Loss of neurofibromin Ras-GAP activity enhances the formation of cardiac blood islands in murine embryos.

Type I neurofibromatosis (NF1) is caused by mutations in the NF1 gene encoding neurofibromin. Neurofibromin exhibits Ras GTPase activating protein (Ras-GAP) activity that is thought to mediate cellular functions relevant to disease phenotypes. Loss of murine Nf1 results in embryonic lethality due to heart defects, while mice with monoallelic loss of function mutations or with tissue-specific inactivation have been used to model NF1. Here, we characterize previously unappreciated phenotypes in Nf1-/- embryos, which are inhibition of hemogenic endothelial specification in the dorsal aorta, enhanced yolk sac hematopoiesis, and exuberant cardiac blood island formation. We show that a missense mutation engineered into the active site of the Ras-GAP domain is sufficient to reproduce ectopic blood island formation, cardiac defects, and overgrowth of neural crest-derived structures seen in Nf1-/-embryos. These findings demonstrate a role for Ras-GAP activity in suppressing the hemogenic potential of the heart and restricting growth of neural crest-derived tissues.

Introduction

NF1 is a common human disorder characterized by benign and malignant tumors of neural crest origin, pigmentation defects, learning disorders, cardiovascular abnormalities and a wide spectrum of other abnormalities including a predilection for leukemia (especially juvenile myelomonocytic leukemia, [JMML]) and vascular defects (Cichowski and Jacks, 2001; Friedman et al., 2002). Some of these phenotypes, including JMML and vascular defects, are shared by patients with related disorders associated with activation of the Ras signaling pathway, which together have been termed the 'RAS-opathies' (Rauen et al., 2010). Neurofibromin contains a protein domain termed the GAP-related domain (GRD) that is homologous to yeast IRA proteins. The NF1 GRD is able to complement yeast IRA mutants and hydrolyze GTP bound to active Ras, thereby down-regulating Ras signaling (Ballester et al., 1990; Xu et al., 1990). Interestingly, however, missense mutations in humans with NF1 have been identified that alter amino acids throughout the protein, suggesting functional domains outside of the GRD (Mattocks et al., 2004). Additional cellular functions for neurofibromin have also been identified, including modulation of protein kinase A (PKA) and cyclic adenosine monophosphate (cAMP) pathways (Brown et al., 2010; The et al., 1997; Wolman et al., 2014). A C-terminal region of neurofibromin has also been shown to interact with a major class of heparan sulfate proteoglycans (Hsueh et al., 2001) while full-length neurofibromin can bind to the scaffolding domain of caveolin-1 (Boyanapalli et al., 2006). Therapeutic strategies for the treatment of NF1 have focused on modulation of the Ras pathway, but the degree to which Ras dysregulation accounts for the diverse aspects of the human disease, or for the equally diverse features of various animal models of NF1, remains a critical question in the field.

Mouse models of NF1 have demonstrated critical developmental functions for neurofibromin in multiple tissues, including neural crest, endothelium, and hematopoietic stem and progenitor cells (HSPCs) (Brossier and Carroll, 2012; Costa and Silva, 2003; Gitler et al., 2003; Bollag et al., 1996; Zhang et al., 1998). HSPCs arise during midgestation from a transient population of endothelial cells called hemogenic endothelium (HE) located in the yolk sac, the dorsal aorta, vitelline and umbilical arteries (Bertrand et al., 2010; Boisset et al., 2010; Chen et al., 2009; Kissa and Herbomel, 2010; Lam et al., 2010; Oberlin et al., 2010; Zovein et al., 2008). HE gives rise to HSPCs through a direct transition of endothelial cells into hematopoietic cells, independent of cell division (Kissa and Herbomel, 2010; Eilken et al., 2009). This endothelial to hematopoietic transition (EHT) was thought to occur only in the major arteries of the embryo, the placenta, and the yolk sac, but recent studies have identified the heart and the head as sites of de novo hematopoiesis (Dzierzak and Speck, 2008; Nakano et al., 2013; Li et al., 2012). In the heart, hemogenic endocardial cells are integrated into the outflow cushion and atria and undergo EHT on embryonic day (E) 9.5. Unlike arterial HE cells that give rise to the full repertoire of hematopoietic cells, hemogenic endocardial cells produce a transient population of hematopoietic cells restricted to the erythroid/myeloid lineage, similar in potential to an early wave of erythroid/myeloid progenitors (EMPs) that emerge starting at E8.5 in the yolk sac (Nakano et al., 2013; Palis et al., 1999).

