Cycles of gene expression and genome response during mammalian tissue regeneration.

Compensatory liver hyperplasia-or regeneration-induced by two-thirds partial hepatectomy (PH) permits the study of synchronized activation of mammalian gene expression, particularly in relation to cell proliferation. Here, we measured genomic transcriptional responses and mRNA accumulation changes after PH and sham surgeries. During the first 10-20 h, the PH- and sham-surgery responses were very similar, including parallel early activation of cell-division-cycle genes. After 20 h, however, whereas post-PH livers continued with a robust and coordinate cell-division-cycle gene-expression response before returning to the resting state by 1 week, sham-surgery livers returned directly to a resting gene-expression state. Localization of RNA polymerase II (Pol II), and trimethylated histone H3 lysine 4 (H3K4me3) and 36 (H3K36me3) on genes dormant in the resting liver and activated during the PH response revealed a general de novo promoter Pol II recruitment and H3K4me3 increase during the early 10-20 h phase followed by Pol II elongation and H3K36me3 accumulation in gene bodies during the later proliferation phase. H3K36me3, generally appearing at the first internal exon, was preceded 5' by H3K36me2; 3' of the first internal exon, in about half of genes H3K36me3 predominated and in the other half H3K36me2 and H3K36me3 co-existed. Further, we observed some unusual gene profiles with abundant Pol II but little evident H3K4me3 or H3K36me3 modification, indicating that these modifications are neither universal nor essential partners to Pol II transcription. PH and sham surgical procedures on mice reveal striking early post-operatory gene expression similarities followed by synchronized mRNA accumulation and epigenetic histone mark changes specific to PH

A wash-free ER marker to monitor micropolarity during ER stress and visualize ER- Golgi transport

Cellular stress plays a key role to regulate and maintain organismal as well as microenvironmental homeostasis. The stress-induced response is also reflected in the micropolarity of any specific cellular compartment, which is essential to be quantified for early disease diagnosis. Alongside, coming up with a biocompatible small-molecule fluorophore NBD-Oct for exclusive endoplasmic reticulum (ER) localization simply driven by its hydrophobicity, in this contribution, we present a quantitative study of micropolarity alteration inside the ER during G1/S and G2/M phases. The cell cycle arrests caused by the induced ER stress led to the enhancement of the ER micropolarity in cells. NBD-Oct is selected among a series of analogous probes based on its fastest diffusion properties demonstrated by fluorescence recovery after the photobleaching experiment. The probe is a versatile staining agent as it could efficiently stain the ER in live/fixed mammalian cells, isolated ER, Caenorhabditis elegans, and mice tissues. Finally, a well-known biological event, ER to Golgi transport is also visualized by live-cell fluorescence microscopy using this probe. We believe this exhaustive investigation of micropolarity using NBD-based dye provides a new avenue to study ER stress that may unravel a deeper understanding of proteostasis in model systems and potentially even fixed patient samples

Number of Poxn-expressing cells per brain hemisphere in embryos, larvae, and adults.

<p>Number of Poxn-expressing cells per brain hemisphere in embryos, larvae, and adults.</p

Projections of <i>Poxn</i>-neurons and axonal tracts labeled by FasII in wild-type and <i>Poxn</i> mutant brains of late third instar larvae.

<p><b>(A-D)</b><i>Poxn</i>-neurons, immunostained for the expression of <i>Poxn-CD8</i>::<i>GFP</i> (green), and neuropils, immunostained for the expression of FasII (red), are shown in brains of <i>w</i>^<i>1118</i>; <i>Poxn-CD8</i>::<i>GFP</i> <b>(A,D)</b> and <i>w</i>^<i>1118</i>; <i>Poxn</i>^{<i>ΔM22-B5</i>} <i>Poxn-Sbl-107</i>; <i>Poxn-CD8</i>::<i>GFP</i> <b>(B,C)</b> late third instar larvae in the red and green channel (A-C) and only in the green channel (D) at 40x magnification. The two <i>Poxn</i> mutant brains display the two types of mutant projection patterns commonly observed: projections from the VC seem to follow the mALT instead of the mlALT (B), or adopt an entirely different path, eventually running parallel to the projections from the DC before they stall (C). Scale bars: 50 μm. <b>(E-H')</b> Central parts of substacks of (A) are shown from posterior to anterior at 10–15 μm (E,E'), 23–28 μm (F,F'), 28–30 μm (G,G'), and 30–35 μm (H,H') as maximum intensity projections of CLSM sections of a Z-stack extending over 85 μm in both channels (E-H) and only in the green channel (E'-H') at 40x magnification. <i>Poxn</i>-neuron tracts that pass through the SEC and do or do not co-express FasII are indicated by arrowheads and arrows, respectively. DC, dorsal cluster of <i>Poxn</i>-neurons; vl, vertical lobe of mushroom bodies; mALT, middle antennal lobe tract; mlALT, mediolateral antennal lobe tract; ml, medial lobe of mushroom bodies; pBU, primordial bulb; VC, ventral cluster of <i>Poxn</i>-neurons.</p

Most <i>Poxn</i>-neurons survive metamorphosis and continue to express Poxn in the adult brain.

