tg perforation: retina Adult regenerating tissue Muller cell RTPCR
shhb WT perforation: retina Adult regenerating tissue retina RTPCR
smo WT perforation: retina Adult regenerating tissue retina RTPCR
mi4Tg perforation: retina Adult regenerating tissue Muller cell RTPCR
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-ptch1 IHC WT perforation: retina Adult regenerating tissue Muller cell
Ab1-shha IHC WT perforation: retina Adult regenerating tissue Muller cell
IHC mi4Tg perforation: retina Adult regenerating tissue Muller cell
Ab2-gli3 IHC WT perforation: retina Adult regenerating tissue Muller cell
Ab2-smo IHC WT perforation: retina Adult regenerating tissue Muller cell
Phenotype Details
Fish Conditions Stage Phenotype
WT chemical treatment: Cyclopamine Day 4 eye fused with eye, abnormal
WT perforation: retina, chemical treatment: Cyclopamine Adult Muller cell cell dedifferentiation decreased occurrence, abnormal
Adult regenerating tissue Muller cell decreased amount, abnormal
Adult regenerating tissue retina her4.1 expression decreased amount, abnormal
Adult regenerating tissue retina ascl1a expression decreased amount, abnormal
Adult regenerating tissue retina lin28a expression decreased amount, abnormal
Adult regenerating tissue retina her4.1 expression decreased amount, abnormal
Adult regenerating tissue retina insm1a expression increased amount, abnormal
Adult regenerating tissue retina insm1a expression increased amount, abnormal
WT amputation: caudal fin, chemical treatment: Cyclopamine Adult blastema decreased size, abnormal
Adult fin regeneration decreased rate, abnormal
WT + MO2-sufu perforation: retina Adult Muller cell cell dedifferentiation increased occurrence, abnormal
Adult regenerating tissue Muller cell increased amount, abnormal
WT + MO5-shha + MO6-shha perforation: retina Adult Muller cell cell dedifferentiation decreased occurrence, abnormal
Adult regenerating tissue Muller cell decreased amount, abnormal
mi4Tg perforation: retina, chemical treatment: Cyclopamine Adult Muller cell cell dedifferentiation decreased occurrence, abnormal
Adult regenerating tissue Muller cell 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.