Zebrafish as a model to study PTPs during development

Protein-tyrosine phosphatases (PTPs) have important roles in signaling, but relatively little is known about their function in vivo. We are using the zebrafish as a model to study the function of PTPs at the organismal, cellular and molecular level. The zebrafish is an excellent experimental model for the analysis of gene function. We have developed methods to quantitatively study effects of PTP knockdown or expression of (mutant) PTPs, particularly with respect to gastrulation cell movements. Moreover, we have studied the phosphoproteome of zebrafish embryos. In this review, we will discuss methods to manipulate the zebrafish genome and techniques that we have developed to assess developmental defects during gastrulation and to assess differences in the phosphoproteome

Noonan and LEOPARD syndrome Shp2 variants induce heart displacement defects in zebrafish

Germline mutations in PTPN11, encoding Shp2, cause Noonan syndrome (NS) and LEOPARD syndrome (LS), two developmental disorders that are characterized by multiple overlapping symptoms. Interestingly, Shp2 catalytic activity is enhanced by NS mutations and reduced by LS mutations. Defective cardiac development is a prominent symptom of both NS and LS, but how the Shp2 variants affect cardiac development is unclear. Here, we have expressed the most common NS and LS Shp2-variants in zebrafish embryos to investigate their role in cardiac development in vivo. Heart function was impaired in embryos expressing NS and LS variants of Shp2. The cardiac anomalies first occurred during elongation of the heart tube and consisted of reduced cardiomyocyte migration, coupled with impaired leftward heart displacement. Expression of specific laterality markers was randomized in embryos expressing NS and LS variants of Shp2. Ciliogenesis and cilia function in Kupffer's vesicle was impaired, likely accounting for the left/right asymmetry defects. Mitogen-activated protein kinase (MAPK) signaling was activated to a similar extent in embryos expressing NS and LS Shp2 variants. Interestingly, inhibition of MAPK signaling prior to gastrulation rescued cilia length and heart laterality defects. These results suggest that NS and LS Shp2 variant-mediated hyperactivation of MAPK signaling leads to impaired cilia function in Kupffer's vesicle, causing left-right asymmetry defects and defective early cardiac development

Arthritis bei Immundefekt

Noonan syndrome (NS) and LEOPARD syndrome (LS) cause congenital afflictions such as short stature, hypertelorism and heart defects. More than 50% of NS and almost all of LS cases are caused by activating and inactivating mutations of the phosphatase Shp2, respectively. How these biochemically opposing mutations lead to similar clinical outcomes is not clear. Using zebrafish models of NS and LS and mass spectrometry-based phosphotyrosine proteomics, we identified a down-regulated peptide of Fer kinase in both NS and LS. Further investigation showed a role for Fer during development, where morpholino-based knockdown caused craniofacial defects, heart edema and short stature. During gastrulation, loss of Fer caused convergence and extension defects without affecting cell fate. Moreover, Fer knockdown cooperated with NS and LS, but not wild type Shp2 to induce developmental defects, suggesting a role for Fer in the pathogenesis of both NS and LS

Distinct and overlapping functions of ptpn11 genes in Zebrafish development

The PTPN11 (protein-tyrosine phosphatase, non-receptor type 11) gene encodes SHP2, a cytoplasmic PTP that is essential for vertebrate development. Mutations in PTPN11 are associated with Noonan and LEOPARD syndrome. Human patients with these autosomal dominant disorders display various symptoms, including short stature, craniofacial defects and heart abnormalities. We have used the zebrafish as a model to investigate the role of Shp2 in embryonic development. The zebrafish genome encodes two ptpn11 genes, ptpn11a and ptpn11b. Here, we report that ptpn11a is expressed constitutively and ptpn11b expression is strongly upregulated during development. In addition, the products of both ptpn11 genes, Shp2a and Shp2b, are functional. Target-selected inactivation of ptpn11a and ptpn11b revealed that double homozygous mutants are embryonic lethal at 5-6 days post fertilization (dpf). Ptpn11a-/-ptpn11b-/- embryos showed pleiotropic defects from 4 dpf onwards, including reduced body axis extension and craniofacial defects, which was accompanied by low levels of phosphorylated Erk at 5 dpf. Interestingly, defects in homozygous ptpn11a-/- mutants overlapped with defects in the double mutants albeit they were milder, whereas ptpn11b-/- single mutants did not show detectable developmental defects and were viable and fertile. Ptpn11a-/-ptpn11b-/- mutants were rescued by expression of exogenous ptpn11a and ptpn11b alike, indicating functional redundance of Shp2a and Shp2b. The ptpn11 mutants provide a good basis for further unravelling of the function of Shp2 in vertebrate development

Comparative mass-spectrometry of pTyr immunoprecipitated zebrafish lysates.

