Shp2 Knockdown and Noonan/LEOPARD Mutant Shp2–Induced Gastrulation Defects

Abstract

Shp2 is a cytoplasmic protein-tyrosine phosphatase that is essential for normal development. Activating and inactivating mutations have been identified in humans to cause the related Noonan and LEOPARD syndromes, respectively. The cell biological cause of these syndromes remains to be determined. We have used the zebrafish to assess the role of Shp2 in early development. Here, we report that morpholino-mediated knockdown of Shp2 in zebrafish resulted in defects during gastrulation. Cell tracing experiments demonstrated that Shp2 knockdown induced defects in convergence and extension cell movements. In situ hybridization using a panel of markers indicated that cell fate was not affected by Shp2 knock down. The Shp2 knockdown–induced defects were rescued by active Fyn and Yes and by active RhoA. We generated mutants of Shp2 with mutations that were identified in human patients with Noonan or LEOPARD Syndrome and established that Noonan Shp2 was activated and LEOPARD Shp2 lacked catalytic protein-tyrosine phosphatase activity. Expression of Noonan or LEOPARD mutant Shp2 in zebrafish embryos induced convergence and extension cell movement defects without affecting cell fate. Moreover, these embryos displayed craniofacial and cardiac defects, reminiscent of human symptoms. Noonan and LEOPARD mutant Shp2s were not additive nor synergistic, consistent with the mutant Shp2s having activating and inactivating roles in the same signaling pathway. Our results demonstrate that Shp2 is required for normal convergence and extension cell movements during gastrulation and that Src family kinases and RhoA were downstream of Shp2. Expression of Noonan or LEOPARD Shp2 phenocopied the craniofacial and cardiac defects of human patients. The finding that defective Shp2 signaling induced cell movement defects as early as gastrulation may have implications for the monitoring and diagnosis of Noonan and LEOPARD syndrome.

Author Summary

Shp2 is a protein-tyrosine phosphatase and mutations in Shp2 cause the related Noonan and LEOPARD syndromes in humans. We used the zebrafish to investigate the cell biological role of Shp2 in early development. Shp2 knockdown and expression of mutant Shp2 that contained mutations corresponding to those found in human Noonan and LEOPARD patients, induced similar convergence and extension cell movement defects during gastrulation without affecting cell specification. Active Src family kinases and active RhoA rescued the Shp2 knockdown, indicating that signaling downstream of Shp2 was mediated by Src family kinases and RhoA. Expression of the Noonan and LEOPARD Shp2s in zebrafish induced craniofacial and cardiac defects that were reminiscent of the symptoms observed in human patients. Coinjections demonstrated that Noonan and LEOPARD Shp2s did not cooperate, which is consistent with the two mutants acting in the same signaling pathway with opposing effects. The finding that defective Shp2 signaling induced cell movement defects as early as gastrulation may have important implications for the monitoring and diagnosis of Noonan and LEOPARD syndromes in humans.