ZFIN ID: ZDB-FIG-180716-28
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. 6

Expression Dynamics and Necessity of Foxn4 during Regeneration

(A and B) RT-PCR (A) and qPCR (B) analysis of injury-dependent foxn4 expression in the retina; n = 6 biological replicates. (C) IF microscopy of a 0.5-μm-thick optical section of retina revealing co-localization of Foxn4 with BrdU+ MGPCs in 4 dpi retina.

(D and E) IF microscopy images of the retina with foxn4 knockdown at 4 dpi (D). The number of PCNA+ MGPCs is quantified in (E).

(F) BF microscopy images of foxn4 mRNA ISH in retinal sections with cyclopamine treatment and shha or gli1 knockdowns.

(G and H) Schematic of foxn4 promoter with a putative Gli-BS cluster, where arrows mark ChIP primers, N.S marks negative control, and capital letters mark putative Gli-BSs (G). A retinal ChIP assay at 4 dpi showing both Gli1 and Gli3 bound to the foxn4 promoter (H).

(I) BF microscopy images of foxn4 mRNA ISH in retinal sections with mmp9 or ascl1a knockdowns.

(J) FISH and IF microscopy images of a 0.5-μm-thick optical section of retina showing co-localization of foxn4 and ascl1a in BrdU+ MGPCs at 4 dpi. Arrowheads mark co-expression of genes in BrdU+ cells.

(K and L) Schematic of the foxn4 promoter with a putative Ascl1a-binding site cluster, where arrows mark ChIP primers, N.S marks negative control, and capital letters mark putative Ascl1a-BS (K). A retinal ChIP assay at 4 dpi showing Ascl1a bound to the foxn4 promoter (L).

(M) Luciferase assay showing foxn4 promoter activity with overexpression or knockdown of ascl1a in 24 hpf embryos.

(N) BF microscopy images of mRNA ISH in retinal sections with foxn4 knockdown showing levels of genes (namely, ascl1a, zic2b, mmp9, and foxn4) at 4 dpi.

(O) Luciferase assay showing ascl1a promoter activity with overexpression or knockdown of foxn4 in 24 hpf embryos.

(P and Q) Schematic of ascl1a and zic2b promoter with a putative Foxn4-binding site cluster, where arrows mark ChIP primers, N.S marks negative control, and capital letters mark putative Foxn4-BS (P). A retinal ChIP assay at 4 dpi showing Foxn4 bound to both the ascl1a and zic2b promoters (Q).

Scale bars represent 10 μm (C, D, F, I, J, and N). Error bars represent SD. p < 0.001 (M); p < 0.04 (O). Biological replicates n = 6 in (M) and O, and n = 3 in (B). Asterisk marks injury spots in (C),(D),(F), (J) and (N). See also Figures S5–S7.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
ascl1a WT perforation: retina Adult regenerating tissue Muller cell ISH
WT + MO3-foxn4 perforation: retina Adult regenerating tissue retina ISH
foxn4 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
WT + MO2-gli1 perforation: retina Adult regenerating tissue retina ISH
WT + MO3-ascl1a perforation: retina Adult regenerating tissue retina ISH
WT + MO3-foxn4 perforation: retina Adult regenerating tissue retina ISH
WT + MO5-shha + MO6-shha perforation: retina Adult regenerating tissue retina ISH
WT + MO7-mmp9 perforation: retina Adult regenerating tissue retina ISH
mmp9 WT perforation: retina Adult regenerating tissue retina ISH
WT + MO3-foxn4 perforation: retina Adult regenerating tissue retina ISH
zic2b WT perforation: retina Adult regenerating tissue retina ISH
WT + MO3-foxn4 perforation: retina Adult regenerating tissue retina ISH
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab12-pcna IHC WT perforation: retina Adult regenerating tissue retina
IHC WT + MO3-foxn4 perforation: retina Adult regenerating tissue retina
Phenotype Details
Fish Conditions Stage Phenotype
WT perforation: retina, chemical treatment: Cyclopamine Adult regenerating tissue retina foxn4 expression decreased amount, abnormal
WT + MO2-gli1 perforation: retina Adult regenerating tissue retina foxn4 expression decreased amount, abnormal
WT + MO3-ascl1a perforation: retina Adult regenerating tissue retina foxn4 expression decreased amount, abnormal
WT + MO3-foxn4 perforation: retina Adult regenerating tissue cell population proliferation decreased occurrence, abnormal
Adult regenerating tissue retina foxn4 expression decreased amount, abnormal
Adult regenerating tissue retina zic2b expression decreased amount, abnormal
Adult regenerating tissue retina ascl1a expression decreased amount, abnormal
Adult regenerating tissue retina mmp9 expression increased amount, abnormal
WT + MO5-shha + MO6-shha perforation: retina Adult regenerating tissue retina foxn4 expression decreased amount, abnormal
WT + MO7-mmp9 perforation: retina Adult regenerating tissue retina foxn4 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.