Neural Crest Migration and Survival Are Susceptible to Morpholino-Induced Artifacts

<div><p>The neural crest (NC) is a stem cell-like embryonic population that is essential for generating and patterning the vertebrate body, including the craniofacial skeleton and peripheral nervous system. Defects in NC development underlie many birth defects and contribute to formation of some of the most malignant cancers in humans, such as melanoma and neuroblastoma. For these reasons, significant research efforts have been expended to identify genes that control NC development, as it is expected to lead to a deeper understanding of the genetic mechanisms controlling vertebrate development and identify new treatments for NC-derived diseases and cancers. However, a number of inconsistencies regarding gene function during NC development have emerged from comparative analyses of gene function between mammalian and non-mammalian systems (chick, frog, zebrafish). This poses a significant barrier to identification of single genes and/or redundant pathways to target in NC diseases. Here, we determine whether technical differences, namely morpholino-based approaches used in non-mammalian systems, could contribute to these discrepancies, by examining the extent to which NC phenotypes in <i>fascin1a (fscn1a)</i> morphant embryos are similar to or different from <i>fscn1a</i> null mutants in zebrafish. Analysis of <i>fscn1a</i> morphants showed that they mimicked early NC phenotypes observed in <i>fscn1a</i> null mutants; however, these embryos also displayed NC migration and derivative phenotypes not observed in null mutants, including accumulation of <i>p53</i>-independent cell death. These data demonstrate that morpholinos can cause seemingly specific NC migration and derivative phenotypes, and thus have likely contributed to the inconsistencies surrounding NC gene function between species. We suggest that comparison of genetic mutants between different species is the most rigorous method for identifying conserved genetic mechanisms controlling NC development and is critical to identify new treatments for NC diseases.</p></div

NC-derived tissues form abnormally in <i>fscn1a</i>-morphant embryos.

<p>(A) Lateral and ventral views of 5 dpf <i>tp53</i>^<i>zdf1</i> embryos injected with <i>coMO</i> or <i>fscn1aMO</i> and stained with Alcian blue. Numbers correspond to pharyngeal arches. Asterisk denotes arches that are severely reduced in size or absent. (B) Lateral views of 3 dpf <i>tp53</i>^<i>zdf1</i> embryos analyzed by whole-mount ISH for <i>th</i>. Arrows denote <i>th</i>-positive neurons of sympathetic ganglia. (C) Lateral views of section of the gut in 4 dpf <i>Tg(phox2b</i>:<i>gfp)</i> embryos injected with <i>tp53MO</i> or <i>tp53MO</i> plus <i>fscn1aMO</i>. Arrows denote <i>phox2b</i>-positive enteric neurons. (D) Lateral views of trunk in 3 dpf Tg(<i>ngn1</i>:<i>gfp</i>) embryos injected with <i>tp53MO</i> or <i>tp53MO</i> plus <i>fscn1aMO</i>. Arrows in top panel highlight <i>ngn1</i>-positive NC-derived dorsal root ganglia (drg) and central nervous system (CNS)-derived Rohon-Beard neurons (rb). In lower panel, arrowhead and asterisk indicate misplaced and absent dorsal root ganglia, respectively. drg; dorsal root ganglia, rb; Rohan-Beard neurons, ye; yolk extension. In all panels, anterior is to the left. All experiments in this figure were performed independently at least three times with similar results. All scale bars in this figure = 100 μm.</p

<i>fscn1aMO</i> reduces NC-cell filopodia formation.

(A) Lateral views of posterior cranial NC streams in 26 hpf Tg(sox10:rfpmb) embryos injected with tp53MO or tp53MO plus fscn1aMO. Numbers correspond to NC streams. Arrows denote filopodia at leading edge of NC streams. Arrowheads mark RFP-positive puncta surrounding NC streams. Asterisk highlights fusion of NC streams 3 and 4 in fscn1a-morphant embryo. ov; otic vesicle. Scale bar = 50μm. (B) Time-lapse confocal images of filopodia at leading edge of NC stream 3 in 26 hpf Tg(sox10:rfpmb) embryos injected with tp53MO or tp53MO plus fscn1aMO. Arrows mark tips of single filopodia throughout the time lapse. Scale bar = 10 μm. (C) Quantitation of mean filopodia number in 20 μm region at leading edge of NC stream 3, mean filopodia length, maximum filopodia length, and change in filopodia length over 45 minutes in 26 hpf Tg(sox10:rfpmb) embryos injected with tp53MO or tp53MO plus fscn1aMO (n = 5 embryos and 25 filopodia for each condition, **p<0.005, ***p<0.001 by an unpaired t-test analysis). In all panels, anterior is to the left. At least three independent experiments were performed in this figure with similar results.</p

Analysis of a translation-blocking <i>fscn1a</i> morpholino.

<p>(A) Schematic of <i>fscn1a</i> genomic locus. Binding sites for translation-blocking <i>fscn1a-ATGMO</i> and splice-blocking <i>fscn1a-i1e2MO</i> are depicted. Arrow indicates translation start site. (B) Bright-field and fluorescent images of 24 hpf <i>tp53</i>^<i>zdf1</i> mutant embryos injected with the indicated amount of <i>fscnMOgfp</i> mRNA and <i>coMO</i> or <i>fscn1a-ATGMO</i>. (C) Immunoblot showing Fscn1a protein levels in 48 hpf <i>tp53</i>^<i>zdf1</i> embryos injected with 1.25 ng of <i>coMO</i> or <i>fscn1a-ATGMO</i>. Values below the blot represent relative band intensity of Fscn1a/GAPDH normalized to <i>coMO</i> sample. (D) Representative bright-field images of 5 dpf <i>tp53</i>^<i>zdf1</i> mutant embryos injected with 1.25 ng of the indicated MOs. Fscn1a morphants show loss of tissue associated with the lower jaw as well as cardiac edema (arrows highlight both phenotypes). (E) <i>tp53</i>^<i>zdf1</i> embryos were injected with the indicated MO and analyzed at 5 dpf for craniofacial morphology. (F) <i>tp53</i>^<i>zdf1</i> embryos were injected with the indicated MO (1.25 ng) and/or mRNA (25 pg) and analyzed at 5 dpf for craniofacial morphology. All experiments in this figure were performed independently at least three times with similar results.</p

<i>Fscn1aMO</i> induced <i>tp53</i>-independent cell death in NC cells.

