ZFIN ID: ZDB-FIG-180716-26
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:
Knockdown Reagents:
Anatomical Terms:
Stage: Adult
PHENOTYPE:
Fish:
Conditions:
Knockdown Reagents:
Observed In:
Stage: Adult

Fig. 4

Shh-Mmp9-Ascl1a Interplay Is Necessary during MG Reprogramming

(A) RT-PCR (top) and qPCR (bottom) analysis of injury-dependent mmp9 expression in the retina; n = 6 biological replicates.

(B–D) BF microscopy images of mmp9 mRNA ISH in the retina at 4 dpi with cyclopamine treatment and shha or sufu knockdown (B), as quantified by qPCR (C), and a luciferase assay in 24 hpf embryos injected with mmp9:GFP-luciferase vector (D).

(E–G) IF microscopy images of 4 dpi retina with Mmp9 blockade using drugs (E) and MO against mmp9 (F). The number of BrdU+ MGPCs is quantified in (G).

(H) FISH and IF microscopy images of a 0.5-μm-thick optical section of retina showing co-localization of mmp9 and ascl1a in BrdU+ MGPCs at 4 dpi. Arrowheads mark co-expression of genes in BrdU+ cells.

(I) BF microscopy images of ascl1a and mmp9 mRNA ISH in ascl1a and mmp9 knockdowns in 4 dpi retina.

(J) Western blotting experiment showing Shh levels in 2 dpi retina with the mmp9 knockdown.

(K) Western blotting assay of Ascl1a in 2 dpi retina with shha or sufu knockdowns.

(L) Western blotting assay of Ascl1a in 2 dpi zebrafish retina injected with recombinant SHH protein.

(M) Western blotting assay of ASCL1 in 6 dpi mouse retina injected with recombinant SHH protein.

(N) Western blotting assay of Shha in DAPT-treated retina at 1dpi.

(O and P) RT-PCR (top) and qPCR (bottom) analysis of ascl1a and mmp9 in DAPT-treated retina, with or without ascl1a or mmp9 knockdown (O), and confirmed by western blotting assay (P).

(Q) FISH and IF microscopy images of a 0.5-μm-thick optical section of retina showed substantial co-exclusion and marginal co-localization of mmp9 with her4.1 at 4 dpi. Arrowheads mark co-expression of the gene, and arrows mark her4.1+ cells.

(R and S) Schematic of the mmp9 promoter with potential Hes/Her-BS binding sites (inside box), and luciferase assay in 24 hpf embryos co-injected with mmp9:GFP-luciferase construct and notch intracellular domain (nicd) mRNA (S).

