Mechanistic target of rapamycin complex 1 signaling regulates cell proliferation, cell survival, and differentiation in regenerating zebrafish fins

[Background]:The mechanistic target of rapamycin complex1 (mTORC1) signaling pathway has been implicated in functions of multicellular processes, including cell growth and metabolism. Although recent reports showed that many signaling pathways, including Activin, Bmp, Fgf, sonic hedgehog, Insulin-like growth factor (IGF), Notch, retinoic acid, and Wnt, are implicated in non-mammalian vertebrate regeneration, also known as epimorphic regeneration, mTORC1 function remains unknown. [Results]:To investigate the role of mTORC1 signaling pathway in zebrafish caudal fin, we examined the activation and function of mTORC1 signaling using an antibody against phosphorylated S6 kinase and a specific inhibitor, rapamycin. mTORC1 signaling is activated in proliferative cells of intra-ray and wound epidermal cells before blastema formation, as well as in proliferative blastema cells, wound epidermal cells, and osteoblasts during regenerative outgrowth. Before blastema formation, proliferation of intra-ray and wound epidermal cells is suppressed, but cell death is not affected by mTORC1 signaling inhibition with rapamycin. Moreover, rapamycin treatment inhibits blastema and wound epidermal cell proliferation and survival during blastema formation and regenerative outgrowth, as well as osteoblast proliferation and differentiation during regenerative outgrowth. We further determined that mTORC1 signaling is regulated through IGF-1 receptor/phosphatidylinositol-3 kinase and Wnt pathways during fin regeneration. [Conclusion]:Taken together, our findings reveal that mTORC1 signaling regulates proliferation, survival, and differentiation of intra-ray cells, wound epidermis, blastema cells, and/or osteoblasts in various fin regeneration stages downstream of IGF and Wnt signaling pathways.This study was supported by grants from Grant-in-Aid for Scientific Research from the JSPS (KAKENHI 23616002) to Y.K., and from Hiroshima University Alumni Association Research Grant & Hiroshima University Support Foundation Research and Grant-in-Aid for Scientific Research from the JSPS (KAKENHI 26 · 6771) to K.H

DEAD-Box Protein Ddx46 Is Required for the Development of the Digestive Organs and Brain in Zebrafish

Spatially and temporally controlled gene expression, including transcription, several mRNA processing steps, and the export of mature mRNA to the cytoplasm, is essential for developmental processes. It is well known that RNA helicases of the DExD/H-box protein family are involved in these gene expression processes, including transcription, pre-mRNA splicing, and rRNA biogenesis. Although one DExD/H-box protein, Prp5, a homologue of vertebrate Ddx46, has been shown to play important roles in pre-mRNA splicing in yeast, the in vivo function of Ddx46 remains to be fully elucidated in metazoans. In this study, we isolated zebrafish morendo (mor), a mutant that shows developmental defects in the digestive organs and brain, and found that it encodes Ddx46. The Ddx46 transcript is maternally supplied, and as development proceeds in zebrafish larvae, its ubiquitous expression gradually becomes restricted to those organs. The results of whole-mount in situ hybridization showed that the expression of various molecular markers in these organs is considerably reduced in the Ddx46 mutant. Furthermore, splicing status analysis with RT-PCR revealed unspliced forms of mRNAs in the digestive organ and brain tissues of the Ddx46 mutant, suggesting that Ddx46 may be required for pre-mRNA splicing during zebrafish development. Therefore, our results suggest a model in which zebrafish Ddx46 is required for the development of the digestive organs and brain, possibly through the control of pre-mRNA splicing

Mechanistic target of rapamycin complex 1 signaling regulates cell proliferation, cell survival, and differentiation in regenerating zebrafish fins

