Cell cycle dynamics during diapause entry and exit in an annual killifish revealed by FUCCI technology

Cellerino, Alessandro; Dolfi, Luca; Ripa, Roberto; Antebi, Adam; Valenzano, Dario Riccardo

doi:10.1101/522417

Cited by 7 publications

(7 citation statements)

References 49 publications

(76 reference statements)

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“…As such, we provide proof of principle evidence that the mScarlet-pcna line can be successfully used as an endogenous cell cycle reporter in a teleost model. Using the visualization of endogenous Pcna for cell cycle phase classification offers an attractive alternative to cell cycle reporters that rely on the insertion of two-colour transgenes, such as the FUCCI system (Sugiyama et al, 2009, Dolfi et al, 2019, Araujo et al, 2016, Bajar et al, 2016, Oki et al, 2014, Sakaue-Sawano et al, 2008. First, by using endogenous fusion proteins there is no requirement for over-expression of cell cycle regulators.…”

Section: Discussionmentioning

confidence: 99%

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

Seleit

Aulehla

Paix

2021

Preprint

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The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology directed repair in a variety of species. Despite its success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient and precise strategy for CRISPR/Cas9 mediated endogenous protein tagging in medaka. We use a cloning-free approach that relies on PCR amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40bp), a synthetic sgRNA and streptavidin tagged Cas9. We generate six novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole Genome Sequencing results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion-protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna knock-in has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.

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Section: Discussionmentioning

confidence: 99%

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

Seleit

Aulehla

Paix

2021

Preprint

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“…A recent publication reported the use of Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) to explore cell cycle dynamics during killifish development. It revealed surprising levels of coordination of cellular dynamics during diapause and provided a reference framework for further analyses of embryonic diapause [23].…”

Section: Systems Biology and Holistic Approachesmentioning

confidence: 98%

New directions to understand and learn from embryonic diapause in mammals

Comizzoli¹

2020

Proc

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Embryonic diapause is a key strategy to extend pregnancy until conditions are ideal for birth and postnatal survival. There is still a lot to discover about this unique phenomenon observed in more than 130 mammalian species. The present review aims at complementing existing research efforts by (1) identifying new directions for a better understanding of embryonic diapause in mammals and (2) considering this complex mechanism as a source of inspiration for other areas in cellular biology. Comparative explorations in different species of new molecular players associated with the use of emerging technologies in the study of embryos (epigenetics for instance), the uterus (immune cells, microbiota, cell-free DNA), and the whole organism (remote sensing, systems biology) will shed some new lights on embryonic diapause characterizations. Collective results of advanced studies should be integrated into the measurement of climate and environmental changes potentially influencing the physiology of females and their arrested embryos. Interestingly, lessons from nonmammalian species using similar strategies (killifish for instance) could also improve our understanding of this unique phenomenon. Furthermore, studying embryonic diapause offers a great opportunity to decipher other cellular mechanisms and develop new applications (stem cell technologies, cancer treatments, contraceptive methods, short-term storage of embryos, or early fetal loss prevention). Overall, these new ideas and directions should define some themes for a future International Symposium on Embryonic Diapause.

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“…In addition to miR-430, we identified other DEMs with essential development-related functions, such as miR-19 being part of the miR-17/92 cluster and associated with endothelial cell differentiation in embryonic stem cells 68 and more generally with the positive control of the cell cycle. Its downregulation is therefore expected given the profound depression of cell cycle in diapausing embryos 69 . Similarly, miR-200 regulating the epithelial/mesenchymal transition during embryonic development 70 and miR-221, which is involved in cell proliferation and angiogenesis 71,72 .…”

Section: Differentially Expressed Mirnas Related To Diapause Regulation In Killifishmentioning

confidence: 99%

Analysis of microRNA expression reveals convergent evolution of the molecular control of diapause in annual fish

Barth

Baumgart

Dolfi

et al. 2021

Preprint

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BackgroundDiapause is a key adaptation that enabled the colonization of ephemeral habitats subject to the alternation of dry and wet seasons by annual killifishes. Upon desiccation of the ponds, killifish embryos remain vital but quiescent in the clay, where they can survive months or even years. Diapause can occur at three different developmental stages, but Diapause II (DII), which occurs at mid-somitogenesis, is the primary point of developmental arrest. However, diapause is not obligatory, and some embryos can proceed to direct development, skipping one or more diapauses. The precise molecular mechanisms that regulate entry and exit from diapause are beginning to be investigated, but this knowledge is yet fragmentary. Diapause has evolved independently several times in killifish clades from Africa and South America, enabling the identification of possible molecular determinants of diapause by comparative expression analysis. MicroRNAs are small RNAs that represent central nodes in the control of gene expression at the post-transcriptional level and are involved in many developmental processes. Here, we compare microRNA expression profiles of annual killifishes during DII with non-annual killifish in a comparable stage of morphological development. ResultsWe used smallRNA-Seq to quantify microRNA expression from four annual- and four non-annual killifish species from three independent clades and from direct-developing embryos of the annual fish Nothobranchius furzeri. We analyzed the expression of broadly conserved microRNAs and microRNAs that appear to have evolved in the killifish lineage.We found several microRNAs that showed convergent regulation in the three different clades, and for some microRNAs also a phenomenon of switch in the prevalent form between 3p and 5p or vice versa was noted. In addition, we detected a significant overlap between the microRNA regulation during diapause and aging. Particularly interesting is the regulation of the miR-430 family. These microRNAs represent the second most expressed microRNA family in the killifish embryos, and diapause is associated with dramatic down-regulation of the prevalent -3p form and up-regulation of the -5p form. Members of the miR-430 family are contained in a large repetitive cluster whose organization is variable among teleost. Analysis of recently sequenced 45 low-coverage killifish genomes revealed that the miR-430 locus contains a lower number of copies in annual- as opposed to non-annual killifish. ConclusionsThe Evolution of diapause is reflected in the convergent evolution of microRNA regulation in killifishes. A prominent feature is a dramatic down-regulation of miR-430 expression that could be partially explained with a reduction of its copy numbers in the genome.

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Cell cycle dynamics during diapause entry and exit in an annual killifish revealed by FUCCI technology

Abstract: Background:

Cited by 7 publications

References 49 publications

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

New directions to understand and learn from embryonic diapause in mammals

Analysis of microRNA expression reveals convergent evolution of the molecular control of diapause in annual fish

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