Adult sox10+ Cardiomyocytes Contribute to Myocardial Regeneration in the Zebrafish

During heart regeneration in the zebrafish, fibrotic tissue is replaced by newly formed cardiomyocytes derived from preexisting ones. It is unclear whether the heart is composed of several cardiomyocyte populations bearing different capacity to replace lost myocardium. Here, using sox10 genetic fate mapping, we identify a subset of preexistent cardiomyocytes in the adult zebrafish heart with a distinct gene expression profile that expanded after cryoinjury. Genetic ablation of sox10+ cardiomyocytes impairs cardiac regeneration, revealing that these cells play a role in heart regeneration.This work has been funded by the Spanish Ministry of Economy and Competitiveness (BFU2014–56970–P), the Swiss National Science Foundation (grant 31003A_159721), the ERC (starting grant 337703–zebra–Heart), Comunidad de Madrid (FIBROTEAM S2010/BMD-2321), and co-funding by Fondo Europeo de Desarrollo Regional (FEDER). I.J.M. was supported by Marie Curie Slodowska fellowship (PIEF-A-2012-330728). D.F. was supported by BtRAIN (European Brain Barriers Training Network) (H2020-MSCA-ITN-2015, n 675619). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovacion y Universidades (MCNU), and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Endogenous mouse Dicer is an exclusively cytoplasmic protein

Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse

Lightning Fast and Highly Sensitive Full-Length Single-cell sequencing using FLASH-Seq

In the last 10 years, single-cell RNA-sequencing (scRNA-seq) has undergone exponential growth. Emulsion droplets methods1–3, such as those commercialized by 10x Genomics, have allowed researchers to analyze tens of thousands of cells in parallel in a robust and reproducible way. However, in contrast to SMART-based full-length sequencing protocols4,5, these methods interrogate only the outer portion of the transcripts and still lack the required sensitivity for analyzing comprehensively the transcriptome of individual cells. Building upon the existing SMART-seq forerunners protocols4,5, we developed FLASH-Seq (FS), a new scRNA-seq method which displays greater sensitivity while decreasing incubation times and reducing the number of processing steps compared to its predecessors. The entire FS protocol - from lysed cells to pooled cDNA libraries - can be performed in ~4.5 hours, is automation-friendly and can be easily miniaturized to decrease costs

Dicer is solely cytoplasmic during embryogenesis.

<p>(A) Anti-HA immunofluorescence of E13.5 <i>Dcr</i>^<i>FH/FH</i> embryo section. Scale bar is 1 mm. (B-I) Confocal microscopy of immunofluorescence staining of wild type and <i>Dcr</i>^<i>FH/FH</i> E13.5 tissue sections with anti-HA antibody. Sections of lung (B) and liver (C) are shown as representatives of endoderm, vertebrae (D) represent mesoderm, forebrain (E), root ganglion (F), lens (G) and epidermis (H) represent ectoderm, placenta (I) is depicted as an example for an extraembryonic tissue. Nuclei are stained with Hoechst. Scale bar is 10 μm.</p

Dicer localizes exclusively to the cytoplasm of embryonic fibroblasts and adult tissues.

<p>(A) Schematic representation of the domain structure of N-terminal Flag-HA₂ tagged murine Dicer. (B) Western blot using anti-HA antibody on serial dilution of whole cell extract from <i>Dcr</i>^<i>FH/FH</i> PMEFs. (C, E and G) Western blot analysis after subcellular fractionation using anti-HA antibody on cytoplasmic (Cy), nuclear (Nu) and whole cell (WC) extracts from wild type and <i>Dcr</i>^<i>FH/FH</i> PMEFs (C), adult testis (E) and thymus (G). Tubulin served as a cytoplasmic marker and histone H3 as a nuclear marker. (D, F and H) Confocal immunofluorescence micrographs showing wild type and <i>Dcr</i>^<i>FH/FH</i> PMEFs (D) as well as sections of adult testis (F) and thymus (H) stained with anti-HA antibody. Nuclei are stained with Hoechst. Scale bars represent 5 μm. (I) Immunoprecipitation using anti-HA beads and mass spectrometry analysis of the Dicer interactome in PMEFs, testis and thymus (each biological duplicates) and all of them pooled (six biological replicates).</p

Dicer does not translocate to the nucleus after DNA damage.

<p>(A) Confocal imaging of anti-HA stained wild type and <i>Dcr</i>^<i>FH/FH</i> PMEFs after irradiation. Cells were irradiated with 20 Gy and then allowed to recover for 30 min. Activation of the DNA damage response was verified with anti-γH2AX staining. Nuclei are stained with Hoechst. Scale bars represent 10 μm. (B) Western blot analysis after irradiation and subcellular fractionation. Anti-HA antibody was used on cytoplasmic (Cy) and nuclear (Nu) extracts of irradiated and non-irradiated cells. Tubulin served as a cytoplasmic marker, histone H3 as a nuclear marker and γH2AX as a marker of DNA damage. (C) Confocal microscopy on anti-HA stained wild type and <i>Dcr</i>^<i>FH/FH</i> PMEFs after irradiation and LMB treatment.</p

Dicer is not a nucleocytoplasmic shuttling protein.

<p>(A) Confocal micrographs showing immunofluorescence anti-HA staining of wild type and <i>Dcr</i>^<i>FH/FH</i> PMEFs without and with LMB-induced inhibition of nuclear export. Cells were treated with 20 nM LMB or solvent for 6 h. Cyclin B1 served as a positive control for the LMB treatment. Nuclei are stained with Hoechst. Scale bars represent 10 μm. (B) Western blot analysis after LMB treatment and subcellular fractionation. Anti-HA antibody was used on cytoplasmic (Cy) and nuclear (Nu) extracts, where tubulin served as a cytoplasmic marker and histone H3 as a nuclear marker.</p

Additional file 1: of Early transcriptional events linked to induction of diapause revealed by RNAseq in larvae of drosophilid fly, Chymomyza costata

Table S1. List of 21,327 putative Chymomyza costata mRNA transcripts obtained by de novo transcriptome assembly of HiSeq2000 (Illumina) sequencing data. (XLSX 5626 kb