The complete mitogenome of nereid worm, <i>Neanthes glandicincta</i> (Annelida: Nereididae)

<p>In this study, the complete mitogenome sequence of nereid worm, <i>Neanthes glandicincta</i> (Annelida: Nereididae), has been decoded for the first time by PCR amplification and Sanger sequencing methods. The overall base composition of <i>N. glandicincta</i> mitogenome is 31.5% for A, 22.2% for C, 14.5% for G and 31.7% for T, and has GC content of 36.7%. The assembled mitogenome, consisting of 16,126 bp, has unique 13 protein-coding genes (PCGs), 22 transfer RNAs and 2 ribosomal RNAs genes. The complete mitogenome of <i>N. glandicincta</i> shows 65% identities to <i>Namalycastis abiuma</i>. All PCGs, tRNA and rRNA genes were encoded on H-strand. The potential D-loop is 1625 bp in length and located between tRNA-Gly and tRNA-Met. The complete mitogenome provides essential and important DNA molecular data for further phylogenetic and evolutionary analysis for Annelida.</p

Sarcomeric length of living and fixed zebrafish, and developing larva at different stages.

<p>(A) Comparison of length of sarcomeres determined from zebrafish living and fixed with paraformaldehyde. The SHG images used to measure the lengths were measured on somites near the head (black, somites 5–8) and the tail (red, somites 21–24). The statistics were calculated based on 21 images obtained from three 72-hpf zebrafish. (B) Representative SHG images of zebrafish measured at three developmental stages (24, 36 and 72 hpf). The images were measured on regions near the head (somites 5∼8) and the tail (somites 21–24). (C) Growth of the sarcomere from 1 to 12 dpf. The SHG images used to evaluate the length were measured on somites near the head (black, somites 5∼8) and the tail (red, somites 21–24). The statistics were calculated based on 21 images obtained from three 72-hpf zebrafish.</p

Sarcomeric length determined with SHG imaging.

<p>(A) A cartoon illustration of a sarcomere and the architectural arrangement of its major constituent filaments. The length of a sarcomere is defined as the separation between two <i>z</i>-disks. (B) A high-resolution SHG image of zebrafish muscles. The image exhibits two characteristic patterns that are denoted as double (gray arrow) and single bands (white arrow), respectively. Scale bar: 5 µm. (C, D) Representative cross-sectional plots of a double band (C) and a single band (D). The length of the sarcomere was determined from the distance between the two dashed lines as shown in the two cross-sectional plots. (E) Comparison of the averaged length of the sarcomere determined from the two sarcomeric patterns. The statistics were calculated based on 12 images obtained from somites near the head (fifth to eighth somite) of three 72-hpf zebrafish.</p

Structural modification of zebrafish muscles induced on treatment with statin.

<p>(A) A representative SHG image of the control (an untreated zebrafish larva, 72 hpf). (B) A representative SHG image of a zebrafish larva (72 hpf) subject to treatment with statin (50 µM) for 12 h. Image size: 140 µm (w)×100 µm (h). (C) Effect of statin treatment on zebrafish with underdeveloped sarcomeres. The zebrafish was subject to a treatment of statin (0.5 µM) at 24 hpf for 12 h, and imaged at 36 hpf (black: somites 5∼8; red: somites 21–24). (D) Effect of statin treatment on zebrafish with fully developed sarcomeres. The zebrafish was subject to a treatment of the same dosage at 72 hpf for 12 h, and the image was taken at 84 hpf (head, somites 5∼8).</p

<b>Knock-down </b><i>foxi3a</i> Expression Severely Reduces Epidermal Ionocyte Progenitor Number and Abolishes the Later Differentiation Program.

<p>(A–G) Interfering with <i>foxi3a</i> and <i>foxi3b</i> functions of epidermal ionocyte differentiation by a morpholino (0.5 mM/embryo) injection. Morphants were fixed at 24 hours post fertilization (hpf) and stained with <i>atp1b1b</i> (green), <i>ca2a</i> (red), and P63 (blue) to detect Na<i>⁺</i>,K<i>⁺</i>-ATPase rich cells (NaRCs), H<i>⁺</i>-ATPase rich cells (HRCs), and epidermal stem cells, respectively. (H–I) A rescue experiment to show the specificity of <i>foxi3a</i> morpholinos. The <i>foxi3a</i> mRNA used for the rescue experiment does not contain a binding site for MO2. (J–L) Comparison of the vital dye uptake ability between the wild type (wt), <i>foxi3a</i> morphants, and <i>foxi3b</i> morphants. NaRCs and HRCs in either wild-types or <i>foxi3b</i> morphants can absorb MitoTracker (red) and Con-A (green) through to their apical openings. For <i>foxi3a</i> morphants, no MitoTracker or Con-A staining was detected due to blockage of the entire differentiation program. (M–O) Detection of the apical opening of epidermal ionocytes in wild-types, <i>foxi3a</i> morphants, and <i>foxi3b</i> morphants by scanning electron microscopy. The apical openings of NaRCs and HRCs in wild-types are shaped as deep holes (green box) or a mesh (red box), respectively. The apical openings were totally undetected in <i>foxi3a</i> morphants due to blockage of the entire differentiation program. For <i>foxi3b</i> morphants, the apical openings for both NaRCs and HRCs were reduced. Embryos in (A–I) were scored at 24 hpf, while in (J–O), they were scored at 72 hpf. In I, embryos are orientated in a dorsal-up and anterior-top position.</p