Later in gestation, the heart is associated with a less-defined second wave of hematopoiesis characterized by aggregates of endothelial and hematopoietic cells called blood islands. Cardiac blood island formation is a prevalent physiological process that has been identified in embryonic mice, chicks, quails and humans, but surprisingly, little is known about the formation of these structures (Hiruma and Hirakow, 1989; Hutchins et al., 1988; Kattan et al., 2004; Ratajska et al., 2006; Red-Horse et al., 2010; Wu et al., 2013; Jankowska-Steifer et al., 2015). What is known about cardiac blood islands comes primarily from histological studies. Blood islands form in the subepicardial space near the interventricular sulci between E11 and E14 and consist primarily of erythroblasts, but have also been associated with megakaryocytes, platelets, and leukocytes (Ratajska et al., 2009; Red-Horse et al., 2010). Clonal and histological analysis suggests that blood islands emerge from the endocardium, protruding into the myocardium where they pinch off, forming blood-filled spheres or tubules that join the coronary plexus (Red-Horse et al., 2010; Jankowska-Steifer et al., 2015). It has been suggested that hematopoietic cells enter cardiac blood islands by diapedesis, but other routes such as circulation or the de novo generation of hematopoietic cells from the endocardium in situ have not been ruled out (Ratajska et al., 2006). Cardiac blood island formation was found to be more robust in Tbx18 null mouse embryos, and thought to be an indirect consequence of aberrant signaling (Wu et al., 2013). Here we show that hyperactive Ras signaling increases cardiac blood island formation, and that endocardial cells of the blood islands have functional characteristics of HE and express Runx1, a marker of HE.

Results

Nf1 deficiency increases yolk sac hematopoiesis but decreases specification of hemogenic endothelium in the dorsal aorta

E 12.5–13.5 Nf1-deficient fetuses were reported to have increased numbers of committed hematopoietic progenitors in the fetal liver (Largaespada et al., 1996; Bollag et al., 1996; Zhang et al., 1998). Since many fetal liver progenitors in the midgestation embryo originate in the yolk sac (Lux et al., 2008), we examined the number of EMPs in the yolk sac of E10.5 Nf1-deficient embryos. Nf1 yolk sacs (Figure 1A) contained significantly more EMPs, specifically due to an increased number of erythroid progenitors (Figure 1B), suggesting that Ras signaling positively regulates EMP numbers. We next examined the impact of Nf1 deficiency on hematopoiesis in the major arteries. The majority of HE cells in the major arteries (dorsal aorta, vitelline and umbilical) undergo EHT between E9.0-–10.5, resulting in the formation of Kit+ CD31+ Runx1+ hematopoietic cells that remain briefly attached as clusters to the luminal wall of the arteries. In contrast to the increase in EMPs observed in the yolk sac, CD31+ Kit+ Runx1+ hematopoietic cluster cells were decreased in the dorsal aortas of E10.5 Nf1-deficient embryos (Figure 1C,D). The decrease in CD31+ Kit+ Runx1+ hematopoietic cluster cells appears to be due to decreased de novo generation, as fewer Runx1+ CD31+ Kit--/low HE cells were present in the dorsal aortas (Figure 1C,D). These data suggest that disruption of neurofibromin function augments the formation of EMPs in the yolk sac, but inhibits the specification of hemogenic endothelium in the dorsal aorta.

Figure 1.

Nf1 deficiency increases yolk sac hematopoiesis but decreases specification of hemogenic endothelium in the dorsal aorta at E10.5.

(A) Quantification of erythroid and myeloid progenitors (EMPs) in the yolk sacs of E10.5 Nf1and Nf1 conceptuses (Nf1 n = 7; Nf1n = 8; Nf1 n = 3). One-way ANOVA and Bonferroni’s multiple comparison test was applied to determine significance, error bars represent the standard deviation (SD) (B) Percent of EMP colony type. Mk: megakaryocyte; Mix: granulocyte-erythroid-monocyte-megokaryocyte; BFU-E: burst forming unit-erythroid; G/M: granulocyte-macrophage colonies. There were significantly more BFU-E progenitors in the yolk sacs of Nf1 compared to Nf1and Nf1littermates, p≤0.0001. (C) Confocal Z-projections (Z intervals = 2 μm) of Nf1 and Nf1 dorsal aortas at E10.5, immunostained for CD31 (red) Runx1 (green) and Kit (cyan). Scale bars = 100 μm. Aortas are oriented with the ventral aspect on the left. (D) Quantification of CD31+ Runx1+ Kit+ hematopoietic cluster cells and CD31+ Runx1+ Kit-/low hemogenic endothelial cells within the dorsal aorta at E10.5, One-way ANOVA and Bonferroni’s multiple comparison test applied to determine significance, error bars represent the SD and n = 3 for all genotypes. ** indicates that p≤0.01.

