Expression of zebrafish ptpn11a and ptpn11b mRNA during embryogenesis.

(a) RNA was isolated from zebrafish embryos at 4 hpf, 10 hpf, 1 dpf, 2 dpf, 3 dpf and 5 dpf. Reverse transcription PCR was performed to detect the indicated genes; reverse transcriptase was omitted from the reaction as a control (-RT). (b) Quantitative PCR for ptpn11a and ptpn11b gene expression was performed from a pool of wild-type embryos (n = 30) collected at 10 hpf, 1 dpf, 2 dpf, 3 dpf and 5 dpf. The values were normalized using Actin as a control and relative expression is depicted here (value at 5 dpf set to 1.0). Error bars indicate standard error of the mean. (c) Whole-mount in situ hybridization using ptpn11a and ptpn11b specific probes of 4 hpf, 10 hpf, 30 hpf, 2 dpf, 3 dpf and 5 dpf embryos. Lateral and dorsal views are given as indicated.

Loss of Shp2 causes severe morphological defects at 5 dpf.

Morphology of 5(a) Representative wild-type, (b) ptpn11a-/-, (c) ptpn11b-/- and (d) double homozygous mutant are depicted. (e) The length was measured from nose to tip of tail at the same magnification. Bars show average length of the studied genotypes (n = 10-20). Statistics were determined using a student′s t-test; * indicates a p value <0.05.

Ptpn11a-/- and double mutant embryos exhibit craniofacial defects at 5 dpf.

Alcian blue staining was done to visualize the cartilage. (a,e) wild-type, (b,f) ptpn11a-/-, (c,g) ptpn11b-/- and (d,h) double homozygous mutant. (i) Average width of the ceratohyal angle (indicated in panel a) was determined for each genotype (n = 10–24). Statistics were determined using a student′s t-test; ** indicates a p value of <0.01, *** indicates a p value <0.001.

Rescue of mutant phenotype by exogenous ptpn11a and ptpn11b.

Morphology of 5(a) ptpn11a-/-ptpn11b-/- double mutant, (b) double mutant expressing exogenous ptpn11a, (c) double mutant expressing exogenous ptpn11b, (d) ptpn11a-/- mutant, (e) ptpn11a-/- mutant expressing exogenous ptpn11b. Representative embryos are depicted here. See Table 2 for quantification.

Impaired Erk/MAPK, but not Akt/PKB signalling in Shp2 double mutant embryos.

WT, ptpn11a-/-, ptpn11b-/- and ptpn11a-/- ptpn11b-/- embryos were lyzed and immunoblotted with phosho-specific antibody for ERK and AKT; membranes were then reprobed for total Erk and Akt expression to control for loading. The blots were quantified and the ratio of phosphorylated protein/total protein from three independent experiments is indicated below each lane.