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Dev Biol
2023 Jun 02;500:31-39. doi: 10.1016/j.ydbio.2023.06.001.
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Downregulation of Yap1 during limb regeneration results in defective bone formation in axolotl.
Bay S
,
Öztürk G
,
Emekli N
,
Demircan T
.
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The Hippo pathway plays an imperative role in cellular processes such as differentiation, regeneration, cell migration, organ growth, apoptosis, and cell cycle. Transcription coregulator component of Hippo pathway, YAP1, promotes transcription of genes involved in cell proliferation, migration, differentiation, and suppressing apoptosis. However, its role in epimorphic regeneration has not been fully explored. The axolotl is a well-established model organism for developmental biology and regeneration studies. By exploiting its remarkable regenerative capacity, we investigated the role of Yap1 in the early blastema stage of limb regeneration. Depleting Yap1 using gene-specific morpholinos attenuated the competence of axolotl limb regeneration evident in bone formation defects. To explore the affected downstream pathways from Yap1 down-regulation, the gene expression profile was examined by employing LC-MS/MS technology. Based on the generated data, we provided a new layer of evidence on the putative roles of increased protease inhibition and immune system activities and altered ECM composition in diminished bone formation capacity during axolotl limb regeneration upon Yap1 deficiency. We believe that new insights into the roles of the Hippo pathway in complex structure regeneration were granted in this study.
Fig. 1. Yap1 was downregulated during axolotl limb regeneration using morpholino (MO). Neotenic axolotl Yap1 mRNA expression levels were 72 times more compared to metamorphic Yap1 mRNA expression at 10 days post amputation (dpa) (A) (0.01<p∗∗≤0.001). For determining YAP1 protein level, neotenic (B′) and metamorphic (B″) blastema tissues were stained with YAP1 antibody at 10 dpa (B). Percentage of YAP1-positive cells was 13,12% in neotenic axolotl while percentage of YAP1-positive cells was 3,03% in metamorphic axolotl at 10 dpa (B‴) (p∗∗∗<0.001). YAP-MO were synthesized for inhibition of axolotl Yap1 mRNAs. After MO injection to blastematissue of neotenic axolotl at 10 dpa, qRT-PCR analysis were performed at 16 dpa. Yap1 mRNA expression in YAP-MO animals decreased 3,48 times compared to control animals (C) (0.05≤p∗≤0.01). Yap1 in control axolotl (D′) and Yap-MO (D″) axolotl were imaged in blastema tissues (D). Yap-MO caused the level of Yap1 protein to decrease from 20,89% to 6,49% at 16 dpa (D‴). Scale bars represent 20 μm.
Fig. 2. YAP-MO caused bone defects during axolotl limb regeneration. After YAP-MO injection, macroscopic imaging's were performed at 10, 17, 24, 31 and 52 dpa. Different regeneration patterns were observed between control axolotls (n = 3) and YAP-MO axolotls (n = 3) as a result of long-term bright field imaging (A). Scale bars represent 1 mm. YAP-MO injection induced the decrease of blastema areas at 16 dpa. At 10 dpa, significant change was not determined between control axolotls (n = 3) and YAP-MO axolotls (n = 3). Blastema areas were decreased in YAP-MO axolotls (n = 3) compared to control axolotls (n = 3) (0.01<p∗∗≤0.001) (B). Macroscopic differences were not detected in the bright-field results after regeneration was completed. Micro-CT analysis was performed to determine whether there was a defect in bone development after YAP-MO injection at 52 dpa. Bone orientation defect and bone loss defect were imaged with Micro-CT (C).
Fig. 3. Expression levels of selected genes. Among the differentially expressed genes, 10 of them were selected for validation of proteomics. Red and blue bars show the fold change based on proteomics and qRT-PCR results, respectively (A). Expression level of osteogenesis and chondrogenesis related genes was compared between control and Yap1-KD group (B). ∗p-value < 0.05, ∗∗p-value < 0.01, ∗∗∗p-value < 0.001, ns; non-significant. Apoa4: Apolipoprotein A4, Bmp2: Bone morphogenetic protein 2, Bmp4:Bbone morphogenetic protein 4, C3: Complement component 3, Idh1: Isocitrate Dehydrogenase (NADP(+)) 1, Kng1: Kininogen-1, Ldb3: LIM domain binding 3, Mybpc3: Myosin-binding protein C 3, Myl4: Myosin Light Chain 4, Prdx2:Peroxiredoxin 2, Runx2: Runt-related transcription factor 2, Smad3: SMAD family member 3, Smad7: SMAD family member 3, Sox6: SRY-Box transcription factor 6, Sox9: SRY-Box transcription factor 9, Tgfb1: Transforming growth factor beta 1, Tnni1: troponin I type 1, Tpm2: Tropomyosin 2.
Fig. 4. Gene ontology analysis of differentially expressed proteins. (A) top 10 GO terms enriched by 208 upregulated proteins in Yap depleted samples compared to the control group. (B) top 10 GO terms enriched by 77 down regulated proteins in Yap depleted samples compared to the control group. (C) Gene-concept network of the top 3 immune-system related biological processes enriched by significantly upregulated proteins in Yap depleted groups. (D) Gene-concept network of the top 5 muscle system related biological processes enriched by significantly downregulated proteins in Yap depleted groups.
Fig. 5. KEGG and gene ontology analyses of identified proteins. (A) Enriched KEGG pathways by upregulated proteins in YAP knocked-down samples compared to control group. (B) Enriched KEGG pathways by downregulated proteins in YAP depleted samples compared to control group. (C) top 10 GO terms enriched by identified proteins using the gene set enrichment analysis. (D) KEGG pathways enriched by the protein list using the gene set enrichment analysis.
Fig. S1. Expression levels of Yap1 gene. Yap1 expression levels in the blastema stage at different time points were evaluated by qRT-PCR. (A) Relative mRNA levels of Yap1 at 1,7, 14, and 21 DPA compared to 0 DPA in neotenic and metamorphic axolotls. (B) Relative mRNA levels of Yap1 at 1,7, 10, 14, and 21 DPA between neotenic and metamorphic axolotls. **p-value < 0.01, ***p-value < 0.001, ns; non-significant.
