Bmp Suppression in Mangrove Killifish Embryos Causes a Split in the Body Axis

<div><p>Bone morphogenetic proteins (Bmp) are major players in the formation of the vertebrate body plan due to their crucial role in patterning of the dorsal-ventral (DV) axis. Despite the highly conserved nature of Bmp signalling in vertebrates, the consequences of changing this pathway can be species-specific. Here, we report that Bmp plays an important role in epiboly, yolk syncytial layer (YSL) movements, and anterior-posterior (AP) axis formation in embryos of the self-fertilizing mangrove killifish, <i>Kryptolebias marmoratus</i>. Stage and dose specific exposures of embryos to the Bmp inhibitor dorsomorphin (DM) produced three distinctive morphologies, with the most extreme condition creating the splitbody phenotype, characterised by an extremely short AP axis where the neural tube, somites, and notochord were bilaterally split. In addition, parts of caudal neural tissues were separated from the main body and formed cell islands in the posterior region of the embryo. This splitbody phenotype, which has not been reported in other animals, shows that modification of Bmp may lead to significantly different consequences during development in other vertebrate species.</p></div

Stage specific inhibition of Bmp in <i>K. marmoratus</i>.

<p>Embryos were exposed to 100 µM dorsomorphin at the 32-cell (<b>B</b>), late blastula (<b>C</b>) and 80% epiboly (<b>D</b>) stages of development. Photographs of the embryos were taken 3 days post-fertilization. <b>A1–3</b>: Control (n = 20/20). <b>B1–3</b>: splitbody (phenotype variation details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084786#pone-0084786-g003" target="_blank">Figure 3</a>), this phenotype is characterised by absence of a distinct tail region (<b>B1</b> arrowhead), morphologically undifferentiated head region (<b>B2</b> arrowhead) and split body axis (<b>B2</b> arrow), and cell islands in the posterior region (<b>B3</b> arrowhead). <b>C1–3</b>: Curled tail (n = 8/10), this phenotype resembles <i>snailhouse</i> seen in zebrafish and is characterised by its curled tail (<b>C3</b> arrowhead). <b>D1–3</b>: Bent tail (n = 12/12, this phenotype primarily displayed a bent tail (<b>D3</b> arrowhead). Overview images are lateral views and head/tail images are dorsal views of the embryos. Scale bars: 250 µm.</p

Dorsomorphin inhibits phosphorylation of Smad1/5.

<p>Bmp signalling activity was quantified by measuring phosphorylation of Smad 1/5 at the late gastrula stage. Embryos were exposed to 100 µM from 32-cell as well as 100 and 200 µM DM from late blastula. These were then frozen at the late gastrula stage and used for Western Blotting. <b>A:</b> Quantification of densitometry results obtained from 3 independent experiments (Mean ± SE), normalised to total Smad and indicated as fold increase over the resting control condition. All 3 treatments were significantly different from the control as indicated by asterisks (P<0.001), but no significant differences were observed between the treatments. <b>B:</b> Representative Western Blot of 3 independent experiments showing the levels of total Smad1/5/8 and phospho-Smad1/5 at late gastrula from the dose and stage specific treatments. These data demonstrate that all 3 treatments equally suppress phospho-Smad1/5 by late gastrula.</p

Somites and the notochord are divided in the splitbody phenotype.

<p><i>K. marmoratus</i> embryos were exposed to 100 µM dorsomorphin at the 32-cell stage and fixed 1 and 4 days post-fertilization in order to stain the notochord by <i>in situ</i> hybridization using a medaka <i>ntl</i> probe (<b>A, B</b>, <b>C</b>), and somites using the myosin antibody MF-20 (<b>D</b>, <b>E</b>). In control embryos at late gastrula, <i>ntl</i> stained axial mesoderm in the dorsal axis (<b>A1</b> arrowhead, n = 10/10), whereas in DM treated embryos these cells appeared to have stayed in the lateral domains (<b>A2</b> arrowhead, n = 10). At day 4, <i>ntl</i> stained the notochord in the tip of the tail for control embryos (<b>B2</b> arrowhead, n = 10/10), whereas splitbody embryos had the tips of both body axes stained with <i>ntl</i> (<b>C2</b> arrowheads, n = 10/10). For control embryos, somites are formed as pairs arranged either side of the neural axis (<b>D1–3</b> arrowheads, n = 10/10). In the splitbody phenotype, somites were unpaired and separated in the two body axes (<b>E1</b> Hoechst staining showing the body split; <b>E2, 3</b> arrowheads, somites are present in both axes, n = 10/10). Photographs were taken at late gastrula for <b>A</b>, and 4 days post-fertilization for <b>B</b>–<b>E</b>. Images in <b>A</b> are lateral views and for <b>B</b>–<b>E</b> dorsal views of the embryos. Scale bars: 250 µm.</p

Bmp inhibition delays epiboly progression in <i>K. marmoratus</i>.

