<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

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

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

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

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

<i>fscn1a</i> cooperates with <i>cxcr4a/cxcl12b signaling</i> to regulate directional cranial NC migration.

<p>(A) Representative confocal images of cranial NC cells from a 26 hpf <i>Tg(sox10:rfpmb)</i> embryo injected with <i>cxcr4a-GFP</i> mRNA. Arrows highlight Cxcr4a-GFP localized to NC filopodia. Scale bar = 10 μm. (B) Lateral view of leading edge of cranial NC stream 1 in 26 hpf uninjected, <i>cxcl12b</i> mRNA-injected or <i>cxcr4aMO</i>-injected <i>Tg(sox10:rfpmb)</i> or <i>Tg(sox10:rfpmb); fscn1a MZ</i> embryos. Scale bar = 25 μm. (C) Quantitation of filopodia number and filopodia length in uninjected, <i>cxcl12b</i> mRNA-injected or <i>cxcr4aMO</i>-injected <i>Tg(sox10:rfpmb)</i> and <i>Tg(sox10:rfpmb); fscn1a MZ</i> embryos (n = 7 <i>Tg(sox10:rfpmb)</i>, n = 9 <i>Tg(sox10:rfpmb)</i> + <i>cxcl12b</i> mRNA, n = 5 <i>Tg(sox10:rfpmb)</i> + <i>cxcr4aMO</i>, n = 7 <i>Tg(sox10:rfpmb); fscn1a MZ</i>, n = 8 <i>Tg(sox10:rfpmb); fscn1a MZ</i> + <i>cxcl12b</i> mRNA, n = 3 <i>Tg(sox10:rfpmb); fscn1a MZ</i> + <i>cxcr4aMO</i>, ***p<0.001, ****p<0.0001). (D) Lateral views of 5 dpf uninjected, <i>cxcl12b</i> mRNA-injected or <i>cxcr4aMO</i>-injected wild type (wt) and <i>fscn1a MZ</i> embryos. Arrowheads indicate reduced jaw. (E) Quantitation of percentage of embryos with normal or abnormal craniofacial development at 5 dpf (n = 43 wt, 104 wt + <i>cxcl12b</i> mRNA, n = 51 wt + <i>cxcr4aMO</i>, n = 165 <i>fscn1a MZ</i>, n = 130 <i>fscn1a MZ</i> + <i>mmMO</i>, n = 123 <i>fscn1a MZ</i> + <i>cxcl12b</i> mRNA, n = 154 <i>fscn1a MZ</i> + <i>cxcr4aMO</i>).</p

Depletion of <i>fscn1a</i>-independent NC cell filopodia by low concentration Latrunculin B treatment does not enhance the severity of <i>fscn1a MZ</i> craniofacial defects.

<p>(A) Lateral views of leading edge of cranial NC stream 1 in 26 hpf DMSO or Lat. B-treated <i>Tg(sox10:rfpmb)</i> or <i>Tg(sox10:rfpmb); fscn1a MZ</i> embryos. Scale bar = 25 μm. (B) Quantitation of filopodia number and filopodia length in DMSO or Lat. B-treated <i>Tg(sox10:rfpmb)</i> and <i>Tg(sox10:rfpmb); fscn1a MZ</i> embryos (n = 9 <i>Tg(sox10:rfpmb)</i> + DMSO, n = 10 <i>Tg(sox10:rfpmb)</i> + Lat. B, n = 10 <i>Tg(sox10:rfpmb); fscn1a MZ</i> + DMSO, n = 10 <i>Tg(sox10:rfpmb); fscn1a MZ</i> + Lat. B, *p = 0.02, **p<0.005, ***p<0.001, ****p<0.0001, ns = not significant). (C) Representative ventral views of 5 dpf DMSO or Lat. B-treated wild type (wt) and <i>fscn1a MZ</i> embryos stained with Alcian blue. (D) Quantitation of percentage of embryos with normal or abnormal craniofacial development at 5 dpf. Numbers above columns indicate number of embryos included in analysis (n = 83 wt + DMSO, 93 wt + Lat. B, n = 86 <i>fscn1a MZ</i> + DMSO, n = 72 <i>fscn1a MZ</i> + Lat. B, *p = 0.02).</p

<i>fscn1a</i> is required for collective migration of the first NC stream.

<p>Dorsal cranial views of wild type and <i>fscn1a MZ</i> embryos analyzed by RNA <i>in situ</i> hybridization for (A) <i>foxd3</i> mRNA at 11 hpf, (B) <i>sox10</i> mRNA at 15 hpf, showing asymmetric dispersion of NC cells in <i>fscn1a MZ</i> (arrow), and (C) <i>dlx2a</i> mRNA at 18, 28 and 36 hpf. Numbers correspond to pharyngeal arches, arrows indicate abnormal NC migration phenotypes observed in <i>dlx2a+</i> NC cells in <i>fscn1a MZ</i> embryos. Anterior is left in all panels. Phenotypes shown in <i>fscn1a MZ</i> panels were observed in 100% (A) or ∼20% (B-C) of embryos examined (n>200).</p

<i>fscn1a</i> is required for development of NC-derived craniofacial cartilage.

<p>(A) Lateral views of wild type (wt) and <i>fscn1a MZ</i> 5 dpf embryos. Arrow indicates reduced jaw in <i>fscn1a MZ</i> embryos. (B) Lateral (left) and ventral (right) views of Alcian blue-stained wt and <i>fscn1a MZ</i> 5 dpf embryos. Numbers correspond to pharyngeal arches. Arrow indicates missing palatoquadrate in <i>fscn1a MZ</i> embryos. (C) Ventral view of dissected Alcian blue-stained pharyngeal skeletons from wt and <i>fscn1a MZ</i> 5 dpf embryos. ch = ceratohyal, cb = ceratobranchial, hm = hyomandibular, hs = hyosymplectic, mk = Meckel’s cartilage, pq = palatoquadrate.</p