Pair-Wise Regulation of Convergence and Extension Cell Movements by Four Phosphatases via RhoA

Various signaling pathways regulate shaping of the main body axis during early vertebrate development. Here, we focused on the role of protein-tyrosine phosphatase signaling in convergence and extension cell movements. We identified Ptpn20 as a structural paralogue of PTP-BL and both phosphatases were required for normal gastrulation cell movements. Interestingly, knockdowns of PTP-BL and Ptpn20 evoked similar developmental defects as knockdown of RPTPα and PTPε. Co-knockdown of RPTPα and PTP-BL, but not Ptpn20, had synergistic effects and conversely, PTPε and Ptpn20, but not PTP-BL, cooperated, demonstrating the specificity of our approach. RPTPα and PTPε knockdowns were rescued by constitutively active RhoA, whereas PTP-BL and Ptpn20 knockdowns were rescued by dominant negative RhoA. Consistently, RPTPα and PTP-BL had opposite effects on RhoA activation, both in a PTP-dependent manner. Downstream of the PTPs, we identified NGEF and Arhgap29, regulating RhoA activation and inactivation, respectively, in convergence and extension cell movements. We propose a model in which two phosphatases activate RhoA and two phosphatases inhibit RhoA, resulting in proper cell polarization and normal convergence and extension cell movements

Somitic contribution to the zebrafish pectoral fin

The pectoral fin muscle is generally understood to be derived from the adjacent somites. The detailed migratory processes that lead to the population of somite derived muscle progenitors within the pectoral fin are not well understood. Here I provide a detailed analysis of pectoral fin muscle progenitors from the somitic compartment into the zebrafish pectoral fin. I find somites 4-6 contribute to the pectoral fin and somite 7 contributes to the Posterior Hypaxial Muscle, both likely through the mechanism of 2D sheet migration. This detailed analysis of muscle progenitors revealed a somitically derived group of cells present within the Apical Ectodermal Ridge. The somitic origin of these cells has been validated using a range of lineage tracing methods, including Cre/Lox, photo-conversions, time-lapse analysis and somite transplantations. Both the timing of this event shortly before the Apical Ectodermal Ridge to Apical Fold transition, and the specific location of these mesoderm derived cells within the Apical Fold, are highly suggestive that these cells are actinotrichia forming cells. As such they can indeed play a crucial role in fin fold formation. Considering the loss of actinotrichia has been considered to be a crucial event in fin the limb transition, these results provide new insight in how fin to limb transition may have occurred

Somitic contribution to the zebrafish pectoral fin

The pectoral fin muscle is generally understood to be derived from the adjacent somites. The detailed migratory processes that lead to the population of somite derived muscle progenitors within the pectoral fin are not well understood. Here I provide a detailed analysis of pectoral fin muscle progenitors from the somitic compartment into the zebrafish pectoral fin. I find somites 4-6 contribute to the pectoral fin and somite 7 contributes to the Posterior Hypaxial Muscle, both likely through the mechanism of 2D sheet migration. This detailed analysis of muscle progenitors revealed a somitically derived group of cells present within the Apical Ectodermal Ridge. The somitic origin of these cells has been validated using a range of lineage tracing methods, including Cre/Lox, photo-conversions, time-lapse analysis and somite transplantations. Both the timing of this event shortly before the Apical Ectodermal Ridge to Apical Fold transition, and the specific location of these mesoderm derived cells within the Apical Fold, are highly suggestive that these cells are actinotrichia forming cells. As such they can indeed play a crucial role in fin fold formation. Considering the loss of actinotrichia has been considered to be a crucial event in fin the limb transition, these results provide new insight in how fin to limb transition may have occurred

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

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

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