SUMMARYNoonan syndrome is a relatively common developmental disorder that is characterized by reduced growth, wide-set eyes and congenital heart defects. Noonan syndrome is associated with dysregulation of the Ras–mitogen-activated-protein-kinase (MAPK) signaling pathway. Recently, two mutations in NRAS were reported to be associated with Noonan syndrome, T50I and G60E. Here, we report a mutation in NRAS, resulting in an I24N amino acid substitution, that we identified in an individual bearing typical Noonan syndrome features. The I24N mutation activates N-Ras, resulting in enhanced downstream signaling. Expression of N-Ras-I24N, N-Ras-G60E or the strongly activating mutant N-Ras-G12V, which we included as a positive control, results in developmental defects in zebrafish embryos, demonstrating that these activating N-Ras mutants are sufficient to induce developmental disorders. The defects in zebrafish embryos are reminiscent of symptoms in individuals with Noonan syndrome and phenocopy the defects that other Noonan-syndrome-associated genes induce in zebrafish embryos. MEK inhibition completely rescued the activated N-Ras-induced phenotypes, demonstrating that these defects are mediated exclusively by Ras-MAPK signaling. In conclusion, mutations in NRAS from individuals with Noonan syndrome activated N-Ras signaling and induced developmental defects in zebrafish embryos, indicating that activating mutations in NRAS cause Noonan syndrome.
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.
Convergence and extension (C&E) cell movements are essential to shape the body axis during vertebrate gastrulation. We have used the zebrafish to assess the role of the receptor protein-tyrosine phosphatases, RPTPalpha and PTPepsilon, in gastrulation cell movements. Both RPTPalpha and PTPepsilon knockdown and ptpra(-/-) embryos show defects in C&E movements. A method was developed to track gastrulation cell movements using confocal microscopy in a quantitative manner and ptpra(-/-) embryos displayed reduced convergence as well as extension speeds. RPTPalpha and PTPepsilon knockdowns cooperated with knockdown of a well known factor in C&E cell movement, non-canonical Wnt11. RPTPalpha and PTPepsilon dephosphorylate and activate Src family kinases in various cell types in vitro and in vivo. We found that Src family kinase phosphorylation was enhanced in ptpra(-/-) embryos, consistent with reduced Src family kinase activity. Importantly, both ptpra(-/-) and RPTPalpha and PTPepsilon knockdown induced C&E defects were rescued by active Fyn and Yes. Moreover, active RhoA rescued the RPTPalpha and PTPepsilon knockdown and ptpra(-/-) induced gastrulation cell movement defects as well. Our results demonstrate that RPTPalpha and PTPepsilon are essential for C&E movements in a signaling pathway parallel to non-canonical Wnts and upstream of Fyn, Yes and RhoA.
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