PTP knockdowns affect C/E and cell polarization.

<p>(a) Zebrafish embryos were microinjected with morpholinos (high concentration) targeting the different phosphatase genes or RNA constructs encoding constitutively active forms of Fyn or Yes at the one cell stage and grown to 1 somite stage. Embryos were fixed and stained for <i>dlx3</i> and <i>hgg1</i> expression using whole mount <i>in situ</i> hybridization, staining the precursors of the hatching gland (<i>hgg1</i>) and the edge of the neural plate (<i>dlx3</i>). Posterior shift of the hatching gland and angle of <i>dlx3</i> staining are measured as shown in inset, the results are plotted in (a) and (b). Pictures of representative embryos used in the quantifications in (a) and (b) are shown in (c). Embryos were microinjected using the same conditions as described above and grown to 8–9 somite stage. Embryos were fixed and stained for <i>krox20</i> and <i>myod</i> using whole mount <i>in situ</i> hybridization. <i>Krox20</i> stains rhombomere 3 and 5, while <i>myod</i> stains the somites. Resulting staining patterns were used to quantify width to ratio by measuring rhombomere width (<i>krox20</i>) and somite length (8 somites, <i>myod</i>). Ratios are plotted in (d), representative embryos are depicted in (e). (f) Zebrafish embryos were micro-injected using the constructs described above, co-injected with RNA encoding YFP-caax and RNA encoding mCherry-H2B at the one cell stage and mounted at shield stage. Embryos were imaged over time at the presomitic mesoderm, representative areas of presomitic mesoderm for each condition are shown. Resulting images were analyzed for cell shape (aspect ratio) by dividing the length of the longest axis by the length of the shortest axis for each cell, average aspect ratios are plotted in (g). The distribution of angles of the longest axis towards the dorsal midline were plotted in rose-plots and shown in (f; bottom). All error bars are standard error of the mean. Student t-tests were performed with non-injected control; no asterisk indicates P>0.05, * indicates 0.05>P>0.001 and ** indicates P<0.001.</p

<i>Ptpn13</i> and <i>ptpn20</i> cooperate with each other and with <i>ptpra</i> and <i>ptpre</i>.

<p>Morpholinos targeting <i>ptpn13</i> and <i>ptpn20</i> were injected in the zebrafish at the one cell stage, and concentrations were titrated down until no phenotype was observed. Normal (red), low (green) concentrations and combined low concentrations of <i>ptpn13</i> and <i>ptpn20</i> morpholino were micro-injected and embryos were grown to 3dpf under normal conditions. Pictures were taken from all embryos and tails were measured using ImageJ imaging software, from the yolk to the tip of the tail, and compared to non-injected control. Average tail length compared to non-injected control is plotted as a percentage deviating from 100% in (a) and representative fish are shown for each condition in (b). Zebrafish embryos were microinjected as described above, using low concentration combined knockdown of <i>ptpra</i> with either <i>ptpn13</i> or <i>ptpn20</i>, or <i>ptpre</i> with either <i>ptpn13</i> or <i>ptpn20</i> and tail lengths are plotted in (c) and (d). (e) Shown are representative fish from the experiments depicted in (c) and (d). All error bars are standard error of the mean. Student t-test was performed where indicated; no asterisk indicates P>0.05, * indicates 0.05>P>0.001 and ** indicates P<0.001. Morpholino concentrations are color coded: red for “full" knockdown, giving full phenotype without being toxic and green for “low" concentration, giving no observable phenotype.</p

Model for PTP signaling in RhoA (in)activation and cell polarization.

<p>(a) RPTPα and PTPε are known activators of the SFKs, Fyn and Yes. Fyn and Yes either directly or indirectly activate NGEF by phosphorylation of Tyr-87 residue, increasing the specificity and activity of NGEF towards RhoA. PTP-BL and Ptpn20 likely indirectly activate Arhgap29 by either ensuring its recruitment or activation in order to inhibit RhoA activity. (b) Model for how enhanced and decreased RhoA activation may induce similar phenotypes. Assuming polarized distribution of RhoA-GTP (red dots) and RhoA-GDP (blue dots), either loss or increase of RhoA activation will result in loss of cell polarity (see text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035913#pone.0035913.s005" target="_blank">Fig. S5</a> for further details).</p

Knockdown of <i>ngef</i> or <i>arhgap29b</i> induces C/E cell movement and cell polarization defects.

<p>Zebrafish embryos were microinjected with morpholinos (high concentration) targeting <i>arhgap29b</i>, <i>arhgap5</i> or <i>ngef</i> at the one cell stage and grown to 1 somite stage. Embryos were fixed and stained for <i>dlx3</i> and <i>hgg1</i> expression using whole mount <i>in situ</i> hybridization. Posterior shift of the hatching gland and angle of <i>dlx3</i> staining are measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035913#pone-0035913-g003" target="_blank">Fig. 3</a>. (a,b). Representative embryos are shown in (c). Embryos were grown to 8–9 somite stage, fixed and stained for <i>krox20</i> and <i>myod</i>. Rhombomere width (<i>krox20</i>) and somite length (8 somites, <i>myod</i>) ratios are plotted in (d); representative embryos are depicted in (e). (f) Representative areas of presomitic mesoderm for the indicated conditions were analyzed for cell shape and the distribution of angles of the longest axis towards the dorsal midline was plotted in rose-plots (f; bottom); aspect ratio plotted in (g). All error bars are standard error of the mean. Student t-tests were performed with non-injected control; no asterisk indicates P>0.05, * indicates 0.05>P>0.001 and ** indicates P<0.001.</p

Identification of <i>ptpn20</i> as a homologue of <i>ptpn13</i>.