<p>Epiboly coverage was recorded at day 1 (<b>A</b>) and day 2 (<b>B</b>) post-fertilization (dpf) in embryos exposed to 100 µM dorsomorphin (DM) at the 32-cell stage. Progression of the yolk syncytial layer (YSL) during gastrulation was assessed via staining of yolk syncytial nuclei (YSN) using Sytox Green. The green fluorescent YSN were observed 1 dpf (<b>C</b>). <b>A1, 2</b>: As control embryos reach <i>c.</i> 70% epiboly (<b>A1</b> arrowhead, n = 10/10), DM treated embryos are delayed with epiboly covering <i>c.</i> 30% of the yolk (<b>A2</b> arrowhead, n = 10/10). <b>B1, 2</b>: Controls reach the otic vesicle formation stage (<b>B1,</b> n = 10/10) whilst exposed embryos are lagging behind around 90% epiboly (<b>B2</b> arrowhead, n = 10/10). <b>C1</b>, <b>2</b>: Shortly after epiboly closure, control embryos enter the eye formation stage (<b>C1,</b> n = 10/10) (embryo and the eye are outlined) and YSN are spread all over the yolk. On the other hand DM exposed embryos are still mid-epiboly and fluorescent YSN are observed near the blastoderm margin (<b>C2</b> arrowhead, n = 10/10), demonstrating that YSN are also delayed by inhibition of Bmp signalling. All images are lateral views of the embryos. Scale bars: 250 µm.</p

The neural tube is separated in embryos of the splitbody phenotype.

<p><i>K. marmoratus</i> embryos were exposed to 100 µM dorsomorphin (DM) at the 32-cell stage (<b>B, C</b>), and 200 µM DM at the late blastula stage (<b>D</b>, <b>E</b>) of development. These embryos were then fixed 3 days post-fertilization and used for <i>in situ</i> hybridization using a <i>sox3</i> probe (stains all neural tissue) (<b>A–E2, A–E4</b>) and Hoechst staining (a blue fluorescent DNA stain) (<b>A–E1, A–E3</b>) in order to examine body contour and split neural tube (<b>A–E1</b>, <b>2</b>), and the nature of the posterior isolated cell lumps or cell islands (<b>A–E3</b>, <b>4</b>). <b>A1–4</b>: Control embryo (n = 20/20). <b>B, D</b>: Splitbody individual with an opened end of the body axis and neural tube split (<b>B1, 2</b> arrowheads n = 19/20, and <b>D1, 2</b> arrowheads n = 12/20). Splitbody individuals with a closed end, as both strands of the body axis and neural tube join in their most posterior region (<b>C1, 2</b> arrowheads n = 1/20, and <b>E1, 2</b> arrowheads n = 8/20). All DM embryos presented here generated cell islands (<b>B-E3</b> arrowheads) with distinct <i>sox3</i> positive staining (<b>B–E4</b> arrowheads). All images are dorsal views of the embryos. Scale bar: 250 µm.</p

Sensory systems and ionocytes are targets for silver nanoparticle effects in fish

<p>Some nanoparticles (NPs) may induce adverse health effects in exposed organisms, but to date the evidence for this in wildlife is very limited. Silver nanoparticles (AgNPs) can be toxic to aquatic organisms, including fish, at concentrations relevant for some environmental exposures. We applied whole mount <i>in-situ</i> hybridisation (<i>WISH</i>) in zebrafish embryos and larvae for a suite of genes involved with detoxifying processes and oxidative stress, including metallothionein (<i>mt2</i>), glutathionine <i>S</i>-transferase pi (<i>gstp</i>), glutathionine <i>S</i>-transferase mu (<i>gstm1</i>), haem oxygenase (<i>hmox1</i>) and ferritin heavy chain 1 (<i>fth1</i>) to identify potential target tissues and effect mechanisms of AgNPs compared with a bulk counterpart and ionic silver (AgNO₃). AgNPs caused upregulation in the expression of <i>mt2, gstp</i> and <i>gstm1</i> and down regulation of expression of both <i>hmox1</i> and <i>fth1</i> and there were both life stage and tissue-specific responses. Responding tissues included olfactory bulbs, lateral line neuromasts and ionocytes in the skin with the potential for effects on olfaction, behaviour and maintenance of ion balance. Silver ions induced similar gene responses and affected the same target tissues as AgNPs. AgNPs invoked levels of target gene responses more similar to silver treatments compared to coated AgNPs indicating the responses seen were due to released silver ions. In the <i>Nrf2</i> zebrafish mutant, expression of <i>mt2</i> (24 hpf) and <i>gstp</i> (3 dpf) were either non-detectable or were at lower levels compared with wild type zebrafish for exposures to AgNPs, indicating that these gene responses are controlled through the Nrf2-Keap pathway.</p