SMALL RNA EXPRESSION DURING PROGRAMMED REARRAGEMENT OF A VERTEBRATE GENOME

The sea lamprey (Petromyzon marinus) undergoes programmed genome rearrangements (PGRs) during embryogenesis that results in the deletion of ~0.5 Gb of germline DNA from the somatic lineage. The underlying mechanism of these rearrangements remains largely unknown. miRNAs (microRNAs) and piRNAs (PIWI interacting RNAs) are two classes of small noncoding RNAs that play important roles in early vertebrate development, including differentiation of cell lineages, modulation of signaling pathways, and clearing of maternal transcripts. Here, I utilized next generation sequencing to determine the temporal expression of miRNAs, piRNAs, and other small noncoding RNAs during the first five days of lamprey embryogenesis, a time series that spans the 24-32 cell stage to the formation of the neural crest. I obtained expression patterns for thousands of miRNA and piRNA species. These studies identified several thousand small RNAs that are expressed immediately before, during, and immediately after PGR. Significant sequence variation was observed at the 3’ end of miRNAs, representing template-independent covalent modifications. Patterns observed in lamprey are consistent with expectations that the addition of adenosine and uracil residues plays a role in regulation of miRNA stability during the maternal-zygotic transition. We also identified a conserved motif present in sequences without any known annotation that is expressed exclusively during PGR. This motif is similar to binding motifs of known DNA binding and nuclear export factors, and our data could represent a novel class of small noncoding RNAs operating in lamprey

Cellular and Molecular Features of Developmentally Programmed Genome Rearrangement in a Vertebrate (Sea Lamprey: \u3cem\u3ePetromyzon marinus\u3c/em\u3e)

The sea lamprey (Petromyzon marinus) represents one of the few vertebrate species known to undergo large-scale programmatic elimination of genomic DNA over the course of its normal development. Programmed genome rearrangements (PGRs) result in the reproducible loss of ~20% of the genome from somatic cell lineages during early embryogenesis. Studies of PGR hold the potential to provide novel insights related to the maintenance of genome stability during the cell cycle and coordination between mechanisms responsible for the accurate distribution of chromosomes into daughter cells, yet little is known regarding the mechanistic basis or cellular context of PGR in this or any other vertebrate lineage. Here we identify epigenetic silencing events that are associated with the programmed elimination of DNA and describe the spatiotemporal dynamics of PGR during lamprey embryogenesis. In situ analyses reveal that the earliest DNA methylation (and to some extent H3K9 trimethylation) events are limited to specific extranuclear structures (micronuclei) containing eliminated DNA. During early embryogenesis a majority of micronuclei (~60%) show strong enrichment for repressive chromatin modifications (H3K9me3 and 5meC). These analyses also led to the discovery that eliminated DNA is packaged into chromatin that does not migrate with somatically retained chromosomes during anaphase, a condition that is superficially similar to lagging chromosomes observed in some cancer subtypes. Closer examination of “lagging” chromatin revealed distributions of repetitive elements, cytoskeletal contacts and chromatin contacts that provide new insights into the cellular mechanisms underlying the programmed loss of these segments. Our analyses provide additional perspective on the cellular and molecular context of PGR, identify new structures associated with elimination of DNA and reveal that PGR is completed over the course of several successive cell divisions

Cellular and Molecular Features of Developmentally Programmed Genome Rearrangement in a Vertebrate (Sea Lamprey: \u3cem\u3ePetromyzon marinus\u3c/em\u3e)

