ZFIN ID: ZDB-FIG-120301-43
Wan et al., 2012 - HB-EGF Is Necessary and Sufficient for Müller Glia Dedifferentiation and Retina Regeneration. Developmental Cell   22(2):334-347 Full text @ Dev. Cell
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibody:
Fish:
Conditions:
Anatomical Terms:
Stage: Adult

Fig. 5

Notch Inhibition Stimulates MG Proliferation and Expands the Zone of Dedifferentiated MG in the Injured Retina via Induction of hb-egfa Gene Expression (A) DAPT treatment of 1016 tuba1a:gfp transgenic fish results in an expansion of the zone of dedifferentiated MG (GFP+/BrdU+) in the injured retina. Scale bar, 50 μm. (B) RT-PCR (gel) and real-time PCR (4 dpi, graph) showing that DAPT induces ascl1a, hb-egfa, and egfr mRNA expression in the injured retina. **p < 0.01 for ascl1a and hb-egfa; *p < 0.05 for egfr. (C) In situ hybridization showing that DAPT expands ascl1a mRNA expression in the injured retina (* marks the injury site). Scale bar, 50 μm. (D) ascl1a:gfp transgenic fish exhibit injury- and Notch-dependent regulation of transgene expression. Scale bar, 50 μm. (E) DAPT stimulates hb-egfa mRNA expression in proliferating MG-derived progenitors (arrows) of the injured retina. Scale bar, 50 μm. (F) MAPK inhibition (PD98059 and SL327) and EGFR inhibition (PD153035) suppress DAPT-dependent expansion of MG proliferation in the injured retina (asterisks in photomicrographs identify injury sites). Graph quantifies data represented in photomicrographs. *p < 0.01. (G) BrdU immunofluorescence on retinal sections reveals BrdU incorporation in the injured retinas of WT and hsp70:gal4;uas:nicd-myc transgenic fish overexpressing NICD. Scale bar, 50 μm. *p < 0.01. (H) In situ hybridization assays for hb-egfa (arrows) and ascl1a mRNA expression in WT and hsp70:gal4;uas:nicd-myc fish overexpressing NICD (* marks the injury site). Scale bar, 50 μm. (I) RT-PCR shows that ascl1a, hb-egfa, and pax6b mRNA expression is inhibited by NICD overexpression. (J) NICD-myc overexpression suppresses HB-EGF-induced cell proliferation in the uninjured retina. BrdU+ cells in HB-EGF-treated retinas overexpressing NICD-myc are generally NICD negative (arrows). Scale bars are 150 μm in low-magnification images and 50 μm in higher-magnification images. Error bars represent SD. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. See also Figure S6.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
ascl1a WT standard conditions Adult retina RTPCR
WT physical alteration: anatomical structure, chemical treatment: DAPT Adult retina ISH
Adult retina RTPCR
WT physical alteration: anatomical structure Adult retina ISH
Adult retina RTPCR
kca3Tg; kca4Tg control Adult Muller cell ISH
EGFP mi30Tg physical alteration: anatomical structure, chemical treatment: DAPT Adult retina IFL
mi30Tg physical alteration: anatomical structure Adult retina IFL
GFP mi14Tg physical alteration: anatomical structure, chemical treatment: DAPT Adult retina IFL
mi14Tg physical alteration: anatomical structure Adult retina IFL
hbegfa WT standard conditions Adult retina RTPCR
WT physical alteration: anatomical structure, chemical treatment: DAPT Adult retina RTPCR
WT physical alteration: anatomical structure Adult retina RTPCR
kca3Tg; kca4Tg control Adult Muller cell ISH
pax6b WT standard conditions Adult retina RTPCR
WT physical alteration: anatomical structure Adult retina RTPCR
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
zpr-1 IHC WT physical alteration: anatomical structure, chemical treatment: DAPT Adult retina
IHC kca3Tg; kca4Tg standard conditions Adult retina
Phenotype Details No data available
Acknowledgments:
ZFIN wishes to thank the journal Developmental Cell for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Cell, 22(2), Wan, J., Ramachandran, R., and Goldman, D., HB-EGF Is Necessary and Sufficient for Müller Glia Dedifferentiation and Retina Regeneration, 334-347, Copyright (2012) with permission from Elsevier. Full text @ Dev. Cell