Preparation of a fish embryo for micromanipulation: staging of development, removal of the chorion and traceability of PGCs in Prochilodus lineatus

The transplantation of primordial germ cells (PGCs) is a valuable tool for gene-banking and reconstitution by means of a germline chimera. For this technology, studies regarding developmental stages and traceability of PGCs are necessary. The objective of this study was to develop a micromanipulation procedure for the future establishment of cryobanks of PGCs in migratory characins. Incubation temperatures were evaluated at 22 ° C, 26 ° C, and 30 ° C in order to synchronize developmental stages. The highest hatching rates and the lowest abnormality rate arose at 26° C, which was considered to be the best incubation temperature. Enzymatic removal of the chorion was determined to be best using 0.05% pronase, in which the embryos presented better survival rates. In order to visualize PGCs in vivo, artificial GFP-nos1 3’UTR mRNA was injected and the migration route was observed in vivo as PGCs were visualized firstly at the segmentation stage (6 to 13 somites). The number of GFP positive cells ranged from 8 to 20 per embryo (mean of 13.8; n = 5). After hatching, GFP-positive cells increased to 14 to 27 embryos (mean of 19.8; n = 5). Visualization of the GFP-positive cells was possible at 10 days post hatching, and at this stage, the cells were positioned in the yolk extension region. This is the first report on PGC visualization in vivo in Neotropical fish; the obtained data provide information on the identification and migration of PGCs. The information presented in this work brings new insights in gene banking in Neotropical species and subsequent reconstitution through a germinal germline chimera.

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Developmental stages, incubation temperature, and in vivo traceability of primordial germ cell in an important aquaculture species Piaractus mesopotamicus

Aquaculture ◽

10.1016/j.aquaculture.2021.736381 ◽

2021 ◽

Vol 535 ◽

pp. 736381

Author(s):

Geovanna Carla Zacheo Coelho ◽

Dilberto Ribeiro Arashiro ◽

Tamiris Disselli ◽

Matheus Pereira-Santos ◽

Tatiana María Mira-López ◽

...

Keyword(s):

Germ Cell ◽

Developmental Stages ◽

Incubation Temperature ◽

Primordial Germ Cell ◽

Piaractus Mesopotamicus

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Dynamics of the transcriptome during chicken embryo development based on primordial germ cells

BMC Research Notes ◽

10.1186/s13104-020-05286-w ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Aleksandra Dunislawska ◽

Agata Szczerba ◽

Maria Siwek ◽

Marek Bednarczyk

Keyword(s):

Gene Expression ◽

Embryo Development ◽

Germ Cells ◽

Developmental Stages ◽

Single Cells ◽

Primordial Germ Cells ◽

Regulation Of Gene Expression ◽

Cell Number ◽

Early Developmental

Abstract Objective Regulation of gene expression during embryo development on the basis of migration of primordial germ cells (PGCs) in vivo has been rarely studied due to limited cell number and the necessity to isolate PGCs from a large number of embryos. Moreover, little is known about the comprehensive dynamics of the transcriptome in chicken PGCs during early developmental stages. The current study investigated transcriptome dynamics of chicken PGCs at key developmental stages: 4.5, 8 and 12 days of embryo incubation. PGCs were collected, and RNA was isolated using a commercial kit for single cells. The isolated RNA was subjected to microarray analysis (Agilent Technologies). Results Between 8 and 12 days of incubation, the highest number of genes was regulated. These data indicate that the most intense biological activity occurs between 8 and 12 days of embryo development. Heat map showed a significant decrease in gene expression on day 8, while it increased on day 12. The development of a precise method to isolate bird PGCs as well as the method to isolate RNA from single cells isolated from one embryo allows for early molecular analysis and detection of transcriptome changes during embryonic development.

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Lamin-B1 contributes to the proper timing of epicardial cell migration and function during embryonic heart development

Molecular Biology of the Cell ◽

10.1091/mbc.e16-06-0462 ◽

2016 ◽

Vol 27 (25) ◽

pp. 3956-3963 ◽

Cited By ~ 6

Author(s):

Joseph R. Tran ◽

Xiaobin Zheng ◽

Yixian Zheng

Keyword(s):

