Identification and analysis of ia2 deletion.

<p>(A) Schematic representation of ia2 deletion in chromosome 1 (in red). All the STS and genes mapping in the region defined by marker chr1_452 (272 bp downstream Chr1_ 450) and LG1Tel6035 were not amplifiable by PCR in homozygous mutants. According with marker positions, we identified in chromosome 1 of <i>Tg(-8</i>.<i>5nkx2</i>.<i>2a</i>:<i>GFP)</i>^<i>ia2</i> embryos a deletion of 15.2 Mb. The black bar displays the position where the -8.5nkx2.2a:GFP transgene was mapped by linkage analysis. (B) STRING analysis of the protein-protein interactions among <i>p53</i> and the products encoded by the zebrafish ia2 deleted genes. A first STRING analysis was performed on the products encoded by the human orthologues of the ia2 deleted genes only (ia2). The virtual addition of p53 determines the formation of a big cluster, with p53 directly linked with 9 ia2 deleted gene products (ia2 + p53). Other important tumor suppressors don’t share the same feature: as an example, the analysis with APC (ia2 + APC) and PTEN (ia2 + PTEN) are reported. Disconnected nodes are not shown.</p

Morphological alterations of ia2/ia2 mutant at 24 hpf.

<p>(A, A’) Head side view: mutant embryos display a relevant delay in head structures development, with just sketchy eye, when visible. (B, B’) Somites side view, showing their poorly defined shape and boundaries in the mutant. Globular cells with impaired adhesion are also visible. (C, C’) Notochord dorsal view: in mutant embryos notochord bending on the coronal plane is evident. (D, D’) Notochord side view, showing how notochord cells structure is also affected in mutant embryos, with globular and disorganized vacuoles, compared to the stretched and flanked ones of the wild-type. (E, E’) Tail terminal portion, side view: the notochord is indistinguishable in the mutant and the mesenchyme seems disaggregated with many non-adherent globular cells. Scale bar 50 μm.</p

Representative histological features of the ia2 abdominal carcinomas.

<p>(A) Abdominal tumors arise beneath the swim bladder, in close proximity with the gut, pancreas and liver. The neoplastic cells occasionally infiltrate the hepatic parenchyma (arrows) and the intestinal wall (arrowhead). (B, C) Cytologically, the tumor is composed of polygonal cells with abundant eosinophilic cytoplasm. In most cases, a sub-population of epithelioid cells with more basophil cytoplasm is also faced (arrow). (D) The abdominal carcinomas consistently express the pan-cytokeratin AE1/AE3 (brown staining). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145178#pone.0145178.g004" target="_blank">Fig 4A</a> for the normal abdominal region.</p

Phenotype of the ia2 mutant at 24 hpf.

<p>The homozygous mutant (C) displays a strongly delayed development and compromised phenotype, while the heterozygote (B) appears indistinguishable from the wild-type (A). The green fluorescence relative to the <i>-8</i>.<i>5nkx2</i>.<i>2a</i>:<i>GFP</i> transgene is present in heterozygous and homozygous ia2 mutant fish, being the <i>GFP</i> gene co-inherited with the ia2 mutant allele. This allows the early and immediate screening of the three genotypes.</p

Incidence rate tumors in heterozygous ia2 mutant (ia2/+).

<p>(A) Cumulative percentages of tumor masses developed by ia2/+ individuals within 9, 12 and 16 months of age are depicted. (B) Representative images of the spontaneous tumor masses developed by heterozygous ia2/+ mutants, arising in the abdomen (top), along the spinal cord (middle) and intrathecally, causing the lateral projection of the eye (bottom).</p

Precocious and supernumerary sensory neuron specification in <i>sox10</i>^<i>baz1</i> mutants.

