Yap-lin28a axis targets let7-Wnt pathway to restore progenitors for initiating regeneration
- Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, China
- Department of Otolaryngology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, China
- Key Laboratory of Stem Cells and Tissue Engineering, Zhongshan School of Medicine, Sun Yat-Sen University, Ministry of Education, China
Figures
Figure 1 with 2 supplements
Exhausted sox2+ progenitors were able to restore quickly post severe injury.
(A, B) ET4;ET20 larvae were treated with neomycin, LY411575 (3dpf-5dpf), or neomycin following LY (LY+neo), and collected at indicated time points post neomycin treatment for sox2 immunostaining. …
Figure 1—figure supplement 1
Severe injury causes damage to HCs, SCs and MCs.
ET4;ET20 larvae were treated with 2 μM LY from 3dpf to 5dpf followed by neomycin, and the number of HCs, SCs and MCs were counted before and right after neo treatment. Results showed that HCs were …
Figure 1—figure supplement 2
More proliferative SCs and MCs were induced post severe injury compared with normal injury.
Larvae treated with neo or LY+neo were incorporated with EdU for different time points and counted for numbers of differentiating cells (EdU+ET4+ cells, HC+), proliferative SCs (EdU+ET4-ET20- cells, …
Figure 2 with 2 supplements
Activated yap upregulate lin28a transcription in atoh1a+ HC precursors post severe injury.
(A) Lin28a was not expressed in the developing lateral line primordium or neuromast. (B) Treatment with Notch inhibitor LY411575 from 3dpf to 5dpf increased expression atoh1a and s100t. More lin28a …
Figure 2—figure supplement 1
lin28a was induced by various kinds of injury and lin28a expression level was proportional to injury size.
(A) RNA-Seq analysis using neomycin-treated neuromast cells revealed that lin28a was highly induced at 1 hr post neo while lin28b expression wa s not changed. (B) lin28a was induced at 1 hr post …
Figure 2—figure supplement 2
Yap was activated immediately post LY+neo.
(A) Wnt inhibition with hs:dkk1 has no effect on lin28a induction post neo treatment. (B) Expression levels of lin28a, yap, taz and Hippo pathway genes, such as lats1 and mob1, were detected by …
Figure 3 with 2 supplements
Yap-lin28a pathway is essentially required for progenitor recovery post severe injury.
(A) The number of SCs and total cells were counted at 48 hr post LY+neo and were significantly decreased in lin28a mutant compared with sibling. (B–E) The ET4;ET20 larvae were incorporated with EdU …
Figure 3—figure supplement 1
Create lin28a mutant using CRISPR.
(A) Diagram shows four exons of lin28a and the position of CIRPSR that was used for creating lin28a mutant. (B–C) Deletion of five nucleotides in the lin28a mutant allele damages the restriction …
Figure 3—figure supplement 2
HCs and sox2+ progenitors were not affected by lin28a deficiency in homeostasis, LY and LY+neo.
We compared numbers of ET4+ HCs and sox2+ progenitors in 5dpf sibling and lin28a-/- and found that they were not changed in lin28-/- in homeostasis (A), LY (B) and LY+neo (C). Scale bar equals 10 μm.
Figure 4 with 1 supplement
Overexpression of lin28a is sufficient to restore the exhausted progenitors.
(A–D) ET4;ET20;hs:lin28a larvae were incorporated with BrdU for 24 hr following heat-shock and/or neomycin treatment. (A, B) Overexpression of lin28a increased number of proliferative SCs (SC+) in …
Figure 4—figure supplement 1
Developing HCs and MCs were not affected in hs:lin28a.
The ET4;ET20;hs:lin28a larvae were heat-shocked at 3dpf or 5dpf and collected after 24 hr for HC and MC counting. Results showed that numbers of HCs and MCs were not significantly changed by …
Figure 5 with 1 supplement
Yap-lin28a pathway promotes sox2 expression in atoh1a+ HC precursors.