Cell Migration ◽

Heart Development ◽

Developmental Stages ◽

Morphological Changes ◽

Nuclear Shape ◽

Epicardial Cells ◽

Biological Phenomena ◽

And Migration

Lamin proteins form a meshwork beneath the nuclear envelope and contribute to many different cellular processes. Mutations in lamins cause defective organogenesis in mouse models and human diseases that affect adipose tissue, brain, skeletal muscle, and the heart. In vitro cell culture studies have shown that lamins help maintain nuclear shape and facilitate cell migration. However, whether these defects contribute to improper tissue building in vivo requires further clarification. By studying the heart epicardium during embryogenesis, we show that Lb1-null epicardial cells exhibit in vivo and in vitro migratory delay. Transcriptome analyses of these cells suggest that Lb1 influences the expression of cell adhesion genes, which could affect cell migration during epicardium development. These epicardial defects are consistent with incomplete development of both vascular smooth muscle and compact myocardium at later developmental stages in Lb1-null embryos. Further, we found that Lb1-null epicardial cells have a delayed nuclear morphology change in vivo, suggesting that Lb1 facilitates morphological changes associated with migration. These findings suggest that Lb1 contributes to nuclear shape maintenance and migration of epicardial cells and highlights the use of these cells for in vitro and in vivo study of these classic cell biological phenomena.

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A phosphoregulated RhoGEF feedback loop tunes cortical flow driven amoeboid migration in vivo

10.1101/2021.10.29.466358 ◽

2021 ◽

Author(s):

Benjamin Lin ◽

Jonathan Luo ◽

Ruth Lehmann

Keyword(s):

Feedback Loop ◽

Primordial Germ Cells ◽

Pdz Domain ◽

Cell Types ◽

Migration Speed ◽

Ph Domain ◽

Cortical Actin ◽

And Migration

Cortical flow driven amoeboid migration utilizes friction from retrograde cortical actin flow to generate motion. Many cell types, including cancer cells, can assemble a cortical flow engine to migrate under confinement and low adhesion in vitro, but it remains unclear whether this engine is endogenously utilized in vivo. Moreover, in the context of a changing environment, it is not known how upstream regulation can set in motion and sustain a mutual feedback between flow and polarity. Here, we establish that Drosophila primordial germ cells (PGCs) utilize cortical flow driven amoeboid migration and that flows are oriented by external cues during developmental homing in vivo. The molecular basis of flow modulation is a phosphoregulated feedback loop involving RhoGEF2, a microtubule plus-end tracking RhoA specific RhoGEF, enriched at the rear of PGCs. RhoGEF2 depletion slows and disorganizes cortical flow, reducing migration speed, while RhoGEF2 activation accelerates cortical flow, thereby augmenting myosin II polarity and migration speed. Both perturbations impair PGC pathfinding, suggesting cortical flows must be tuned for accurate guidance. We surprisingly find that RhoGEF2 polarity and activation are independent of upstream canonical Gα12/13 signaling. Instead, its PDZ domain and conserved RhoA binding residues in its PH domain are required to establish a positive feedback loop that augments its basal activity. Upstream regulation of this feedback loop occurs via AMPK dependent multisite phosphorylation near a conserved EB1 binding SxIP motif, which releases RhoGEF2 from EB1 dependent inhibition. Thus, we reveal cortical flows as versatile, tunable engines for directed amoeboid migration in vivo.

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Effects of transferring pink (Oncorhynchus gorbuscha) and chum (Oncorhynchus keta) salmon embryos at different developmental stages to a low incubation temperature

Canadian Journal of Zoology ◽

10.1139/z87-015 ◽

1987 ◽

Vol 65 (1) ◽

pp. 96-105 ◽

Cited By ~ 11

Author(s):

Terry D. Beacham ◽

Clyde B. Murray

Keyword(s):

Chum Salmon ◽

Developmental Stages ◽

Incubation Temperature ◽

Pink Salmon ◽

Survival Rates ◽

Oncorhynchus Gorbuscha ◽

Oncorhynchus Keta ◽

Cell Stage ◽

Embryo Survival ◽

Eye Pigmentation

We transferred embryos of pink (Oncorhynchus gorbuscha) and chum (Oncorhynchus keta) salmon from 8 to 2 °C at five stages of development: 16-cell stage, blastula stage, completion of epiboly, early eye pigmentation, and late eye pigmentation. Survival rates of the embryos increased the later in development that they were transferred to 2 °C. All pink salmon blastulas transferred died, whereas 50% of chum salmon blastulas transferred survived until hatching. After epiboly was complete, survival rates of the embryos subsequently transferred to 2 °C were usually in excess of 75%. Chum salmon embryos had higher survival rates than did pink salmon embryos at all transfer stages. Significant differences were found in embryo survival rates among stocks within species and among families within stocks. Fry from early-spawning chum salmon took longer for exogenous yolk absorption ("button-up") than did those from late-spawning ones. Alevins and fry from early transfers were generally smaller than those from later ones. Different trends in embryo and alevin survival rates and alevin and fry size among stocks within species and among families within stocks were assumed to be indicative of adaptive variation to variable natural incubation environments.

