scholarly journals Two Different Transgenes to Study Gene Silencing and Re-Expression During Zebrafish Caudal Fin and Retinal Regeneration

2006 ◽  
Vol 6 ◽  
pp. 65-81 ◽  
Author(s):  
Ryan Thummel ◽  
Christopher T. Burket ◽  
David R. Hyde
Keyword(s):  
Tissue Regeneration ◽  
Transgene Expression ◽  
Egfp Expression ◽  
Transgenic Zebrafish ◽  
Retinal Regeneration ◽  
Caudal Fin ◽  
Fin Regeneration ◽  
Differentiated Cells ◽  
Neuronal Progenitors ◽  
Wound Epithelium

We used the 500-bpXenopusef1-α promoter and the 2-kb zebrafish histone2A.F/Zpromoter to generate several independent transgenic zebrafish lines expressing EGFP. While both promoters drive ubiquitous EGFP expression in early zebrafish development, they are systematically silenced in several adult tissues, including the retina and caudal fin. However, EGFP expression is temporarily renewed in the adult during either caudal fin or retinal regeneration. In the Tg(H2A.F/Z:EGFP)ntline, EGFP is moderately expressed in both the wound epithelium and blastema of the regenerating caudal fin. In the Tg(ef1-α:EGFP)ntline, EGFP expression is reinitiated and restricted to the blastema of the regenerating caudal fin and colabels with BrdU, PCNA, andmsxc-positive cells. Thus, these two ubiquitous promoters drive EGFP transgene expression in different cell populations during caudal fin regeneration. We further analyzed the ability of theef1-α:EGFPtransgene to label nonterminally differentiated cells during adult tissue regeneration. First, we demonstrated that the transgene is highly methylated in adult zebrafish caudal fin tissue, but not during fin regeneration, implicating methylation as a potential means of transgene silencing in this line. Next, we determined that theef1-α:EGFPtransgene is also re-expressed during adult retinal regeneration. Specifically, theef1-α:EGFPtransgene colabels with PCNA in the Müglia, a specialized cell that is the source of neuronal progenitors during zebrafish retinal regeneration. Thus, we concluded that Tg(ef1-α:EGFP)nt line visually marks nonterminally differentiated cells in multiple adult regeneration environments and may prove to be a useful marker in tissue regeneration studies in zebrafish.

scholarly journals Cellular diversity of the regenerating caudal fin

2020 ◽  
Vol 6 (33) ◽  
pp. eaba2084
Author(s):  
Yiran Hou ◽  
Hyung Joo Lee ◽  
Yujie Chen ◽  
Jiaxin Ge ◽  
Fujr Osman Ibrahim Osman ◽  
...  
Keyword(s):  
State Transitions ◽  
Specific Cell ◽  
Caudal Fin ◽  
Wound Site ◽  
Fin Regeneration ◽  
Differentiated Cells ◽  
Mesenchymal Differentiation ◽  
Cell Type Specific ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Zebrafish faithfully regenerate their caudal fin after amputation. During this process, both differentiated cells and resident progenitors migrate to the wound site and undergo lineage-restricted, programmed cellular state transitions to populate the new regenerate. Until now, systematic characterizations of cells comprising the new regenerate and molecular definitions of their state transitions have been lacking. We hereby characterize the dynamics of gene regulatory programs during fin regeneration by creating single-cell transcriptome maps of both preinjury and regenerating fin tissues at 1/2/4 days post-amputation. We consistently identified epithelial, mesenchymal, and hematopoietic populations across all stages. We found common and cell type–specific cell cycle programs associated with proliferation. In addition to defining the processes of epithelial replenishment and mesenchymal differentiation, we also identified molecular signatures that could better distinguish epithelial and mesenchymal subpopulations in fish. The insights for natural cell state transitions during regeneration point to new directions for studying this regeneration model.

scholarly journals In vivo monitoring of tissue regeneration using a ratiometric lysosomal AIE probe

2020 ◽  
Vol 11 (12) ◽  
pp. 3152-3163 ◽  
Author(s):  
Xiujuan Shi ◽  
Neng Yan ◽  
Guangle Niu ◽  
Simon H. P. Sung ◽  
Zhiyang Liu ◽  
...  
Keyword(s):  
Tissue Regeneration ◽  
Caudal Fin ◽  
Fin Regeneration ◽  
Lysosomal Ph ◽  
In Vivo Monitoring ◽  
Ratiometric Probe ◽  
First Time

An AIE-active ratiometric probe for the first time achieved the long-term quantification of lysosomal pH during the medaka larva's caudal fin regeneration.

scholarly journals Inhibition of BMP and FGF signaling prior to wound epithelium formation leads to an aberrant regenerative response in teleost fish Poecilia latipinna

2021 ◽  
Author(s):  
Isha Ranadive ◽  
Sonam Patel ◽  
Siddharth Pai ◽  
Kashmira Khaire ◽  
Suresh Balakrishnan
Keyword(s):  
Cell Death ◽  
Teleost Fish ◽  
The Other ◽  
Fgf Signaling ◽  
Caudal Fin ◽  
Fin Regeneration ◽  
Blastema Formation ◽  
Messenger Molecules ◽  
Wound Epithelium