Scale bars represent 10 μm (H and Q) and 20 μm (B, E, F, and I). Asterisk indicates the injury site (B, E, F, H, I and Q). Error bars represent SD. p < 0.001 (C, D, G, and S). Biological replicates n = 6 in (C) and (G), and n = 3 in (D) and (S). See also Figures S3, S4, 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 RTPCR
Adult regenerating tissue retina ISH
WT perforation: retina, chemical treatment: DAPT Adult regenerating tissue retina RTPCR
WT + MO3-ascl1a perforation: retina Adult regenerating tissue retina ISH
WT + MO3-ascl1a perforation: retina, chemical treatment: DAPT Adult regenerating tissue retina RTPCR
WT + MO7-mmp9 perforation: retina Adult regenerating tissue retina ISH
WT + MO7-mmp9 perforation: retina, chemical treatment: DAPT Adult regenerating tissue retina RTPCR
her4.1 WT perforation: retina Adult regenerating tissue retina ISH
mmp9 WT perforation: retina Adult regenerating tissue Muller cell ISH
Adult regenerating tissue retina RTPCR
Adult regenerating tissue retina ISH
WT perforation: retina, chemical treatment: DAPT Adult regenerating tissue retina RTPCR
WT perforation: retina, chemical treatment: Cyclopamine Adult regenerating tissue retina RTPCR
Adult regenerating tissue retina ISH
WT + MO2-sufu perforation: retina Adult regenerating tissue retina RTPCR
Adult regenerating tissue retina ISH
WT + MO3-ascl1a perforation: retina Adult regenerating tissue retina ISH
WT + MO3-ascl1a perforation: retina, chemical treatment: DAPT Adult regenerating tissue retina RTPCR
WT + MO5-shha + MO6-shha perforation: retina Adult regenerating tissue retina ISH
Adult regenerating tissue retina RTPCR
WT + MO7-mmp9 perforation: retina Adult regenerating tissue retina ISH
WT + MO7-mmp9 perforation: retina, chemical treatment: DAPT Adult regenerating tissue retina RTPCR
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-shha IHC WT perforation: retina, chemical treatment: DAPT Adult regenerating tissue retina
IHC WT + MO7-mmp9 perforation: retina Adult regenerating tissue retina
Ab3-ascl1 IHC WT perforation: retina, chemical treatment: DAPT Adult regenerating tissue retina
IHC WT + MO2-sufu perforation: retina Adult regenerating tissue retina
IHC WT + MO3-ascl1a perforation: retina, chemical treatment: DAPT Adult regenerating tissue retina
IHC WT + MO5-shha + MO6-shha perforation: retina Adult regenerating tissue retina
IHC WT + MO7-mmp9 perforation: retina, chemical treatment: DAPT Adult regenerating tissue retina
WB WT perforation: retina Adult regenerating tissue retina
Phenotype Details
Fish Conditions Stage Phenotype
WT perforation: retina, chemical treatment: DAPT Adult regenerating tissue retina mmp9 expression increased amount, abnormal
Adult regenerating tissue retina Ab1-shha labeling increased amount, abnormal
Adult regenerating tissue retina ascl1a expression increased amount, abnormal
Adult regenerating tissue retina Ab3-ascl1 labeling increased amount, abnormal
WT perforation: retina, chemical treatment: Cyclopamine Adult regenerating tissue retina mmp9 expression increased amount, abnormal
Adult regenerating tissue retina mmp9 expression increased amount, abnormal
WT perforation: retina, chemical treatment: salvianolic acid B Adult Muller cell cell dedifferentiation decreased occurrence, abnormal
Adult regenerating tissue Muller cell decreased amount, abnormal
WT perforation: retina, chemical treatment: matrix metalloproteinase inhibitor Adult Muller cell cell dedifferentiation decreased occurrence, abnormal
Adult regenerating tissue Muller cell decreased amount, abnormal
WT + MO2-sufu perforation: retina Adult regenerating tissue retina mmp9 expression decreased amount, abnormal
Adult regenerating tissue retina mmp9 expression decreased amount, abnormal
Adult regenerating tissue retina Ab3-ascl1 labeling increased amount, abnormal
WT + MO3-ascl1a perforation: retina Adult regenerating tissue retina mmp9 expression increased amount, abnormal
WT + MO3-ascl1a perforation: retina, chemical treatment: DAPT Adult regenerating tissue retina Ab3-ascl1 labeling decreased amount, abnormal
Adult regenerating tissue retina ascl1a expression increased amount, abnormal
Adult regenerating tissue retina mmp9 expression increased amount, abnormal
WT + MO5-shha + MO6-shha perforation: retina Adult regenerating tissue retina Ab3-ascl1 labeling decreased amount, abnormal
Adult regenerating tissue retina mmp9 expression increased amount, abnormal
Adult regenerating tissue retina mmp9 expression increased amount, abnormal
WT + MO7-mmp9 perforation: retina Adult Muller cell cell dedifferentiation decreased occurrence, abnormal
Adult regenerating tissue Muller cell decreased amount, abnormal
Adult regenerating tissue retina ascl1a expression decreased amount, abnormal
Adult regenerating tissue retina Ab1-shha labeling decreased amount, abnormal
WT + MO7-mmp9 perforation: retina, chemical treatment: DAPT Adult regenerating tissue retina ascl1a expression decreased amount, abnormal
Adult regenerating tissue retina Ab3-ascl1 labeling decreased amount, abnormal
Adult regenerating tissue retina mmp9 expression increased 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.