DOI: http://dx.doi.org/10.7554/eLife.07780.003

Nf1 deficiency results in ectopic cardiac blood island formation

Nf1 deficiency results in embryonic lethality by midgestation (approximately E13) due to cardiac defects. These defects include enlarged endocardial cushions and a malformed outflow tract (Brannan et al., 1994; Jacks et al., 1994; Lakkis and Epstein, 1998). Despite midgestation lethality, E11.5 Nf1 embryos appeared grossly normal (Figure 2A). However, blood -filled protrusions were often visible on the ventricles of Nf1 embryos (Figure 2B arrowheads). Whole-mount confocal analyses revealed that the protrusions are blood island-like structures budding from the ventricular endocardium, as they express CD31 and the hematopoietic markers CD41 and Runx1 (Figure 2C arrowheads). The blood islands are concentrated laterally on both ventricles of Nf1 embryos (Figure 2C), in contrast to wild-type embryos, in which it was reported that blood islands are generally located on the dorsal surface in the interventricular sulcus (Jankowska-Steifer et al., 2015). Cardiac blood island formation was more robust in Nf1-deficient embryos compared to Nf1and Nf1 littermates; an average of 63.7 ± 7.6 blood islands could be identified via confocal microscopy on the ventricles of E11.5 Nf1 embryos, whereas Nf1and Nf1littermates averaged 0.3 ± 0.6 and 1.2 ± 1.3 blood islands, respectively (Figure 2D). At E10.5, only 25% (1/4) of Nf1-deficient embryos displayed small budding cardiac blood islands, whereas 92% (11/12) of E11.5 embryos had robust blood island formation, indicating that cardiac blood islands arise between E10.5 and 11.5 in Nf1embryos.

Figure 2.

Ectopic formation of cardiac blood islands in Nf1 embryos.

(A) Gross view of E11.5 Nf1 and Nf1embryos (B) Isolated hearts from embryos in (A) Black arrowheads point to two examples of blood-filled protrusions. (C) Confocal Z-projections (Z interval = 5 μm) of CD31 (red), CD41 (cyan) and Runx1 (green) immunostained Nf1 and Nf1 E11.5 embryos. Blood island-like structures (arrowheads) are visible on the ventricle of the Nf1 embryo. Scale bars = 500 μm. (D) Quantification of blood islands on the ventricles of E11.5 embryos, One-way ANOVA and Bonferroni’s multiple comparison test applied to determine significance, error bars represent SD. (E) Number of erythroid and myeloid progenitors per flushed E11.5 ventricles. One-way ANOVA and Bonferroni’s multiple comparison test applied to determine significance, error bars represent the standard SD. Nf1 n = 10, Nf1 n = 33, and Nf1 n = 6. *** indicates that p≤0.001. CFU-C: colony-forming units-culture; V: ventricle; A: atrium; FL: fetal liver; Mk: megakaryocyte; Mix: granulocyte-erythroid-monocyte-megakaryocyte; BFU-E: burst forming unit-erythroid; G/M: granulocyte-macrophage colonies.

DOI: http://dx.doi.org/10.7554/eLife.07780.004

To determine if the ectopic cardiac blood islands harbored functional hematopoietic progenitors we performed colony-forming assays. To eliminate circulating blood cells, the atrium was removed and circulating blood flushed from the ventricles before the ventricles were dissociated and plated in methylcellulose supplemented with cytokines. Nf1 ventricles contained significantly more EMPs than their Nf1+/+ and Nf1littermates (Figure 2E). This suggests that the phenotypic hematopoietic cells in the blood islands are functional erythroid and myeloid progenitors.

We used CD31, Runx1, Kit and CD41 whole-mount immunofluorescence and confocal microscopy to examine the structure of cardiac blood islands in Nf1-deficient ventricles at E11.5. Single optical projections through the blood islands indicate that they are cystic structures that consist of a layer of CD31+ endocardial cells that is continuous with the endocardium lining the ventricular trabeculae (Figure 3A,C). A layer of 3–4 CD31 bright cells with morphology between a flat endocardial cell and a rounded hematopoietic cell lined the base of most blood islands (Figure 3A–D). Some of these cells express the hemogenic endothelial marker Runx1 but they do not express high levels of the early hematopoietic markers CD41 and Kit, suggesting that they are hemogenic endocardial cells that have not yet initiated the transition into hematopoietic cells (Figure 3B,D, arrows). Within the cystic structure of most blood islands, there are also rounded cells that are CD31+ Kit+ Runx1+ or CD31+ CD41+ Runx1+; these cells are phenotypic and morphological HSPCs (Figure 3B,D, arrowheads). These data suggest that blood islands are derived from the endocardium of the ventricle and that the endocardium of blood islands has a latent HE potential that is held in check by Ras-GAP activity.

