The neurofibromin GAP-related domain rescues endothelial but not neural crest development in Nf1–/– mice
Fraz A. Ismat, Junwang Xu, Min Min Lu, Jonathan A. Epstein
Fraz A. Ismat, Junwang Xu, Min Min Lu, Jonathan A. Epstein
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Research Article Cardiology

The neurofibromin GAP-related domain rescues endothelial but not neural crest development in Nf1–/– mice

Abstract

Neurofibromatosis type I (NF1; also known as von Recklinghausen’s disease) is a common autosomal-dominant 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–GTPase-activating 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 tissue-specific 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.

Authors

Fraz A. Ismat, Junwang Xu, Min Min Lu, Jonathan A. Epstein

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Figure 4

HA-GRD does not rescue abnormal neural crest–derived tissues lacking Nf1.

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HA-GRD does not rescue abnormal neural crest–derived ...
(A–C) Adrenal medullary enlargement in HA-GRD rescued newborns. Sagittal sections of adrenal glands (ad); Nf1–/–, HA-GRDk/+, CMV-cre+ (B) and the Nf1 neural crest–specific knockout, Nf1flox/–, Pax3-cre+ (C) had overgrown adrenal medullae compared with wild-type (A) with cortical effacement and tumor-like medullary protrusion (arrowheads). k, kidney. (D–I) Enlarged peripheral ganglia in HA-GRD rescued newborns. Sagittal sections of Nf1+/– (D); Nf1–/–, HA-GRDk/+, CMV-cre+ (E, G–I); and Nf1flox/–, Pax3-cre+ (F). at, cardiac atrium. Peripheral ganglia (arrows) were massively enlarged in Nf1–/–, HA-GRDk/+, CMV-cre+ newborns (E and G) compared with an Nf1+/– (D) littermate and similar the Nf1 neural crest–specific knockout (F). (H) Positive staining for neurofilament, with connections (arrowheads) to dorsal root ganglia (drg). (I) Negative staining for the glial marker S-100. (J–L) HA-GRD is expressed with CMV-cre. In situ hybridization of cross-sections of Nf1–/– E12.5 embryos with CMV-cre transgene (J), HA-GRD knock-in (K), or both (L) show expression of HA-GRD only with both. Dorsal root ganglia (arrowheads) had notably robust expression. (M–O) HA-GRD dose did not alter abnormalities of neural crest–derived tissue in Nf1–/– embryos. Cross-sections of wild-type (M); Nf1–/–, HA-GRDk/–, CMV-cre+ (N); and Nf1–/–, HA-GRDk/k, CMV-cre+ (O) E12.5 embryos highlighting sympathetic ganglia (red arrows), enlarged in both Nf1–/– genotypes and no smaller with 2 copies of HA-GRD (O) than with 1 copy (N). Magnification, ×4 (A–I); ×10 (J–O).