The sea lamprey (Petromyzon marinus) represents one of the few vertebrate species known to undergo large-scale programmatic elimination of genomic DNA over the course of its normal development. Programmed genome rearrangements (PGRs) result in the reproducible loss of ~20% of the genome from somatic cell lineages during early embryogenesis. Studies of PGR hold the potential to provide novel insights related to the maintenance of genome stability during the cell cycle and coordination between mechanisms responsible for the accurate distribution of chromosomes into daughter cells, yet little is known regarding the mechanistic basis or cellular context of PGR in this or any other vertebrate lineage. Here we identify epigenetic silencing events that are associated with the programmed elimination of DNA and describe the spatiotemporal dynamics of PGR during lamprey embryogenesis. In situ analyses reveal that the earliest DNA methylation (and to some extent H3K9 trimethylation) events are limited to specific extranuclear structures (micronuclei) containing eliminated DNA. During early embryogenesis a majority of micronuclei (~60%) show strong enrichment for repressive chromatin modifications (H3K9me3 and 5meC). These analyses also led to the discovery that eliminated DNA is packaged into chromatin that does not migrate with somatically retained chromosomes during anaphase, a condition that is superficially similar to lagging chromosomes observed in some cancer subtypes. Closer examination of “lagging” chromatin revealed distributions of repetitive elements, cytoskeletal contacts and chromatin contacts that provide new insights into the cellular mechanisms underlying the programmed loss of these segments. Our analyses provide additional perspective on the cellular and molecular context of PGR, identify new structures associated with elimination of DNA and reveal that PGR is completed over the course of several successive cell divisions

Highly conserved molecular pathways, including Wnt signaling, promote functional recovery from spinal cord injury in lampreys

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Scientific Reports 8 (2018): 742, doi:10.1038/s41598-017-18757-1.In mammals, spinal cord injury (SCI) leads to dramatic losses in neurons and synaptic connections, and consequently function. Unlike mammals, lampreys are vertebrates that undergo spontaneous regeneration and achieve functional recovery after SCI. Therefore our goal was to determine the complete transcriptional responses that occur after SCI in lampreys and to identify deeply conserved pathways that promote regeneration. We performed RNA-Seq on lamprey spinal cord and brain throughout the course of functional recovery. We describe complex transcriptional responses in the injured spinal cord, and somewhat surprisingly, also in the brain. Transcriptional responses to SCI in lampreys included transcription factor networks that promote peripheral nerve regeneration in mammals such as Atf3 and Jun. Furthermore, a number of highly conserved axon guidance, extracellular matrix, and proliferation genes were also differentially expressed after SCI in lampreys. Strikingly, ~3% of differentially expressed transcripts belonged to the Wnt pathways. These included members of the Wnt and Frizzled gene families, and genes involved in downstream signaling. Pharmacological inhibition of Wnt signaling inhibited functional recovery, confirming a critical role for this pathway. These data indicate that molecular signals present in mammals are also involved in regeneration in lampreys, supporting translational relevance of the model.We gratefully acknowledge support from the National Institutes of Health (R03NS078519 to OB; R01GM104123 to JJS; R01NS078165 to JRM), The Feinstein Institute for Medical Research and The Marine Biological Laboratory, including the Charles Evans Foundation Research Award, the Albert and Ellen Grass Foundation Faculty Research Award, and The Eugene and Millicent Bell Fellowship Fund in Tissue Engineering

Morphology of anaphase lagging chromatin in intact embryonic cells.

<p>Immunolabeling and hybridization of intact, PACT-cleared, embryos (2 dpf). (A) immunolabeling with anti-beta-tubulin, lagging chromatin is oriented along the polar microtubules. (B) Confocal image of an anaphase labeled with a centromere specific probe (Cot1 FISH). Centromeres of lagging chromosomes are oriented toward the poles of mitotic spindle. (C) Confocal image of an anaphase from an embryo at 1dpf: lagging chromosomes form equatorial contacts, bridging between the poles of the mitotic spindle. (D) An anaphase with multiple bridging chromosomes, hybridized with a probe to repetitive DNA (Cot2 FISH). Punctate signals corresponding to centromeres are oriented toward the spindle poles but lag behind retained chromosomes. Sites of apparent contact between sister chromatids hybridize strongly to Cot2 DNA, suggesting that repetitive DNA may participate in establishing these contacts. (E) Fluorescence <i>in situ</i> hybridization of the <i>Germ1</i> probe to an anaphase with several bridging chromosomes. <i>Germ1</i> signals are symmetrical, further supporting the interpretation that bridging features consist of pairs of sister chromatids, though notably, <i>Germ1</i> signals do not appear to overlap with the zone of contact between sister chromatids.</p

Lagging chromosomes are abundant during early stages of embryonic development.