The BMP and FGF pathways play a pivotal role in the successful regeneration of caudal fin of teleost fish. Individual inhibition of these pathways led to impaired caudal fin regeneration until the pharmacologic inhibitor of FGF (SU5402) and BMP (LDN193189) were metabolized off. Therefore, in the current study both these pathways were inhibited collectively wherein inhibition of BMP and FGF during the wound epithelium formation led to stalling of the process by bringing down the established levels of shh and runx2. In members of the treatment group, it was observed that, each blastema grows crouched rather than linear and the regrown lepidotrichia therefore remain tilted down. Amongst the other irregularities observed, the transition from epithelial to mesenchymal cells was found hindered due to down-regulation of snail and twist, brought about by BMP and FGF inhibition. Compromised expression of Snail and twist deranged the normal levels of cadherins causing disruption in the transition of cells. Lastly, blocking BMP and FGF delayed blastema formation and proliferation due to diminished levels of fgf2, fgf8, fgf10 and bmp6, while casp3 and casp9 levels remained heightened causing accelerated cell death. This study not only highlights the axial role of BMP and FGF pathways in regeneration but also accentuates the collaboration amongst the two. This ingenious coordination of signalling further reinforces the involvement of relaying messenger molecules between these crucial pathways.

scholarly journals NRG1/ErbB signalling controls the dialogue between macrophages and neural crest-derived cells during zebrafish fin regeneration

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Béryl Laplace-Builhé ◽  
Audrey Barthelaix ◽  
Said Assou ◽  
Candice Bohaud ◽  
Marine Pratlong ◽  
...  
Keyword(s):  
Neural Crest ◽  
Transgenic Zebrafish ◽  
Critical Function ◽  
Caudal Fin ◽  
Fin Regeneration ◽  
Blastema Formation ◽  
Zebrafish Larvae ◽  
Single Cell Rna Sequencing ◽  
Chemical Inhibition ◽  
Macrophage Subsets

AbstractFish species, such as zebrafish (Danio rerio), can regenerate their appendages after amputation through the formation of a heterogeneous cellular structure named blastema. Here, by combining live imaging of triple transgenic zebrafish embryos and single-cell RNA sequencing we established a detailed cell atlas of the regenerating caudal fin in zebrafish larvae. We confirmed the presence of macrophage subsets that govern zebrafish fin regeneration, and identified a foxd3-positive cell population within the regenerating fin. Genetic depletion of these foxd3-positive neural crest-derived cells (NCdC) showed that they are involved in blastema formation and caudal fin regeneration. Finally, chemical inhibition and transcriptomic analysis demonstrated that these foxd3-positive cells regulate macrophage recruitment and polarization through the NRG1/ErbB pathway. Here, we show the diversity of the cells required for blastema formation, identify a discrete foxd3-positive NCdC population, and reveal the critical function of the NRG1/ErbB pathway in controlling the dialogue between macrophages and NCdC.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

Vitamin C supplementation improves growth performance and caudal fin regeneration in zebrafish ( Danio rerio )

2021 ◽  
Author(s):  
Pedro Luiz Pucci Figueiredo Carvalho ◽  
Pedro Henrique Ventura Almeida ◽  
William dos Santos Xavier ◽  
Igor Simões Tiagua Vicente ◽  
Matheus Gardim Guimarães ◽  
...  
Keyword(s):  
Vitamin C ◽  
Growth Performance ◽  
Danio Rerio ◽  
Caudal Fin ◽  
Fin Regeneration ◽  
Vitamin C Supplementation

Glycomic and glycotranscriptomic profiling of mucin-type O-glycans in planarian Schmidtea mediterranea

Glycobiology ◽  
2021 ◽  
Author(s):  
Sabarinath Peruvemba Subramanian ◽  
Vairavan Lakshmanan ◽  
Dasaradhi Palakodeti ◽  
Ramaswamy Subramanian
Keyword(s):  
Tissue Regeneration ◽  
Cell Biology ◽  
Schmidtea Mediterranea ◽  
Ligand Interaction ◽  
Differentiated Cells ◽  
Cell Matrix ◽  
Composition And Structure ◽  
Enzyme Class ◽  
Cell Cell

Abstract O-Glycans on cell surfaces play important roles in cell-cell, cell-matrix, and receptor-ligand interaction. Therefore, glycan-based interactions are important for tissue regeneration and homeostasis. Free-living flatworm Schmidtea mediterranea, because of its robust regenerative potential, is of great interest in the field of stem cell biology and tissue regeneration. Nevertheless, information on the composition and structure of O-glycans in planaria is unknown. Using mass spectrometry and in silico approaches, we characterized the glycome and the related transcriptome of mucin-type O-glycans of planarian S. mediterranea. Mucin-type O-glycans were composed of multiple isomeric, methylated, and unusually extended mono- and di-substituted O-GalNAc structures. Extensions made of hexoses and 3-O methyl hexoses were the glycoforms observed. From glycotranscriptomic analysis, sixty genes belonging to five distinct enzyme classes were identified to be involved in mucin-type O-glycan biosynthesis. These genes shared homology with those in other invertebrate systems. While a majority of the genes involved in mucin-type O-glycan biosynthesis was highly expressed during organogenesis and in differentiated cells, a few select genes in each enzyme class were specifically enriched during early embryogenesis. Our results indicate a unique temporal and spatial role for mucin-type O-glycans during embryogenesis and organogenesis and in adulthood. In summary, this is the first report on O-glycans in planaria. This study expands the structural and biosynthetic possibilities in cellular glycosylation in the invertebrate glycome and provides a framework towards understanding the biological role of mucin-type O-glycans in tissue regeneration using planarians.

