Comparative Transcriptomic Analysis and Structure Prediction of Novel Newt Proteins

Notophthalmus viridescens (Red-spotted Newt) possess amazing capabilities to regenerate their organs and other tissues. Previously, using a de novo assembly of the newt transcriptome combined with proteomic validation, our group identified a novel family of five protein members expressed in adult tissues during regeneration in Notophthalmus viridescens. The presence of a putative signal peptide suggests that all these proteins are secretory in nature. Here we employed iterative threading assembly refinement (I-TASSER) server to generate three-dimensional structure of these novel Newt proteins and predicted their function. Our data suggests that these proteins could act as ion transporters, and be involved in redox reaction(s). Due to absence of transgenic approaches in N. viridescens, and conservation of genetic machinery across species, we generated transgenic Drosophila melanogaster to misexpress these genes. Expression of 2775 transcripts were compared between these five newly identified Newt genes. We found that genes involved in the developmental process, cell cycle, apoptosis, and immune response are among those that are highly enriched. To validate the RNA Seq. data, expression of six highly regulated genes were verified using real time Quantitative Polymerase Chain Reaction (RT-qPCR). These graded gene expression patterns provide insight into the function of novel protein family identified in Newt, and layout a map for future studies in the field

Total RNA Extraction from Transgenic Flies Misexpressing Foreign Genes to Perform Next Generation RNA Sequencing

Due to absence of transgenic approaches in Notopthalmus Viridescens (newt), and conservation of genetic machinery across species, we generated transgenic Drosophila melanogaster to misexpress unique genes from newt. Novel newt genes cloned, and inserted at attP site in Drosophila were misexpressed ubiquitously using tubulin Gal-4. Sample (total RNA) for RNA sequencing was collected at 3rd instar larval stage during which major developmental events takes place in Drosophila. Total RNA was extracted, and purified using RNA clean and ConcentratorTM. RNA quality was quantitated by calculating absorbance at 260 nm (A260) and 280 nm (A280) wavelengths using Nanodrop 2000 spectrophotometer. Good quality samples had A260/ A280 ratio greater than 2 and a peak at 260 nm. Our results show that following this protocol high quality of RNA was obtained. These high quality RNA samples were used for downstream processes e.g. Next generation RNA sequencing. Of the total 36,099 transcripts in Drosophila, 34,967 transcripts were detected, and 2775 transcripts were significantly regulated by misexpressing foreign gene (Unique gene from newt) in Drosophila . Genes involved in the developmental process, cell cycle, apoptosis, and immune response are among those that are highly enriched. Wingless/Wnt was one of the important evolutionarily conserved pathway that was differentially regulated

Comparative transcriptomic analysis and structure prediction of novel Newt proteins.

Notophthalmus viridescens (Red-spotted Newt) possess amazing capabilities to regenerate their organs and other tissues. Previously, using a de novo assembly of the newt transcriptome combined with proteomic validation, our group identified a novel family of five protein members expressed in adult tissues during regeneration in Notophthalmus viridescens. The presence of a putative signal peptide suggests that all these proteins are secretory in nature. Here we employed iterative threading assembly refinement (I-TASSER) server to generate three-dimensional structure of these novel Newt proteins and predicted their function. Our data suggests that these proteins could act as ion transporters, and be involved in redox reaction(s). Due to absence of transgenic approaches in N. viridescens, and conservation of genetic machinery across species, we generated transgenic Drosophila melanogaster to misexpress these genes. Expression of 2775 transcripts were compared between these five newly identified Newt genes. We found that genes involved in the developmental process, cell cycle, apoptosis, and immune response are among those that are highly enriched. To validate the RNA Seq. data, expression of six highly regulated genes were verified using real time Quantitative Polymerase Chain Reaction (RT-qPCR). These graded gene expression patterns provide insight into the function of novel protein family identified in Newt, and layout a map for future studies in the field

Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells

呼吸器の発生をつかさどるメカニズムの解明 --発生現象の発見に基づくES細胞から気管組織の作出へ--. 京都大学プレスリリース. 2020-09-03.The periodic cartilage and smooth muscle structures in mammalian trachea are derived from tracheal mesoderm, and tracheal malformations result in serious respiratory defects in neonates. Here we show that canonical Wnt signaling in mesoderm is critical to confer trachea mesenchymal identity in human and mouse. At the initiation of tracheal development, endoderm begins to express Nkx2.1, and then mesoderm expresses the Tbx4 gene. Loss of β-catenin in fetal mouse mesoderm causes loss of Tbx4+ tracheal mesoderm and tracheal cartilage agenesis. The mesenchymal Tbx4 expression relies on endodermal Wnt activation and Wnt ligand secretion but is independent of known Nkx2.1-mediated respiratory development, suggesting that bidirectional Wnt signaling between endoderm and mesoderm promotes trachea development. Activating Wnt, Bmp signaling in mouse embryonic stem cell (ESC)-derived lateral plate mesoderm (LPM) generates tracheal mesoderm containing chondrocytes and smooth muscle cells. For human ESC-derived LPM, SHH activation is required along with WNT to generate proper tracheal mesoderm. Together, these findings may contribute to developing applications for human tracheal tissue repair

β-Catenin inactivation is a pre-requisite for chick retina regeneration.