<p>(<b>A–C</b>) The DC of <i>Poxn</i>-neurons, visualized by immunofluorescent staining for β-Gal (red) and GFP (green), is shown in the brain of a <i>w</i>^<i>1118</i> <i>UAS-Flp/</i>+; <i>tub-Gal80</i>^<i>ts</i><i>/+</i>; <i>Act5C>polyA>lacZ</i>.<i>nls1 Poxn-Gal4-13-1/Poxn-CD8</i>::<i>GFP</i> female. A maximum intensity projection of the same CLSM sections of an entire Z-stack is shown in the red channel (A), green channel (B), and in both channels (C) at 63x magnification. β-Gal-positive nuclei (red) belong to cells that expressed Poxn at the time of heat shock during the third larval instar (feeding stage). The membrane-associated CD8::GFP fusion protein labels Poxn-expressing cells at the time of fixation. (<b>D</b>–<b>F</b>) Maximum intensity projections of substacks at 0–5 μm (D), 5–10 μm (E), and 10–18 μm (F) of the Z-stack extending from 0 (anterior) to 31 μm (posterior) shown in (C), which includes all <i>Poxn</i>-nuclei. Virtually all β- Gal-labeled neurons also express GFP. Similar results were obtained for the VC of <i>Poxn</i>-neurons (data not shown). Scale bar: 20 μm.</p

At least half of the Poxn-expressing cells in the adult brain completed their last S-phase during embryogenesis.

<p>BrdU fed throughout larval development and incorporated into DNA was analyzed in an adult <i>Ore-R</i> brain in CLSM sections at 63x magnification. <i>Poxn</i>-neurons of one brain hemisphere, stained for Poxn (red) and incorporated BrdU (green), are shown in 1 μm sections at 4–5 μm (A), 7–8 μm (B), 10–11 μm (C), and 13–14 μm (D) of a Z-stack extending from 0 (anterior) to 30 μm (posterior). Colocalization of BrdU with Poxn was analyzed in each Poxn-labeled nucleus by careful inspection of single confocal layers. Arrowheads point at the 20 and 12 largest <i>Poxn</i>-nuclei of the DC and VC, respectively, all of which are free of BrdU, and thus are thought to correspond to the 20 (DC) and 12 (VC) <i>Poxn</i>-neurons that express Poxn during embryogenesis and lack Pros in late third instar larval brains (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176002#pone.0176002.s006" target="_blank">S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176002#pone.0176002.s008" target="_blank">S7A and S7B</a> Figs, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176002#pone.0176002.t001" target="_blank">Table 1</a>). Note that some of these <i>Poxn</i>-nuclei appear smaller than other BrdU-negative <i>Poxn</i>-nuclei in the same section but are actually larger, as evident from adjacent sections (not shown). Scale bar: 20 μm.</p

Projection pattern of <i>Poxn</i>-neurons with respect to glia in late third instar larval brain.

<p>(<b>A,B</b>) Left (A) and right (B) hemispheres of <i>w</i>^<i>1118</i>; <i>UAS-CD2</i>; <i>repo-Gal4/Poxn-CD8</i>::<i>GFP</i> late third instar larval brain, immunostained for GFP (green), labeling <i>Poxn</i>-neurons, and CD2 (red) in glial membranes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176002#pone.0176002.ref060" target="_blank">60</a>]. The axons of the <i>Poxn</i>-neurons project along the lateral region of the larval AL (surrounded by dashed orange line in A), exhibit a striking ‘swelling’ at the developing adult AL (surrounded by dashed white line) but no arborization, and follow the mlALT to target the LH. No arborization is detected at the larval AL. To optimize the visibility of the axon tracts of the <i>Poxn</i>-neurons from the VC along the larval AL and developing adult AL to the LH, substacks extending from 30 μm (anterior) to 56 μm (posterior) (A) and from 48 μm (anterior) to 76 μm (posterior) (B) of a Z-stack, extending over 85 μm, are shown as maximum intensity projections of CLSM sections at 40x magnification. Although these substacks exclude the cell bodies of the <i>Poxn</i>-neurons, they include all neurite projections. Scale bar: 50 μm.</p

Cells expressing Poxn in larval and adult brains are post-mitotic neurons.