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The effect of water pH on the incubation and larviculture of curimbatá Prochilodus lineatus (Valenciennes, 1837) (Characiformes: Prochilodontidae)

Neotropical Ichthyology ◽

10.1590/1982-0224-20130127 ◽

2015 ◽

Vol 13 (1) ◽

pp. 179-186 ◽

Cited By ~ 3

Author(s):

David Augusto Reynalte-Tataje ◽

Bernardo Baldisserotto ◽

Evoy Zaniboni-Filho

Keyword(s):

Developmental Stages ◽

Survival Rates ◽

Total Mortality ◽

Alkaline Ph ◽

Prochilodus Lineatus ◽

Ph Values ◽

Survival And Development ◽

Effects Of Ph ◽

Water Ph ◽

Eggs And Larvae

Different pH levels of water in the incubation and larviculture of 'curimbatá' (Prochilodus lineatus) were tested to evaluate the effect of this variable on the survival and development of eggs and larvae. During incubation, pH of 5.0, 6.0, 7.0, 8.0, and 8.5 was tested. To observe the effects of pH on larvae with different hatchery histories, the following experimental design was utilised (pH incubation - pH larviculture): L 6-6; L 6-7; L 7-6; L 7-7; L 7-8; L 8-7; L 8-8; L 8.5-7, and L 8.5-8.5. At pH 5.0 and four hours after incubation, an almost total mortality of incubated eggs was seen. Eggs incubated at pH 6.0 to 8.5 had similar fertilization and survival rates at the end of incubation, although eggs and larvae incubated at pH 6.0 had a smaller diameter and total length than larvae incubated at neutral and alkaline pH. During larviculture, it was observed the highest post-larvae survival rates with incubation at pH values of 7.0 (93 ± 7%) and the largest post-larvae lengths from the L 8.5-7 treatment (9.29 ± 0.8 mm). It can be concluded that hatchery and larviculture pH directly affects the initial developmental stages of P. lineatus.

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In vivo storage of oocytes leads to lower survival, increased abnormalities and may affect the ploidy status in the yellowtail tetra Astyanax altiparanae

Zygote ◽

10.1017/s0967199418000527 ◽

2018 ◽

Vol 26 (6) ◽

pp. 471-475 ◽

Cited By ~ 2

Author(s):

Nivaldo Ferreira do Nascimento ◽

Talita Maria Lázaro ◽

Nathalia Raissa de Alcântara ◽

Rocha ◽

José Augusto Senhorini ◽

...

Keyword(s):

Developmental Stages ◽

Treatment Time ◽

Survival Rates ◽

Positive Control ◽

Control Group ◽

Lower Survival ◽

Small Aliquot ◽

Ploidy Status ◽

Hatching Rates

SummaryIn this study we analyzed whether the in vivo storage of oocytes (time after ovulation until fertilization) affects the survival and the ploidy status of the yellowtail tetra Astyanax altiparanae. Fish were induced to spawn and, after ovulation, a small aliquot was stripped and immediately fertilized (positive control group). Subsequently, aliquots (~150 oocytes) were stripped and fertilized at various time points of 60, 120, 180 or 240 min. Developmental stages, abnormalities, survival and the ploidy status of the hatched larvae were examined. As expected, in the control group, 100% of the larvae were diploid. Conversely, triploid individuals were observed just at the 60 min treatment time point (0.6%). In vivo storage of oocytes also influenced the survival rates (P < 0.05); the 180 and 240 min samples, respectively, presented lower survival rates at gastrula (50.10±6.26% and 40.92±5.32%), and somite (17.80±5.14% and 4.41±2.76%) stages and lower hatching rates (12.01±4.04% and 4.41±2.76%). A higher percentage (99.27±0.40%) of normal larvae and only a few abnormal larvae (0.73±0.40%) were observed in the control group (P = 0.0000). This observation did not differ from that observed at the 60 min treatment point (P = 0.9976). A significant increase in the percentage of abnormalities was observed in the other treatments, and, after 240 min, the highest percentage of abnormal larvae was seen (P=0.0024; 83.33±16.67%). In conclusion, we showed that oocyte ageing had a significant effect on survival and may affect the ploidy status in A. atiparanae.

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Clinical Prospects for Neural Grafting Therapy for Hippocampal Lesions and Epilepsy

Neurosurgery ◽

10.1227/01.neu.0000047825.91205.e6 ◽

2003 ◽

Vol 52 (3) ◽

pp. 632-644 ◽

Cited By ~ 30

Author(s):

Dennis A. Turner ◽

Ashok K. Shetty

Keyword(s):