[Background]:The mechanistic target of rapamycin complex1 (mTORC1) signaling pathway has been implicated in functions of multicellular processes, including cell growth and metabolism. Although recent reports showed that many signaling pathways, including Activin, Bmp, Fgf, sonic hedgehog, Insulin-like growth factor (IGF), Notch, retinoic acid, and Wnt, are implicated in non-mammalian vertebrate regeneration, also known as epimorphic regeneration, mTORC1 function remains unknown. [Results]:To investigate the role of mTORC1 signaling pathway in zebrafish caudal fin, we examined the activation and function of mTORC1 signaling using an antibody against phosphorylated S6 kinase and a specific inhibitor, rapamycin. mTORC1 signaling is activated in proliferative cells of intra-ray and wound epidermal cells before blastema formation, as well as in proliferative blastema cells, wound epidermal cells, and osteoblasts during regenerative outgrowth. Before blastema formation, proliferation of intra-ray and wound epidermal cells is suppressed, but cell death is not affected by mTORC1 signaling inhibition with rapamycin. Moreover, rapamycin treatment inhibits blastema and wound epidermal cell proliferation and survival during blastema formation and regenerative outgrowth, as well as osteoblast proliferation and differentiation during regenerative outgrowth. We further determined that mTORC1 signaling is regulated through IGF-1 receptor/phosphatidylinositol-3 kinase and Wnt pathways during fin regeneration. [Conclusion]:Taken together, our findings reveal that mTORC1 signaling regulates proliferation, survival, and differentiation of intra-ray cells, wound epidermis, blastema cells, and/or osteoblasts in various fin regeneration stages downstream of IGF and Wnt signaling pathways.This study was supported by grants from Grant-in-Aid for Scientific Research from the JSPS (KAKENHI 23616002) to Y.K., and from Hiroshima University Alumni Association Research Grant & Hiroshima University Support Foundation Research and Grant-in-Aid for Scientific Research from the JSPS (KAKENHI 26 · 6771) to K.H

Phenotype of the <i>mor^ha4</i> mutant.

<p>(A–F) Lateral (A–D) and dorsal (E, F) views of live WT and <i>mor^ha4</i> larvae at 5.5 dpf. The swim bladder failed to inflate (arrows in A, B), the intestine lacked folds (arrowheads in C, D), and the retinae were reduced in size (brackets in E, F) in the <i>mor^ha4</i> mutant. Conversely, somite formation in the <i>mor^ha4</i> mutant appeared normal (arrowheads in A, B). (G–L) Sagittal sections of 5.5-dpf larvae stained with hematoxylin and eosin. The intestine lacked folds and was thin walled (arrowheads in G, H), and the exocrine pancreas (blue dotted lines in I, J) and liver (blue dotted lines in K, L) were small in the <i>mor^ha4</i> mutant. In contrast, the endocrine pancreas (blue dotted lines in I, J) in WT larvae was indistinguishable from that in <i>mor^ha4</i> larvae. Scale bars, 50 µm. (M–P) Dorsal views, anterior to the top (M, N). Lateral views, anterior to the left (O, P). Apoptotic cells were detected using the TUNEL method. An increase in apoptotic cells was evident in the brain, retinae, and posterior intestine of the <i>mor^ha4</i> larvae (white arrowheads in O, P) compared to WT larvae, but not in the <i>mor^ha4</i> somite (white arrows in O, P). en, endocrine pancreas; ex, exocrine pancreas.</p

<i>Ddx46</i> deficiency affects pre-mRNA splicing in the digestive organs and brain.

<p>(A–H) Scheme of the <i>dla</i>, <i>her6</i>, <i>ptf1a</i>, and <i>fabp10a</i> pre-mRNA regions analyzed for splicing (boxes, exons; lines, introns; arrows, primers) (A, C, E, G). The splicing status of <i>dla</i>, <i>her6</i>, <i>ptf1a</i>, and <i>fabp10a</i> pre-mRNA was monitored using RT-PCR with the primers indicated in scheme A, C, E, and G, respectively. Unspliced <i>dla</i>, <i>her6</i>, <i>ptf1a</i>, and <i>fabp10a</i> mRNAs were retained in the <i>Ddx46^{hi2137/hi2137}</i> mutant (mut) larvae compared to the control (con) larvae (arrowheads in B, D, F, H). Unspliced and spliced PCR products were verified by sequencing. +RT refers to the validation reaction itself, and −RT represents the respective control reaction without reverse transcriptase. <i>actb1</i> is a loading control by using primers designed in the exon 6. M, DNA size markers (sizes in bp); the asterisks point to nonspecific PCR products. Control larvae were sibling WT or <i>Ddx46^hi2137/+</i> larvae and had normal phenotypes.</p

<i>Ddx46</i> expression in the developing zebrafish.

