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.

Protein tyrosine phosphatase activity in the neural crest is essential for normal heart and skull development

“…Morpholinomediated SHP2 knockdown resulted in early defects in gastrulation, precluding detailed studies of organogenesis; nonetheless, it was apparent that loss of function led to defective cellular movement. Similarly, expression of either LS or NS mutants also resulted in defects in cell convergence and extension, and the embryos also displayed both craniofacial and cardiovascular defects (39). Our data confirm that SHP2 levels are functionally important in the NCCs, modulating ERK1/2 activity and affecting development of the OFT and frontal skull structures.…”

“…Recently, both activating and inactivating SHP2 mutations have been studied in zebrafish (39). Morpholinomediated SHP2 knockdown resulted in early defects in gastrulation, precluding detailed studies of organogenesis; nonetheless, it was apparent that loss of function led to defective cellular movement.…”

Inhibition of neural crest migration underlies craniofacial dysmorphology and Hirschsprung's disease in Bardet–Biedl syndrome

“…The zebrafish has become a valid model for the study of CF development (14). Similar to previously reported zebrafish models of human genetic diseases, observed phenotypes are often more pronounced likely because of the rapid development and associated sensitivity to genetic perturbation (23,24).…”

Involvement of EphA2-mediated tyrosine phosphorylation of Shp2 in Shp2-regulated activation of extracellular signal-regulated kinase

“…21,22 By using the zebrafish model of LEOPARD syndrome, we first tested the significance of phosphorylation of Tyr542 and Tyr580 of Shp2 and second examined the involvement of EphA2 in the abnormal phenotype. The percentage of cardiac defects of the embryos injected with Shp2T468M/2YF mRNAs was less than those injected with Shp2T468M mRNAs, but more than those injected with wildtype Shp2 mRNAs and those uninjected (Figures 7a and b).…”