<p>(A-D) Images of paraffin sections from lamprey embryos at 1 dpf. Anti-beta-tubulin immunolabeling: (A) metaphase, (B) anaphase A, (C) anaphase B with conglomerated chromatin in the equatorial area, (D) anaphase B with longitudinally stretched lagging chromatin. (E-F) Fluorescence <i>in situ</i> hybridization of the <i>Germ1</i> probe to embryo cells at 2 dpf from paraffin sections. (E) Anaphase with lagging chromosomes contain multiple signals for <i>Germ1</i>, somatic chromosomes retain a single pair of <i>Germ1</i> signals. (F) Late anaphase with two <i>Germ1</i>-positive micronuclei situated between condensing daughter nuclei. (G) Interphase cells with a single pair of <i>Germ1</i> signals in their main nuclei and additional signals in micronuclei.</p

Timing of chromatin elimination.

<p>(A-D) PACT-cleared embryos, stained to highlight DNA (SYTO-24). (A) Cells from a 30-cell embryo at 1 dpf. Interphase cells lack micronuclei, and no lagging chromosomes are visible. (B) Cells from a 78-cell embryo at 1 dpf showing numerous anaphases with lagging chromatin. (C) Cells from an embryo showing anaphases with lagging chromatin and interphase cells with micronuclei. (D) Cells from an embryo at 2.5 dpf with few visible micronuclei and anaphases without lagging chromatin (presumably reflecting the completion of programmed genome rearrangement during earlier cell divisions). (E) Observed proportions of anaphases with lagging chromatin across early developmental stages. (F) Observed proportions of interphase cells containing micronuclei across early developmental stages.</p

Variation in the content and form of eliminated DNA indicates stepwise loss of DNA.

<p>(A-C) FISH of the <i>Germ1</i> probe to anaphase chromosomes from PACT-cleared embryos. (A) A representative anaphase spread from 1 dpf (right panel: red—<i>Germ1</i>, blue—DAPI; left panel: DAPI). A majority of signals corresponding to the <i>Germ1</i> repeat co-migrate with retained chromosomes, and a relatively small conglomerate of chromatin is localized to the equatorial region. (B) A representative anaphase spread from 2 dpf (right panel: red—<i>Germ1</i>, blue—DAPI; left panel: DAPI). Lagging chromosomes are enriched with <i>Germ1</i> while retained chromosomes have only a single pair of <i>Germ1</i> signals. (C) <i>Germ1</i>-negative lagging chromatin at 2 dpf (right panel: red—<i>Germ1</i>, blue—DAPI; left panel: DAPI). <i>Germ1</i> hybridizes to a pair of signals on retained chromosomes that are associated with a relatively small conglomerate of lagging chromatin that lacks <i>Germ1</i> hybridization. (D-F) Late mitotic events in cells undergoing chromosome elimination. (D) Cytokinetic cells from 1 dpf possess dense and presumably heterochromatic structures located near the cleavage furrow with filamentous extensions oriented toward the enveloped nuclei (green—SYTO-24 stained DNA). Cells with multiple micronuclei from 1 dpf (E) and 2 dpf (F) stained with SYTO-24.</p

Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS

C9orf72 mutations are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) produced by unconventional translation of the C9orf72 repeat expansions cause neurodegeneration in cell culture and in animal models. We performed two unbiased screens in Saccharomyces cerevisiae and identified potent modifiers of DPR toxicity, including karyopherins and effectors of Ran-mediated nucleocytoplasmic transport, providing insight into potential disease mechanisms and therapeutic targets.status: publishe