<p>(a) Protein structures are shown encoded by <i>ptpn20</i> homologue and the immediately 5′ upstream <i>FRMPD2</i>, as currently annotated in five fish genomes, the human genome and the mouse genome. In some cases like Fugu and Tetraodon a single known coding transcript exists besides separate transcripts encoding the PTP domain and the “FRMPD" part. For comparison the protein structure encoded by human <i>ptpn13</i> (PTPBL) is added below. (b) Primers were designed as indicated, leaving approximately 100 bp known coding sequence for the purpose of alignment of generated sequences. PCR products with forward primers on the second to last known exon of human and zebrafish <i>FRMPD2</i> and reverse oligos on the second exon of <i>PTPN20</i>. A schematic representation of retrieved sequences blasted to the genome are indicated in green (not to scale). (c) Generated PCR products on human (top) and zebrafish (bottom) cDNA libraries using the described primer sets. Generated band sizes are consistent with expected values based on homology with the <i>ptpn13</i> gene.</p

Arhgap29 and NGEF act downstream of distinct PTPs.

<p>(a) Low dose combined knockdowns of <i>ptpn13</i> or <i>ptpn20</i> and <i>arhgap29b</i> were performed by injecting indicated amounts of morpholino at the one cell stage. Tail lengths were measured at 3dpf and plotted. Co-knockdowns with <i>arhgap5</i> were included as a control. (b) Similar co-knockdowns as in (a) but with <i>ptpra</i> and <i>ptpre</i> knockdown instead of <i>ptpn13</i> and <i>ptpn20</i> knockdown. (c) Zebrafish embryos were micro-injected with morpholinos targeting the different phosphatases in low concentrations together with low dose <i>arhgef27</i> (<i>ngef</i>) morpholino. Embryos were grown to 3 dpf and tail lengths were determined and plotted as a percentage of non-injected control. All error bars are standard error of the mean. Student t-test was performed where indicated; no asterisk indicates P>0.05, * indicates 0.05>P>0.001 and ** indicates P<0.001.</p

Cullin box domain promotes induction of <i>hes1</i> gene <i>in vitro</i>.

<p>nTera-d1 cells were co-transfected with <i>hes1-luciferase</i> (<i>hes1</i>) or <i>hes1-luciferase</i> lacking the conserved CSL-binding site (<i>hes1-RBPdel</i>) and <i>myc-tag</i> (MT) as a control, or myc-tagged <i>d-asb11</i> full length (MT-Asb11) or myc-tagged <i>asb11^cul</i> (MT-Asb11^cul) cDNA. Hes1-dependent Notch activity was analyzed by luciferase measurement.</p

<i>her4::gfp</i> transactivation and premature differentiation of neural cells in <i>asb11^cul</i>.

<p>(<b>A</b>), the <i>her4::gfp</i> reporter was co-injected with <i>myc-tag</i> (MT) mRNA as a control, myc-tagged <i>d-asb11</i> full length (MT-Asb11) or myc-tagged <i>asb11^cul</i> (MT-Asb11^cul) mRNA in zebrafish embryos. Injected embryos were treated with (+) (n = 25) or without (−) (n = 25) DAPT, from 1.5 hpf. At 14 hpf, embryos were analyzed for <i>her4</i> transactivation based on the intensity of the GFP signal. Positive embryos were counted and percentages of embryos presenting weak (blue), medium (green) or strong (red) signal were given. (<b>B</b>), Wild type (<i>left panel</i>) and mutant (<i>middle panel</i>) embryos at 12 hpf were analyzed for WISH using probe against <i>ngn1</i>. (<i>right</i>) Graph quantifies expression of <i>ngn1</i> using qPCR. (<b>C</b>) Wild type (<i>left panel</i>) and mutant (<i>right panel</i>) polster of embryos at 16 hpf were analyzed for WISH using probe against <i>islet1</i>.</p

Cullin box is essential for DeltaA degradation and for maintaining a cell proliferating state <i>in vivo</i>.

<p>(<b>A</b>) Zebrafish embryos were injected with Myc-tagged <i>deltaA</i> (MT-DeltaA) and <i>d-asb11</i> (Asb11) or <i>asb11^cul</i> (Asb11^cul) mRNA at one-cell stage. (<i>lower panel)</i> Lysates of 12 hpf embryos were analyzed by immunoblotting for the presence of DeltaA. (higher panel) Graph quantifies 2 individual experiments, each with 30 injected embryos/group. (<b>B</b>), Fluorescent whole-mount antibody labeling of wild type (WT) and <i>asb11^cul</i> embryos at 24 hpf for the mitotic marker anti-phosphohistone-3 (PH 3) antibody (<i>green</i>) and the neuronal marker Hu(C). Graph shows the number of positive cells per area (5 somites from beginning of yolk extension) of 5 embryos for each genotype.</p

Phenotypic assays on wild type and <i>asb11^cul</i> embryos.

<p>(<b>A</b>), Morphological analysis of wild type and mutant embryos at 48 and 72hpf. (<b>B</b>), (<i>left</i>) Anterior view of wild type and mutant embryos at 10hpf after whole mount <i>in situ</i> hybridization, WISH, using probe against <i>d-asb11</i>. (<i>right</i>) Graph shows the quantification of the respective expressions using qPCR. (<b>C</b>), (<i>left</i>) Endogenous d-Asb11 in wild type (WT), heterozygous (asb11+/−) and mutant (<i>asb11^cul</i>) embryos at 12 hpf was detected by immunoblotting using anti-d-Asb11 antibody. (<i>right</i>) Graph quantifies 3 individual experiments, with 30 embryos/genotype/experiment.</p