<p>(<b>A–D</b>) Colocalization (white) in nuclei of Poxn (red) and Elav (green) proteins, visualized by immunofluorescent staining, is shown in wild-type (<i>Ore-R</i>) brain hemispheres of first (A), second (B), and late third instar larvae (C), and of adults (D). Staining of all <i>Poxn</i>-nuclei with Elav was corroborated by visual inspection in single layers of the Z-stacks. Panels show maximum intensity projections of CLSM sections of Z-stacks extending over 43 μm (A), 45 μm (B), and 50 μm (C) at 63x magnification, and of a Z-stack extending over 26 μm at 40x magnification (D). Scale bars: 10 μm (A–C) and 20 μm (D).</p

Formation of ellipsoid body depends strongly on <i>Poxn</i> function.

<p>Brains of <i>w</i>^<i>1118</i>; <i>Poxn-CD8</i>::<i>GFP</i> (<b>A-H</b>) or <i>w</i>^<i>1118</i>; <i>Poxn</i>^{<i>ΔM22-B5</i>} <i>Poxn-Sbl-107</i>; <i>Poxn-CD8</i>::<i>GFP</i> (<b>A’-H’</b>) pupae, immunostained for GFP at 0 h APF (A,A’), 10 h APF (B,B’), 15 h APF (C,C’), 20 h APF (D,D’), 30 h APF (E, E’), 40 h APF (F,F’), 45 h APF (G,G’), and 50 h APF (H,H’), are shown as maximum intensity projections of confocal Z-stacks at 20x magnification (panels except E, G, and G’ show substacks that remove some of the cell bodies but improve the visibility of projections). Z-stacks extended over 89 μm (A), 67 μm (B), 82 μm (C), 52 μm (D), 72 μm (E), 54 μm (F), 79 μm (G), 51 μm (H), 44 μm (A’), 65 μm (B’), 90 μm (C’), 88 μm (D’), 56 μm (E’), 46 μm (F’), 102 μm (G’), and 66 μm (H’). Arrows point to the bilateral arc-like mlALTs visible at all pupal stages. Filled arrowheads point to the arborizations at the developing bulbs, while arrowheads point to the midline of the forming ellipsoid body neuropil. Note that arborizations at the lateral horn (LH) and antennal lobe (AL) become prominent around 40 h APF in the wild-type brain (F-H), while they are slightly delayed in mutant brains where they occur at the end of the stalled projections (G’,H’). Pupal stages, measured as hours APF, imply a developmental temporal variation corresponding to about 5%. Scale bars: 50 μm.</p

Projection patterns of <i>Poxn</i>-neurons in wild type and <i>Poxn</i> mutants during larval development.

<p>(<b>A–H</b>) <i>Poxn</i>-neurons are visualized by the expression of <i>Poxn-CD8</i>::<i>GFP</i> and immunofluorescent staining for GFP in first (A,E), late second (B,F), and late third instar (C,D,G,H) brains of <i>w</i>^<i>1118</i>; <i>Poxn-CD8</i>::<i>GFP</i> (A-C), <i>w</i>^<i>1118</i>; <i>Poxn</i>^{<i>ΔM22-B5</i>}; <i>Poxn-CD8</i>::<i>GFP</i> (E-G), <i>w</i>^<i>1118</i>; <i>Poxn</i>^{<i>ΔM22-B5</i>} <i>Poxn-SuperA-158</i>; <i>Poxn-CD8</i>::<i>GFP</i> (D), and <i>w</i>^<i>1118</i>; <i>Poxn</i>^{<i>ΔM22-B5</i>} <i>Poxn-Sbl-107</i>; <i>Poxn-CD8</i>::<i>GFP</i> (H) larvae. Note that the projection patterns of the latter two resemble those in wild-type (C) and <i>Poxn</i> mutant brains (G), respectively. Arrowheads in (A-C) point to arc-like projections of the VC and their targets in the lateral protocerebrum, and arrow in (C) points to tracts of the SEC, emanating from the DC of wild-type brains. Asterisks in (G) mark aberrant projections from both <i>Poxn</i> clusters in a <i>Poxn</i> mutant brain. Dashed lines indicate midlines of the flattened brains viewed along the anteroposterior axis. Panels show maximum intensity projections of CLSM sections of Z-stacks extending over 41 μm (A), 50 μm (B), 53 μm (C), 49 μm (D), 75 μm (E), 36 μm (F), 48 μm (G), and 77 μm (H) at 20x magnification. Panel H shows the same brain as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176002#pone.0176002.g006" target="_blank">Fig 6B</a>. DC, dorsal cluster of <i>Poxn</i>-neurons; LH, lateral horn; VC, ventral cluster of <i>Poxn</i>-neurons. Scale bars: 20 μm.</p