Low Temperature Mitigates Cardia Bifida in Zebrafish Embryos

<div><p>The coordinated migration of bilateral cardiomyocytes and the formation of the cardiac cone are essential for heart tube formation. We investigated gene regulatory mechanisms involved in myocardial migration, and regulation of the timing of cardiac cone formation in zebrafish embryos. Through screening of zebrafish treated with ethylnitrosourea, we isolated a mutant with a hypomorphic allele of <i>mil</i> (<i>s1pr2</i>)/<i>edg5</i>, called <i>s1pr2^as10</i> (<i>as10</i>). Mutant embryos with this allele expressed less <i>mil</i>/<i>edg5</i> mRNA and exhibited cardia bifida prior to 28 hours post-fertilization. Although the bilateral hearts of the mutants gradually fused together, the resulting formation of two atria and one tightly-packed ventricle failed to support normal blood circulation. Interestingly, cardia bifida of <i>s1pr2^as10</i> embryos could be rescued and normal circulation could be restored by incubating the embryos at low temperature (22.5°C). Rescue was also observed in <i>gata5</i> and <i>bon</i> cardia bifida morphants raised at 22.5°C. The use of DNA microarrays, digital gene expression analyses, loss-of-function, as well as mRNA and protein rescue experiments, revealed that low temperature mitigates cardia bifida by regulating the expression of genes encoding components of the extracellular matrix (<i>fibronectin 1</i>, <i>tenascin-c</i>, <i>tenascin-w</i>). Furthermore, the addition of N-acetyl cysteine (NAC), a reactive oxygen species (ROS) scavenger, significantly decreased the effect of low temperature on mitigating cardia bifida in <i>s1pr2^as10</i> embryos. Our study reveals that temperature coordinates the development of the heart tube and somitogenesis, and that extracellular matrix genes (<i>fibronectin 1</i>, <i>tenascin-c</i> and <i>tenascin-w</i>) are involved.</p></div

Additional file 2: Figure S1. of Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development

Separation of IBs and ribs of M. amblycephala from 1 to 2 year old by SDS-PAGE. Figure S2. The basic information statistics of proteome in this study. Figure S3. The repeatability analysis of data obtained from iTRAQ in different comparison groups based on CV (Coefficient of Variation) analysis. Figure S4. Functional classification of identified proteins. (DOCX 1365 kb

Additional file 3: Table S1. of Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development

Functional annotation information for the 2,342 identified proteins in IBs and Ribs of M. amblycephala. Table S2. Proteins information annotated with GO functions in IBs and Ribs of M. amblycephala. Table S3. KEGG pathway analysis of identified proteins in IBs and Ribs of M. amblycephala. Table S4. Detailed information for five pathways proteins of M. amblycephala. Table S5. Detailed information for proteins associated with bone cell of M. amblycephala. Table S6. Differential expressed proteins information in 1-IB-vs-1-Rib and 2-IB-vs-2-Rib. Table S7. Differential expressed proteins information in 1-IB-vs-1-IB and 1-Rib-vs-1-Rib. Table S8. The transition information of two comparison group of target proteins. (XLSX 20640 kb

Morphological changes in DA neuron patterning.

<p>In situ hybridization with tyrosine hydroxylase anti-sense mRNA probe. (<b>A</b>) Control. (<b>B</b>) 5 ng <i>ddc</i> tMO1 treated. (<b>C</b>) 100 pg <i>ddc</i> mRNA treated. (<b>D</b>) Co-injected mixture with 5 ng <i>ddc</i> tMO1 and 50 pg <i>ddc</i> mRNA with non-tMO1 binding site. Numbers show the site of dopaminergic neuron clusters 1-7 and 8, which indicated spots in retina, locus coeruleus (LC), pre-tectum (PrC, arrowhead) and olfactory bulb (OB, arrow). Compared to tyrosine hydroxylase expression pattern in control larvae, several tissue parties including olfactory bulb, pre-tectum and DA cluster were malpositioned or absent in the <i>ddc</i> morphant brains.</p

Eye movement in 5 dpf morphant significant reduced during rotation.

<p>Both frequency and angle of rotation decline compared with control and morpahant. (A) Schematic representation of the method used to obtain eye movement recordings from embedded larvae with 3% methylcellulose. Calculate with the angle θ to plane of eyeball and body axis. (B) Statistics analysis for eye rotation frequency during 120 seconds. (C) Statistics anaylsis for average of eye movement angle. Left: left eyeball, Right: right eyeball. The bars represent changes in different treatment of 5 dpf larvae in three independent experiments. n=7 for each treated larvae. Results were expressed as mean±SD. **<i>p</i><0.01, ***<i>p</i><0.001 compared to control larvae.</p