(A–B) Larvae were stained with sox2 antibody and atoh1a RNA probe at 6 hr post neo or LY+neo treatment. The number of atoh1a+sox2+ cells was significantly increased in LY+neo group. (C–D) The atoh1a:…
Figure 5—figure supplement 1
Atoh1a+ cells became SCs and MCs post severe injury.
(A) The Neo-treated atoh1a:TdTomato;brn3c:GFP larvae was imaged for time lapse. Results showed that one Tomato+ cell turned into two HCs with membrane-bound GFP post neo (blue dots). (B) The …
Figure 6 with 1 supplement
MicroRNA let7 acts downstream of lin28a to activate Wnt pathway for promoting progenitor regeneration.
(A) We created hs:let7 transgenic line and found that it recapitulates the phenotype of lin28a-/- by decreasing proliferative SCs post LY+neo. (B) The induction of EdU+ proliferative cells was …
Figure 6—figure supplement 1
Notch and Fgf pathways were not affected by lin28a overexpression.
In situ results show that expression of her4 and dkk2, Notch pathway genes, and fgf3, a Fgf pathway gene, were not affected in hs:lin28a post neo.
Figure 7
Schematic model illustrates how Yap-lin28a pathway regulates progenitor regeneration post severe injury.
(A) It’s known that sox2+ progenitors that are preserved post neo-induced HC injury divide and differentiate to regenerate HCs. To simulate the situation in mammalian inner ear where the progenitors …
Author response image 1
Some SCs and MCs in neuromast are negative with sox2 expression.
(A)ET4;ET20 Larvae were treated with neo and immediately collected for sox2 immunostaining. The white arrowheads pointed out SCs and MCs that were sox2 negative. (B) Total number of SCs or MCs and …
Author response image 2
Sox2+ progenitors were eliminated and atoh1a+ HC precursors dominated post severe injury.
(A) Larvae were treated with LY, neo or LY+neo and collected immediately after neo treatment for sox2 immunostaining. Sox2+ cell numbers were counted, and the results showed that very few sox2+ …
Videos
Video 1
ET4;ET20;cldnB:H2Amcherry larvae treated with LY+neo were processed for time lapse.
Results showed the intensive cell divisions (CDs) during severe-injury-induced regeneration. Scale bar equals 10 μm.
Video 2
The neuromast of atoh1a:TdTomato;brn3c:GFP larvae treated with neo was imaged for time lapse.
Result showed that one Tomato+ cell divided and turned into two GFP+hair cells post neo. Scale bar equals 10 μm.
Video 3
The neuromast of atoh1a:TdTomato;sox2:GFP larvae treated with LY+neo was imaged for time lapse.
One Tomato+ cell (blue dot) divided and turned into two HCs that are GFP negative in the center. The other two Tomato+ cells (white dots) divided and converted into four sox2+ progenitors. Scale bar …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Danio rerio) | Et(krt4:EGFP)sqet20ET | Parinov et al., 2004 | sqET20; RRID:ZFIN_ZDB-ALT-070628-20 | |
| Genetic reagent (Danio rerio) | Et(krt4:EGFP)sqet4ET | Parinov et al., 2004 | sgET4; RRID:ZFIN_ZDB-GENO-110323-1 | |