<p><b>(A)</b> Lateral and dorsal cranial views of 28 hpf <i>tp53</i>^<i>zdf1</i> mutant embryos injected with <i>coMO</i> or <i>fscn1aMO</i> and stained with AO. Arrowheads highlight AO-positive cells adjacent to neural tube. <b>(B)</b> Lateral view of 24 hpf <i>Tg(sox10</i>:<i>rfpmb)</i> embryo injected with <i>tp53MO</i> plus <i>fscn1aMO</i> and stained with AO. Numbers correspond to NC streams. Arrows indicate regions of RFP-positive/AO-positive cells. e; eye, nt; neural tube. In all lateral views or dorsal cranial views, anterior is to the left or bottom, respectively. Experiments in this figure were performed independently at least three times with similar results.</p

Genetic diagram of Bad-mediated radiosensitization of zebrafish neural tissue.

<p>IR activates the pro-apoptotic activity of Bad in a pathway that is either downstream of or parallel to p53 (dotted lines indicate that it is unclear whether this step occurs in a p53-dependent or –independent manner). Bad and Puma are dependent upon each other to promote IR-induced apoptosis. However, based on Bad’s established role as a sensitizer BH3-only protein and Puma’s reported role as an activator BH3-only protein, Bad likely functions upstream of Puma to induce IR-mediated apoptosis through the mitochondrial pathway.</p

Late-stage NC-cell migration is disrupted in <i>fscn1a</i>-morphant embryos.

<p>(A-B) Dorsal cranial views of <i>tp53</i>^<i>zdf1</i> embryos injected with <i>coMO</i> or <i>fscn1aMO</i> and analyzed by whole-mount <i>in situ</i> hybridization (ISH) for (A) <i>foxd3</i> mRNA at 10 hpf and (B) <i>sox10</i> mRNA at 15 hpf. (C) Dorsal cranial and lateral views of 26 hpf <i>tp53</i>^<i>zdf1</i> embryos injected with <i>coMO</i> or <i>fscn1aMO</i> and analyzed by whole-mount ISH for <i>dlx2a</i>. Numbers correspond to pharyngeal arches. Arrow denotes reduction in <i>dlx2a</i>-positive cranial NC cells in <i>fscn1a</i> morphants. (D) Lateral views of cranial NC streams in 22, 25, 28 and 36 hpf <i>Tg(sox10</i>:<i>gfp)</i> embryos injected with <i>tp53MO</i> or <i>tp53MO</i> plus <i>fscn1aMO</i>. Numbers correspond to NC streams. Arrows highlight NC cells migrating independently of NC streams in <i>fscn1a</i> morphants. e; eye, ov; otic vesicle. In all panels, anterior is to the left. All experiments in this figure were performed independently at least three times with similar results. All scale bars in this figure = 100 μm.</p

Bad is required for IR-induced apoptosis in zebrafish embryonic neural tissue.

<p>(A) Shown are lateral views of 27-hpf embryos (head is top left in each panel) either uninjected or injected with 200 nmol of <i>bad ATG</i>, <i>bad e2i2</i> or mismatch (mm) MO. Half of each group of embryos were exposed to 15 Gy IR, and all were analyzed by the Casp3 assay. In control embryos (no inj and mm), IR-induced apoptosis occurs predominantly in the brain and all along the spinal cord (white arrowheads), whereas in <i>bad</i>-deficient embryos (ATG and e2i2), residual apoptosis is only observed in the head (arrowheads). (B) Fluorescence intensity, reflecting level of Caspase 3 activity, was measured in the spinal cords of at least 10 embryos from each group in (A) as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088151#pone.0088151-Sorrells1" target="_blank">[34]</a>. The fluorescence intensity in irradiated mismatch-MO-injected embryos was normalized to 1. (C) One-cell stage zebrafish embryos were injected with 100 nmol of <i>bad ATG</i>, <i>bad e2i2</i> or mm MO as indicated (“++” indicates that 200 nmol was injected to keep total concentration of MO constant between experimental groups) and irradiated and analyzed as in (A-B). Data represent one experiment, and the experiment was independently performed three times with similar results.</p

The ideal eight quality dimensions of 3D printed bone implants.

The ideal eight quality dimensions of 3D printed bone implants.</p

Bad does not augment p53 transcriptional activity.

<p>p53 transcriptional activity was analyzed in embryos injected with 50<i>mcherry</i> (cntl) or <i>hBAD</i>. Embryos were exposed to 8 Gy (or not) at 24 hpf. RNA was harvested from each group at 27 hpf and analyzed for gene expression changes by qPCR. Expression of the <i>gapdh</i> gene was measured to normalize <i>puma</i> and <i>p21</i> mRNA levels. All data was compared to unirradiated wild-type control-mRNA-injected data, which was adjusted to a value of 1. Control-injected <i>p53</i> mutant embryos irradiated at 24 hpf and harvested at 27 hpf were included as a negative control for p53-mediated transcriptional induction. Data represent one experiment, but the experiment was independently performed three times with similar results.</p