<p>A,B) <i>neurod1</i> expression is seen in more cells (close-ups in left panels; arrowheads indicate a subset of <i>neurod1</i>^<i>+</i> cells) and extending more posteriorly (right panels; arrowhead marks posteriormost <i>neurod1</i>^<i>+</i> DRG) in <i>baz1</i> mutants compared with WT siblings at both 36 and 45 hpf. C) Counts of <i>neurod1</i>^<i>+</i> cells on one side of embryo at 36 and 45 hpf embryos (N = 11 for all conditions except 36 hpf <i>baz1</i>, where N = 13). <i>baz1</i> mutants significantly different to WT siblings (Student’s <i>t</i> test; ***, p<0.0001. In this and all subsequent images, embryos are shown in lateral view with dorsal to the top and anterior to the left, unless otherwise stated. Scale bar, 100 μm.</p

Notch signaling is active in nascent DRGs.

<p>Neural crest cells in the DRGs of 48 hpf embryos (arrows) were readily identified by expression of mRFP reporter in <i>sox10</i>:<i>mRFP</i> fish (A). These overlapped with eGFP expression from the <i>12xNRE</i>:<i>eGFP</i> (Notch signaling) reporter (B), as shown in superimposed image (C and D). Close-ups of individual DRGs labelled with asterisk are shown in panels E and F. Panel G shows DRGs (white arrows, yellow signals) as single plane acquisitions, in lateral view (top left), transversal view (top right), dorsal view (bottom left) and mini global view (bottom right). Note that in all panels eGFP is also strongly visible in blood vessels, as expected for the Notch reporter. nc, notochord; sc, spinal cord.</p

deltaA and deltaD gene expression overlaps with neurog1 in the nascent DRGs.

<p>A-C) <i>deltaA</i> expression (red) clearly overlaps with <i>neurog1</i> (green) in the nascent DRG (arrows) at 30 hpf. D-F) At 38 hpf, <i>deltaA</i> expression is clearly seen in the DRGs, but weaker signals make it difficult to discern if expression is in the same cells as express <i>neurog1</i> or simply in other cells of the ganglia. G-I) <i>deltaD</i> expression (red) clearly overlaps with <i>neurog1</i> (green) in the nascent DRG (arrows) at 38 hpf. All main panels are confocal images of fluorescent dual-color <i>in situ</i> hybridisations in lateral view, with insets showing <i>y-z</i> planes (left) and <i>x-z</i> planes (above) for each. Insets in the bottom right of panels C, F and I show enlargements of the double-labeled cells indicated by the arrows. nc, notochord; sc, spinal cord.</p

Quantification of Notch reporter differences in m618 and baz1 mutants at 48 hpf.

<p>A-F) confocal acquisitions of WT control (A,D) and mutant (B, <i>m618</i>; E, <i>baz1</i>) trunk regions, followed by Notch reporter (NRE:EGFP) fluorescence analysis (C,F). A slight increase of reporter signal is detected in the neural tube (nt) and dorsal root ganglia (drg, arrowhead) of <i>m618</i> mutants (B) compared to controls (A), while a decrease of signal is detected in the same regions of <i>baz1</i> mutants (compare E with D). Relative fluorescence intensity (RFI) in aorta (a) and intersomitic vessels (v) appears unmodified in all conditions. n = 6 measurements per condition. n.s. = not significant; ** = p<0.01; *** = p<0.001.</p

Precocious and supernumerary sensory neuron specification in <i>sox10</i>^<i>baz1</i> mutants.

<p>A,B) <i>neurod1</i> expression is seen in more cells (close-ups in left panels; arrowheads indicate a subset of <i>neurod1</i>^<i>+</i> cells) and extending more posteriorly (right panels; arrowhead marks posteriormost <i>neurod1</i>^<i>+</i> DRG) in <i>baz1</i> mutants compared with WT siblings at both 36 and 45 hpf. C) Counts of <i>neurod1</i>^<i>+</i> cells on one side of embryo at 36 and 45 hpf embryos (N = 11 for all conditions except 36 hpf <i>baz1</i>, where N = 13). <i>baz1</i> mutants significantly different to WT siblings (Student’s <i>t</i> test; ***, p<0.0001. In this and all subsequent images, embryos are shown in lateral view with dorsal to the top and anterior to the left, unless otherwise stated. Scale bar, 100 μm.</p