| Genetic reagent (Danio rerio) | Tg(atoh1a: TdTomato)nns8 | Wada et al., 2010 | nns8; RRID:ZFIN_ZDB-GENO-120530-1 | |
| Genetic reagent (Danio rerio) | sox2-2a-sfGFPstl84 | Shin et al., 2014 | stl84; RRID:ZFIN_ZDB-GENO-150721-11 | |
| Genetic reagent (Danio rerio) | Tg(brn3c:GAP43-GFP) s356t | Xiao et al., 2005 | S356t; RRID:ZFIN_ZDB-GENO-200218-3 | |
| Genetic reagent (Danio rerio) | Tg(hsp70l:dkk1-GFP)w32 | Stoick-Cooper et al. (2007) | W32; RRID:ZFIN_ZDB-ALT-070403-1 | |
| Genetic reagent (Danio rerio) | Tg(hsp70l:myc-notch1a;cryaa:Cerulean)fb12 | Zhao et al. (2014) | fb12; RRID:ZFIN_ZDB-ALT-140522-5 | |
| Genetic reagent (Danio rerio) | Tg(hsp70:atoh1a)x20 | Millimaki et al. (2010) | X20; RRID:ZFIN_ZDB-GENO-110315-10 | |
| Genetic reagent (Danio rerio) | yapmw48 | Miesfeld et al., 2015 | Ms48; RRID:ZFIN_ZDB-ALT-160122-5 | |
| Genetic reagent (Danio rerio) | Tg(hsp70:lin28a-P2Amcherry;cmlc:GFP)psi30 | this paper | Details in Fish strain information | |
| Genetic reagent (Danio rerio) | Tg(hsp70:let7-P2AGFP;cryaa:Venus) | this paper | Details in Fish strain information | |
| Genetic reagent (Danio rerio) | lin28apsi37 | this paper | Details in Fish strain information | |
| Antibody | anti-sox2 (Rabbit, polyclonal) | Abcam | Cat# Ab97959; RRID:AB_2341193 | IF(1:200) |
| Antibody | anti-yap (Rabbit, polyclonal) | CST | Cat# 4912, RRID:AB_2218911 | IF(1:200) |
| Antibody | anti-taz (Rabbit, polyclonal) | Abcam | Cat# Ab84927; RRID:AB_1925489 | IF (1:200), WB (1:500) |
| Antibody | anti-GFP (Mouse, monoclonal) | Invitrogen | Cat# A11120; RRID:AB_221568 | IF(1:500) |
| Antibody | Anti-yap (Mouse, monoclonal) | Santa Cruz | Cat# sc-271134; RRID:AB_10612397 | WB (1:1000) |
| Antibody | Anti-lin28a (Rabbit, polyclonal) | CST | Cat# 3978; RRID:AB_2297060 | WB (1:500) |
| Antibody | Anti-lats (Rabbit, monoclonal) | CST | Cat# 3477; RRID:AB_2133513 | WB (1:500) |
| Antibody | Anti-p-mob1 (Rabbit, monoclonal) | CST | Cat# 8699; RRID:AB_11139998 | WB (1:500) |
| Antibody | Anti-β-actin (Mouse, monoclonal) | Sigma | Cat# A1978; RRID:AB_476692 | WB (1:2000) |
| Antibody | Anti-digoxingenin POD (sheep, polyclonal) | Roche | 11207733910; RRID:AB_514500 | 1:2000 |
| Antibody | Anti-fluorescein POD(sheep, polyclonal) | Roche | 11426346910; RRID:AB_840257 | 1:2000 |
| Chemical compound, drug | EdU | Carbosynth | NE08701 | 3.3 mM |
| Chemical compound, drug | LY411575 | Santa Cruz | sc-364529 | 2 μM |
| Chemical compound, drug | Neomycin sulfate | Sigma | N6386 | 300 μM |
| Chemical compound, drug | Alexa Fluor-594 Azide | Thermo Fisher Scientific | N6386 | |
| Chemical compound, drug | Verteporfin | Selleckchem | S1786 | 5 μM |
| Chemical compound, drug | Copper sulfate | Sigma | 451657 | 50 μM |
| Chemical compound, drug | cisplatin | Sigma | 33342 | 500 μM |
| Commercial assay or kit | TSA-Cyanine 3 Reagent | PerkinElmer | SAT704A001EA | |
| Commercial assay or kit | TSA-FITC Reagent | PerkinElmer | SAT704A001EA | |
| Commercial assay or kit | dynabeads | Invitrogen | 10015D |