In the present study we explored the role of β-catenin in mediating chick retina regeneration. The chick can regenerate its retina by activating stem/progenitor cells present in the ciliary margin (CM) of the eye or via transdifferentiation of the retinal pigmented epithelium (RPE). Both modes require fibroblast growth factor 2 (FGF2). We observed, by immunohistochemistry, dynamic changes of nuclear β-catenin in the CM and RPE after injury (retinectomy). β-Catenin nuclear accumulation was transiently lost in cells of the CM in response to injury alone, while the loss of nuclear β-catenin was maintained as long as FGF2 was present. However, nuclear β-catenin positive cells remained in the RPE in response to injury and were BrdU-/p27+, suggesting that nuclear β-catenin prevents those cells from entering the cell cycle. If FGF2 is present, the RPE undergoes dedifferentiation and proliferation concomitant with loss of nuclear β-catenin. Moreover, retinectomy followed by disruption of active β-catenin by using a signaling inhibitor (XAV939) or over-expressing a dominant negative form of Lef-1 induces regeneration from both the CM and RPE in the absence of FGF2. Our results imply that β-catenin protects cells of the CM and RPE from entering the cell cycle in the developing eye, and specifically for the RPE during injury. Thus inactivation of β-catenin is a pre-requisite for chick retina regeneration

Inhibiting β-catenin/LEF/TCF transcriptional activity is sufficient to induce chick retina regeneration.

<p>(A) Schematic of a dominant negative <i>lef1</i> gene that was cloned into an RCAS vector. (B) RT-PCR confirms the RCAS DN-Lef1-HA construct is successfully expressing the HA tag in electroporated CM explants from E4 eyes. The amplified 127 bp region is shown in (A). (C) AMV-3C2 immunohistochemistry shows the presence of viral protein in RCAS DN-Lef1-HA electroporated CM explants after 48 hours. (D) RT-qPCR data shows the level of β-catenin/Lef1/TCF target genes Musashi-1 (<i>msi1</i>) and cyclin D1 (<i>cD1</i>) in RCAS DN-Lef1-HA electroporated CM explants compared to the RCAS GFP electroporated controls (<i>p</i> values shown represent significance). (E–F) Whole eye images show the amount of regeneration in the presence of RCAS GFP (E) and RCAS DN-Lef1-HA (F) at 3 d PR; arrows indicate the regenerating neuroepithelium growing out of the eye, which happens in some cases during regeneration. (G–I) AMV-3C2 immunohistochemistry shows the presence of viral proteins in RCAS GFP infected eyes (G) and in the regenerating neuroepithelium from the CM (H) and RPE transdifferentiation (I) in RCAS DN-Lef1-HA infected eyes. (J) Quantitative analysis shows the difference in amount of regeneration observed in histological sections of RCAS DN-Lef1-HA infected eyes and RCAS GFP infected eyes (<i>p</i> values shown represent significance). (K–L) Histological sections of RCAS GFP and RCAS DN-Lef1-HA infected eyes at 3 d PR. Cr = ciliary regeneration; Td = transdifferentiation; L = lens; RPE: retina pigmented epithelium. Scale bars in (C), (K) and (L) represent 200 µm; Scale bars in (E) and (F) represent 1 mm; Scale bar in (G, H and I) represents 100 µm. Error bars in (D) and (J) represent S.E.M.</p

Dynamic changes of nuclear β-catenin in the chick eye after injury.

<p>(A–L) Presence of nuclear β-catenin (Nu β-cat) and Sox2 in the CM (A, B, E, F, I, J) and RPE (C, D, G, H, K, L) at E5 (A–D), at 1 d PR (E–H) and at 3 d PR (I–L). Panels A, C, E, G, I, K include DIC overlay and are equivalent to B, D, F, H, J, and L respectively. NPE: non-pigmented epithelium; PE: pigmented epithelium; OCL: optic cup lip; RPE: retinal pigmented epithelium; R: retina. DAPI stains the nuclei of the cells in B, D, F, H, J and L. Scale bar in (A) represents 100 µm and applies to all panels.</p