Noonan syndrome is a common human autosomal dominant birth defect, characterized by short stature, facial abnormalities, heart defects and possibly increased risk of leukemia. Mutations of Ptpn11 (also known as Shp2), which encodes the protein-tyrosine phosphatase Shp2, occur in approximately 50% of individuals with Noonan syndrome, but their molecular, cellular and developmental effects, and the relationship between Noonan syndrome and leukemia, are unclear. We generated mice expressing the Noonan syndrome-associated mutant D61G. When homozygous, the D61G mutant is embryonic lethal, whereas heterozygotes have decreased viability. Surviving Ptpn11(D61G/+) embryos ( approximately 50%) have short stature, craniofacial abnormalities similar to those in Noonan syndrome, and myeloproliferative disease. Severely affected Ptpn11(D61G/+) embryos ( approximately 50%) have multiple cardiac defects similar to those in mice lacking the Ras-GAP protein neurofibromin. Their endocardial cushions have increased Erk activation, but Erk hyperactivation is cell and pathway specific. Our results clarify the relationship between Noonan syndrome and leukemia and show that a single Ptpn11 gain-of-function mutation evokes all major features of Noonan syndrome by acting on multiple developmental lineages in a gene dosage-dependent and pathway-selective manner.

Neurofibromatosis type 1 (NF1) or von Recklinghausen neurofibromatosis is a genetic disorder that occurs in 1 of 4000 births and is characterized by benign and malignant tumors. Cardiovascular defects also contribute to NF1, though the pathogenesis is still unclear. Deficiency in neurofibromin (encoded by Nf1) in mice results in mid-embryonic lethality owing to cardiac abnormalities previously thought to be secondary to cardiac neural-crest defects. Using tissue-specific gene inactivation, we show that endothelial-specific inactivation of Nf1 recapitulates key aspects of the complete null phenotype, including multiple cardiovascular abnormalities involving the endocardial cushions and myocardium. This phenotype is associated with an elevated level of ras signaling in Nf1(-/-) endothelial cells and greater nuclear localization of the transcription factor Nfatc1. Inactivation of Nf1 in the neural crest does not cause cardiac defects but results in tumors of neural-crest origin resembling those seen in humans with NF1. These results establish a new and essential role for Nf1 in endothelial cells and confirm the requirement for neurofibromin in the neural crest.

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Background-Neurofibromatosis type I (NF1) is a common autosomal dominant disorder with a broad array of clinical manifestations, including benign and malignant tumors, osseous dysplasias, and characteristic cutaneous findings. In addition, NF1 patients have an increased incidence of cardiovascular diseases, including obstructive vascular disorders. In animal models, endothelial expression of the disease gene, NF1, is critical for normal heart development. However, the pathogeneses of the more common vascular disorders are not well characterized. Methods and Results-To examine the role of NF1 in vascular smooth muscle, we generated mice with homozygous loss of the murine homolog Nf1 in smooth muscle (Nf1smKO). These mice develop and breed normally. However, in response to vascular injury, they display a marked intimal hyperproliferation and abnormal activation of mitogenactivated protein kinase, a downstream effector of Ras. Vascular smooth muscle cells cultured from these mice also display enhanced proliferation and mitogen-activated protein kinase activity. Smooth muscle expression of the NF1 Ras-regulatory domain (GTPase activating protein-related domain) rescues intimal hyperplasia in Nf1smKO mice and normalizes vascular smooth muscle cell Ras effector activity and proliferation in vitro, similar to blockade of downstream effectors of Ras. Conclusions-In this in vivo model of NF1 obstructive vascular disease, we have shown that Nf1 regulation of Ras plays a critical role in vascular smooth muscle proliferation after injury. These results suggest opportunities for targeted therapeutics in the prevention and treatment of NF1-related vascular disease and in the treatment of neointimal proliferation in other settings.

Abstract-Hop is an unusual homeobox gene expressed in the embryonic and adult heart. Hop acts downstream of Nkx2-5 during development, and Nkx2-5 mutations are associated with cardiac conduction system (CCS) defects. Inactivation of Hop in the mouse is lethal in half of the expected null embryos. Here, we show that Hop is expressed strongly in the adult CCS. Hop Ϫ/Ϫ adult mice display conduction defects below the atrioventricular node (AVN) as determined by invasive electrophysiological testing. These defects are associated with decreased expression of connexin40. Our results suggest that Hop functions in the adult CCS and demonstrate conservation of molecular hierarchies between embryonic myocardium and the specialized conduction tissue of the mature heart. (Circ Res. 2005;96:898-903.) Key Words: Hop Ⅲ conduction Ⅲ connexin40 Ⅲ Nkx2-5 Ⅲ mouse mutants S ynchronized contraction of the atrial and ventricular chambers is essential for normal cardiac function, and the cardiac conduction system (CCS) is required for mediating this delicate interplay. However, little is known about the molecular cascades regulating CCS development and function. Several studies suggest that certain components of the avian CCS are locally derived from bipotential cardiomyogenic cells. 1 Endothelin, neuregulin-1, and Nkx2-5 have each been implicated as contributors to the transition between ventricular and conductive cell lineages in animals. 2-5 Additionally, patients with mutations in Nkx2-5 exhibit congenital heart defects and conduction system disease, notable for progressive atrioventricular block. 6 Complete loss of Nkx2-5 leads to loss of the atrioventricular node (AVN) anlage, 5 and a mouse model in which Nxk2-5 has been selectively removed from the ventricular myocardium demonstrates hypoplasia and progressive loss of cells from the AVN along with atrioventricular block. 4 We and others recently identified Hop, a novel homeodomain protein in the embryonic heart functioning downstream of 8 Hop encodes a 73 amino acid protein that contains a domain (the 60 amino acid homeodomain) homologous to those seen in homeobox (Hox) transcription factors. Unlike all other known Hox transcription factors, Hop does not directly bind DNA. 9 Hop is known to interact with serum response factor (SRF) and interfere with SRF-mediated gene transcription. 7,8 Mice lacking Hop (Hop Ϫ/Ϫ ) are born at Ϸ50% of the expected frequency, with loss of embryos between E10.5 and E12.5. 7 Hop expression also continues into the postnatal period and transgenic overexpression in adult myocardium leads to cardiac hypertrophy and heart failure. 9 In this study, we present evidence that Hop is highly expressed in the adult mouse CCS and is required for proper function during adulthood.

Neurofibromatosis type I (NF1; also known as von Recklinghausen's disease) is a common autosomaldominant condition primarily affecting neural crest-derived tissues. The disease gene, NF1, encodes neurofibromin, a protein of over 2,800 amino acids that contains a 216-amino acid domain with Ras-GTPaseactivating protein (Ras-GAP) activity. Potential therapies for NF1 currently in development and being tested in clinical trials are designed to modify NF1 Ras-GAP activity or target downstream effectors of Ras signaling. Mice lacking the murine homolog (Nf1) have mid-gestation lethal cardiovascular defects due to a requirement for neurofibromin in embryonic endothelium. We sought to determine whether the GAP activity of neurofibromin is sufficient to rescue complete loss of function or whether other as yet unidentified functions of neurofibromin might also exist. Using cre-inducible ubiquitous and tissuespecific expression, we demonstrate that the isolated GAP-related domain (GRD) rescued cardiovascular development in Nf1 -/-embryos, but overgrowth of neural crest-derived tissues persisted, leading to perinatal lethality. These results suggest that neurofibromin may possess activities outside of the GRD that modulate neural crest homeostasis and that therapeutic approaches solely aimed at targeting Ras activity may not be sufficient to treat tumors of neural crest origin in NF1.

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