<p>Zebrafish embryos were injected at the 1-cell stage with synthetic mRNA constructs encoding WT Shp2, NS (D61G) Shp2 or LS (A462T) Shp2 and co-injected with mRNA encoding eGFP. Lysates were subjected to mass spectrometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106682#s2" target="_blank">Materials and Methods</a>. Normalized ratios (Log2 scale) based on total levels of non-phosphorylated peptides are given.</p><p>*Protein name based on BLAST sequence of peptide. Accession numbers from BLAST hits are used for non-annotated peptides.</p><p>**pRS score <75, phosphorylation site could not be determined but the most commonly identified site from Phsophosite.org is used. a-n: indicators for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106682#pone-0106682-g001" target="_blank">Figure 1C</a>.</p><p>Comparative mass-spectrometry of pTyr immunoprecipitated zebrafish lysates.</p

Partial knockdown of Fer cooperated with suboptimal expression of NS- and LS- but not WT Shp2 to induce developmental defects.

<p>A. Embryos were injected with control MO, Fer e9i9 MO and NS, LS or WT Shp2 mRNA as indicated, and imaged at 4 dpf. B. Alcian blue staining showing craniofacial defects scored on severity (green: wild type, yellow: mild phenotype, orange: moderate phenotype, red: severe phenotype). C. Embryos were scored based on craniofacial defect upon alcian blue staining. The number of embryos per condition is indicated. D. The angle of the ceratohyal was quantified as a measure of craniofacial defects (* p<0.005, Student's t-test).</p

Fer expression in zebrafish embryos and Fer MO induced splicing defects.

<p>A. Alignment of zebrafish Fer kinase sequence encompassing the autophosphorylation site (black) that was identified by phosphoproteomics with the mammals <i>M. musculus</i>, <i>R. norvegicus</i>, <i>H. sapiens</i>, the avian <i>G. gallus</i>, the amphibian <i>Xenopus tropicalis</i>, and the fish <i>Tetraodon nigrovirides, Takifugu rupripes</i> and <i>Oryzias latipes</i>. B. <i>fer</i> expression during the first 24 hpf and at 3 dpf and 4 dpf was observed using <i>in situ</i> hybridization with an antisense <i>fer</i> probe. C. ISH with negative control sense <i>fer</i> probe. D. RT-PCR showing altered splicing of <i>fer</i> in MO injected embryos. Da. Model of altered splicing by Fer MO i5e6. Exons 5, 6 and 7 are indicated in red, blue and green, respectively. Primers used for RT-PCR are indicated as arrows. MO is indicated in red. Due to splice blocking, intron 5 is not spliced out of the processed mRNA. Db. RT-PCR showing <i>gapdh</i> control in Nacre (control) MO and Fer MO injected embryos. RT-PCR showing <i>Fer</i> product in Nacre control MO and Fer i5e6 MO injected embryos, and genomic DNA as a positive control. Dc. Sanger sequencing showing the inclusion of intron 5 and the normal splicing of exon 6 in the RT-PCR product. Dd. Model of altered splicing by Fer MO e9i9. Exons 8, 9 and 10 are indicated in red, blue and green, respectively. Primers used for RT-PCR are indicated as arrows. MO is indicated in red. Due to defective splicing, exon 9 is spliced out of the processed mRNA. De. RT-PCR showing <i>gapdh</i> control in Nacre (control) MO and Fer e9i9 MO injected embryos. RT-PCR showing <i>Fer</i> product in Nacre control MO and Fer e9i9 MO injected embryos with a decrease in size of the product in e9i9 MO injected embryos. Df. Sanger sequencing showing the exclusion of exon 9.</p

Comparative pTyr mass spectrometry on 1 day old zebrafish embryos expressing wild type, NS (D61G) or LS (A462T) Shp2.