<p>(A–K) <i>Ddx46</i> expression was examined using whole-mount <i>in situ</i> hybridization in WT embryos or larvae at the 128-cell (A), 6-somite (B), 1-dpf (C), 2-dpf (D–G), and 4-dpf (H–K) stages. Lateral view, animal pole to the top (A). Lateral views, anterior to the left (B, C, D, F, H, J). Dorsal views, anterior to the top (E, G, I, K). The <i>Ddx46</i> transcript was maternally supplied and continued to be expressed ubiquitously during the somitogenesis stages (A, B). By 1 dpf, <i>Ddx46</i> expression became restricted to the head region (C). At 2 dpf, strong <i>Ddx46</i> expression was prominent in the head, pectoral fin bud, and digestive organs (D–G). At 4 dpf, <i>Ddx46</i> expression was further restricted to the retina, telencephalon, midbrain, midbrain-hindbrain boundary, branchial arch, esophagus, liver, pancreas, and intestinal bulb (H–K). No <i>Ddx46</i> transcript was detected in the somite (arrowhead in H). (L, M) <i>Ddx46</i> expression was examined using whole-mount <i>in situ</i> hybridization in WT larvae at 3 dpf. Dorsal views, anterior to the top (L). A transverse section was cut at the level indicated by the black dotted line in L. The section revealed <i>Ddx46</i> expression in the intestine and exocrine pancreas, but not in the endocrine pancreas (M). b, branchial arches; e, esophagus; en, endocrine pancreas; ex, exocrine pancreas; i, intestine; ib, intestinal bulb; L, liver; m, midbrain; mhb, midbrain-hindbrain boundary; p, pancreas; pf, pectoral fin bud; r, retina; t, telencephalon.</p

Expression of molecular markers for digestive organs and brain is reduced in the <i>Ddx46^{hi2137/hi2137}</i> mutant.

<p>(A–D) The expression of <i>dla</i> and <i>her6</i> was examined using whole-mount <i>in situ</i> hybridization at 3 dpf. All lateral views, anterior to the left. (E–L) The expression of <i>fabp2</i>, <i>fabp10a</i>, <i>ptf1a</i>, and <i>ins</i> was examined using whole-mount <i>in situ</i> hybridization at 3.5 dpf. All dorsal views, anterior to the top. In the <i>Ddx46^{hi2137/hi2137}</i> mutants, the intensity and area of <i>dla</i>, <i>her6</i>, <i>fabp2</i>, <i>fabp10a</i>, and <i>ptf1a</i> expression were markedly reduced at 3 or 3.5 dpf (A–J; arrowheads in H, J). In contrast, the <i>ins</i> expression in the <i>Ddx46^{hi2137/hi2137}</i> mutant did not change at these developmental stages (K, L). (M–P) Transverse sections of 3.5-dpf <i>Ddx46^{hi2137/hi2137}</i> mutant larvae stained with hematoxylin and eosin. The transverse sections were cut at the levels indicated by black dotted lines in E–L. The tissues in the intestinal bulb, liver, and exocrine pancreas were still present in the <i>Ddx46^{hi2137/hi2137}</i> mutant larvae at 3.5 dpf. Scale bars, 50 µm. en, endocrine pancreas; ex, exocrine pancreas; ib, intestinal bulb; L, liver. Control larvae were sibling WT or <i>Ddx46^hi2137/+</i> larvae and had normal phenotypes.</p