scholarly journals Regrowth of zebrafish caudal fin regeneration is determined by the amputated length

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Toshiaki Uemoto ◽  
Gembu Abe ◽  
Koji Tamura
Keyword(s):  
Caudal Fin ◽  
Fin Regeneration

scholarly journals PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

2016 ◽  
Vol 113 (16) ◽  
pp. E2306-E2315 ◽  
Author(s):  
Vashe Chandrakanthan ◽  
Avani Yeola ◽  
Jair C. Kwan ◽  
Rema A. Oliver ◽  
Qiao Qiao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Tissue Regeneration ◽  
Somatic Cells ◽  
Dependent Manner ◽  
Cell Plasticity ◽  
Differentiation Potential ◽  
Multipotent Stem Cells ◽  
Differentiated Cells

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

240 QUALITY AND VIABILITY OF IVF BOVINE EMBRYOS AFTER INTRACYTOPLASMIC INJECTION OF DNA–LIPOSOME COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 232
Author(s):  
L. N. Moro ◽  
G. Vichera ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Dna Fragmentation ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Egfp Expression ◽  
Bovine Embryos ◽  
Exogenous Dna ◽  
Intracytoplasmic Injection

Transgenic animals have important applications in agriculture and human medicine; nevertheless the available techniques still remain inefficient and technically difficult. We have recently developed a novel method to transfect bovine embryos that consists of intracytoplasmic injection of exogenous DNA–liposome complexes (eDNA-LC) in IVF zygotes. This study was designed to evaluate the quality and viability of IVF bovine embryos, after intracytoplasmic injection of pCX-EGFP–liposome complexes (EGFP-LC) or pBCKIP2.8-liposome complexes (plasmid that codifies the human insulin gene, HI-LC). First, we evaluated embryo development and enhanced green fluorescent protein (EGFP) expression of IVF embryos injected with both plasmids separately. This treatment was analysed by Fisher's Exact test (P ≤ 0.05). Cleavage rates for EGFP-LC, HI-LC and IVF embryos injected with liposomes alone (IVF-L) and IVF control (IVF-C) were 62% (63/102), 67% (67/100), 66% (67/101) and 79% (98/124); blastocysts rates were 17% (17/102), 21% (21/100), 21% (21/101) and 23% (28/124), respectively. No statistical differences were seen among groups. The percentage of EGFP-positive embryos (EGFP+) after EGFP-LC injection was 42.9% after 3 days of culture and 41.8% at the blastocyst stage. In the second experiment, the blastocysts obtained, EGFP+ or EGFP-negative (EGFP–), were analysed by TUNEL assay at Day 6 (Bd6), 7 (Bd7) and 8 (Bd8) of in vitro culture, in order to evaluate the effect of the transgene and culture length, on DNA fragmentation. This treatment was analysed by the difference of proportions test (P ≤ 0.05) using statistical INFOSTAT software. All EGFP+ blastocysts showed TUNEL positive cells (T+). The percentage of T+ in Bd6, Bd7 and Bd8 were 91, 73.7 and 99.5%, respectively (P ≤ 0.05). EGFP– blastocysts showed lower fragmented nuclei (0, 44.6 and 85%, respectively; P ≤ 0.05). Groups IVF-L and IVF-C were also evaluated. In both groups, there was no evidence of DNA fragmentation in Bd6 and Bd7, but T+ were detected in Bd8 (66.4 and 85.8%, respectively; P ≤ 0.05). In the third experiment, bovine blastocysts obtained from the HI-LC group were individually transferred to recipient cows after 6 (n = 11), 7 (n = 5) and 8 (n = 5) days of culture post-IVF and HI-LC injection. The pregnancies obtained were from Bd6 [18.2% (2/11)] and Bd7 [40% (2/5)], although none of the recipients receiving Bd8 were diagnosed pregnant. Two pregnancies developed to term, one derived from Bd6 and the other from Bd7. Analysis by PCR determined that none of the born cows were transgenic. In summary, IVF bovine embryos could be easily transfected after the injection of eDNA-LC and the technique did not affect offspring viability. The results indicate that extended time in in vitro culture increases the percentage of fragmented nuclei in blastocysts. Moreover, this parameter increases in blastocysts with transgene expression compared with those without expression. Finally, more transfers are required in order to obtain the real efficiency of this new technique and to overcome the drawbacks generated by in vitro culture length and transgene expression.

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