<p>A. Work flow depicting the mass spectrometry approach. Approximately 2000 zebrafish embryos per condition were injected at the 1-cell stage and sorted for GFP expression. Embryos were lysed, trypsinised and labeled using the dimethyl labeling method. WT, NS and LS samples were combined and immunoprecipitated using pTyr specific antibodies. Immunoprecipitate was subjected to MS and peptides were identified and quantified based on MS2 and MS1 spectra, respectively. B. 1 dpf zebrafish embryos expressing WT, D61G and A462T Shp2. Body axis length, craniofacial defects and heart edema in D61G and A462T Shp2 expressing zebrafish are indicated with arrowheads. C. Normalized plot of quantified phosphopeptides Log2 ratios. Peptide ratios with Log2 ratios >−1 and <1 are indicated in black. Peptides with more that 1× Log2 difference are annotated with a-n (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106682#pone-0106682-t001" target="_blank">Table 1</a> for reference). Peptides changed with Log2 ratios <−1 in either NS or LS are indicated in green and Log2 ratios >1 in either NS or LS are indicated in red. See text for further details.</p

Fer knockdown induced craniofacial defects in zebrafish embryos.

<p>A. Embryos were injected at the 1-cell stage with 1 ng Fer i5e6 MO, 2.5 ng e9i9 MO or 2.5 ng Nacre MO as a negative control. Additionally, embryos were injected with 150 ng WT Shp2 RNA, 150 ng NS Shp2 RNA or 50 ng LS Shp2 RNA. B. Embryos were injected at the 1-cell stage with 2.5 ng Nacre control MO, 1.0 ng Fer i5e6 MO, 2.5 ng Fer e9i9 MO or in combination with 2.5 ng P53 MO. C. Craniofacial structures were imaged using <i>Tg(-4.9sox10:EGFP)^ba2</i> embryos expressing eGFP in neural crest cells that also form the cartilage. Embryos were injected with Fer MO e9i9 at the 1 cell stage and imaged at 4dpf. Ceratohyal is indicated with a red asterisk and Meckel's cartilage with a white asterisk. Both moderate and severe phenotypes are depicted together with non-injected controls (NIC). D. Embryos were injected at the 1-cell stage with suboptimal concentrations of MO (0.5 ng i5e6 MO; <i>n</i> = 110 and 1.0 ng e9i9 MO; <i>n</i> = 99). High levels of both MO's (1.0 ng i5e6 MO; <i>n</i> = 122 and 2.5 e9i9 MO; <i>n</i> = 135) or low levels of both MO's were co-injected (<i>n</i> = 115). Embryos were imaged at 3 dpf and 4 dpf and grouped by having a WT appearance (green), a craniofacial defect alone (yellow) a heart defect alone (orange) or both (red) at 4 dpf. Relative levels of phenotypes are depicted. E. Embryos were injected at the 1-cell stage with normal dose of MO (1.0 ng i5e6 MO; <i>n</i> = 61 and 2.5 e9i9 MO; <i>n</i> = 53), low doses of MO (0.5 ng i5e6 MO; <i>n</i> = 57 and 1.0 ng e9i9 MO; <i>n</i> = 50) or a co-injected with low doses of MO (<i>n</i> = 70). Morphology at 4 dpf is depicted. Embryos were fixed and stained with Alcian blue at 4 dpf and imaged laterally and ventrally. For quantification, the angle of the ceratohyal (F) and the ratio of the distance from the back of the head to Meckel's cartilage and the width of the head was determined (G) (* indicates significance, Student's t-test p<0.005).</p

Fer knockdown resulted in C&E defects but not in changes in cell fate.

<p>A. <i>Krox20/myoD in situ</i> hybridization as a method to quantify C&E defects. <i>Krox20</i> (red) staining for rhombomeres 3 and 5 was used to measure the width, which correlates with convergence cell movements in the embryo. <i>MyoD</i> (light blue) staining for the somites was used to measure the length, <i>i.e.</i> extension of the embryo. The ratio of <i>Krox20/myoD</i> correlates directly with convergence & extension cell movements during gastrulation. B. Flatmounts of <i>krox20/myoD</i> stained NIC (<i>n</i> = 70), control MO (<i>n</i> = 44) and Fer MO (<i>n</i> = 40) embryos. C. The ratio of the width of a krox20-positive rhombomere and the length of 8 somites was determined (* indicates significance, Student's t-test p<0.005). D–X. Embryos were injected at the 1-cell stage with control MO or Fer e9i9 MO and subjected to ISH for various markers of cell fate determination. Note that the staining of the <i>gsc, pax2, six3</i> and <i>cyc</i> probes show broader and shorter expression in Fer knockdown embryos than in controls.</p