Stem Cells ◽

Survival Rates ◽

Embryonic Cell ◽

Long Distance ◽

Neural Grafting ◽

Hippocampal Lesions ◽

Clinical Benefits ◽

Late Treatment ◽

And Migration

Abstract OBJECTIVE Hippocampal lesions and epilepsy may be potential clinical targets for neural grafting. We hypothesized that neural grafting could be a restorative therapy either acutely, adding unformed neural elements, or chronically, treating postlesioning epilepsy. The goal of this review was to assess the clinical reality of this hypothesis of neural grafting and to determine the problems that remain to be resolved before grafting can be applied clinically. METHODS We quantitatively defined graft integration within the host, on a cellular basis, by directly assessing survival of the transplanted neurons, graft cell dispersion and migration, neuronal differentiation and development, and establishment of appropriate local and long-distance synaptic connectivity. RESULTS Embryonic hippocampal suspension grafts demonstrate excellent survival rates (20–80%). Embryonic axons exhibit extensive, appropriate, local and long-distance connectivity, can facilitate reconstruction of excitatory and inhibitory cortical circuitry, and can prevent the formation of aberrant circuitry. Immature neural stem cells demonstrate lesser degrees of integration, likely because of a paucity of positional cues in the lesioned brain for the differentiation of stem cells into region-specific neuronal phenotypes. Labeled grafted cells may be selectively and noninvasively removed from the host with triggerable stealth toxins, for the late treatment of unanticipated graft problems. CONCLUSION Neural grafting with appropriate embryonic neurons may provide significant clinical benefits. However, embryonic cell availability is severely limited, and alternative sources of cells, such as stem cells, require significant additional research into the induction and maintenance of neuronal commitment and the ability of the cells to form functional synaptic connections in vivo.

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Targeting Matrix Metalloproteinases in Atherosclerosis and Cardiovascular Dysfunction

Revista de Chimie ◽

10.37358/rc.19.2.6992 ◽

2019 ◽

Vol 70 (2) ◽

pp. 718-720

Author(s):

Lucia Corina Dima-Cozma ◽

Sebastian Cozma ◽

Delia Hinganu ◽

Cristina Mihaela Ghiciuc ◽

Florin Mitu

Keyword(s):

Smooth Muscle ◽

Matrix Metalloproteinases ◽

Cholesterol Synthesis ◽

Balloon Dilation ◽

Aortic Aneurysms ◽

Arterial Lesions ◽

Smooth Muscle Cell Migration ◽

And Migration

Matrix metalloproteinases (MMPs) are the primary mediators of extracellular remodeling and their properties are useful in diagnostic evaluation and treatment. They are zinc-dependent proteases. MMPs have been involved in the mechanisms of atherosclerosis in various arterial areas, ischemic heart disease and myocardial infarction, atrial fibrillation and aortic aneurysms. Recently, MMP9 has been implicated in dyslipidemia and cholesterol synthesis by the liver. Increased MMP expression and activity has been associated with neointimal arterial lesions and migration of smooth muscle cells after arterial balloon dilation, while MMP inhibition decreases smooth muscle cell migration in vivo and in vitro.

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Circulatory Cells as Tumortropic Carrier for Targetability Improvement

Current Drug Delivery ◽

10.2174/1567201817666201124122344 ◽

2020 ◽

Vol 17 ◽

Author(s):

Dan Zou ◽

Yajun Weng ◽

Ping Yang

Keyword(s):

Drug Delivery ◽

Drug Delivery System ◽

Delivery System ◽

Cell Function ◽

Incubation Temperature ◽

Survival Rates ◽

Controlled Drug Release ◽

Membrane Receptors ◽

Circulation Time ◽

Targeting Efficiency

Background: How to achieve high targeting efficiency for drug delivery system is still one of the most important issues that tumor diagnosis and non-surgical therapies faced. Although nanoparticle-based drug delivery system made an amount of progress in extending circulation time, improving targetability, controlled drug release etc., yet the targeting efficiency remained low, and the development was limited to reduce side effects with overall survival rates unchanged or improved a little. Objective: This paper aims to review current researches on the cell-driven drug delivery systems, and discuss the potential obstacles and directions for cell-based cancer therapies and diagnosis. Methods: More than one hundred references were collected, and this paper focused on red blood cells, monocytes, macrophages, neutrophils, natural killer cells, T lymphocytes, mesenchymal stem cells, cell membrane, artificial cells and extracellular vesicles, then summarized 1) the utilizable properties, 2) balancing cargo-loading amounts and cell function, 3) cascade strategies for targetability improvement. Main findings: circulatory cells and their derivatives were featured by good biocompatibility, long circulation time in blood, unique chemo-migration and penetration ability. On the base of backpack and encapsulation approach, cargo loading amounts and cell function could be balanced through regulating membrane receptors, particle material/size/shape/structure and incubation temperature, etc. The cell-driven drug delivery system met most of the demands that nanoparticle-based delivery system failed to for effective tumortropic delivery. Conclusion: Despite of new challenges, cell-driven drug delivery system generally brought great benefits to and shed a light on for cancer therapy and diagnosis.

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