Figure 3.

Nf1 cardiac blood islands.

(A) Single optical projection through the ventricle of an E11.5 Nf1 embryo immunostained for CD31 (red), CD41 (cyan) and Runx1 (green). Blood islands (arrowheads) are visible sprouting from the ventricles of Nf1 embryos. (B) Single optical projection through blood islands on the ventricles of E11.5 Nf1 embryos. Runx1+ endocardial cells are visible in the blood islands (arrows). Arrowheads indicate examples of CD31+ CD41+ Runx1+ hematopoietic cells. (C) Single optical projection through the ventricle of an E11.5 Nf1 embryo immunostained for CD31 (red), Kit (cyan) and Runx1 (green). (D) Single optical projection through blood islands. Arrows indicate Runx1+ endocardial cells. Arrowheads indicate examples of CD31+Kit+ Runx1+ hematopoietic cells. Scale bars = 50 μm.

DOI: http://dx.doi.org/10.7554/eLife.07780.005

In addition to robust cardiac blood island formation, E11.5 Nf1 embryos have enlarged fetal livers populated by Runx1+ and CD41+ hematopoietic cells (Figure 2C), consistent with previous studies that found significantly higher numbers of fetal liver clonogenic progenitors (Zhang et al., 1998; Largaespada et al., 1996; Bollag et al., 1996). Furthermore, competitive repopulation assays comparing Sca1+lin–/dim cells isolated from the fetal livers of Nf1 and Nf1embryos demonstrated that Nf1cells have a growth advantage, particularly in the myeloid compartment (Bollag et al., 1996). Thus, the enlargement of the fetal liver may be due to elevated proliferation of Nf1 hematopoietic cells.

Creation of Nf1 R1276P GRD mice

In order to determine if the increase in cardiac blood islands seen in Nf1 embryos is due specifically to loss of the Ras-GAP activity of neurofibromin, we engineered a missense mutation within the GRD. Arginine 1276 has been shown to be the 'arginine finger' of the GRD and is critical for catalytic activity. Mutation of this residue to proline was identified in a family with NF1, and crystal structures of related GAP domains were consistent with empiric studies showing loss of GAP activity following R1276P mutagenesis (Ahmadian et al., 1997; Scheffzek et al., 1997; Klose et al., 1998; Hiatt et al., 2004). We generated 'knockin' mice in which arginine 1276 was mutated to proline (R1276P) and designated these mice Nf1(Figure 4—figure supplement 1). We generated an additional line of engineered mice in order to control for minor changes to intronic genomic sequences necessitated by the gene targeting and selection strategy (see Materials and methods and Figure 4—figure supplement 1). For these control mice, designated Nf1, we utilized the identical targeting strategy but arginine 1276 was left intact. Appropriate targeting in several ES cell clones for each of the Nf1or Nf1constructs was demonstrated by Southern blotting (Figure 4—figure supplement 1). These were used to generate chimeric animals that were then bred for germ line transmission.

Figure 4—figure supplement 1.

Generation of Nf1and Nf1 mouse lines.

(A) Schematic diagram outlining the targeting strategy to develop the Nf1GRD mouse line by modifying the endogenous mouse Nf1 locus with a mutation corresponding to the human NF1 R1276P missense allele. This mutation abrogates neurofibromin GAP activity without impairing secondary or tertiary protein structure or reducing cellular levels of neurofibromin (Klose, et al., 1998). (B) Strategy to develop the Nf1knock-in 'control' mouse by targeting the endogenous mouse Nf1 locus with a construct identical to that used to target the NF1 R1276P mutation in (A) with the exception that no mutation is introduced. Knock-in Nf1mice generated from this construct are a stringent control for Nf1animals. For both (A) and (B) asterisks denote regions where additional DNA sequences are identically introduced into introns as part of the targeting process. The 'an' cassette imparts G418 resistance and is self-excised in the male germ line. N = NcoI restriction endonuclease site. (C) Southern blots of genomic DNA from embryonic stem (ES) cell clones targeted with either the Nf1or Nf1allele display a 13 kb wild type (WT) band as well as a 5.5 kb mutant (MT) band. Five and three positive clones were isolated with genotype Nf1or Nf1, respectively, as shown. (D) DNA products from PCR reactions performed with primers specific for the Nf1 wildtype, Nf1 knockout (KO), or Nf1 GRD alleles using template DNA isolated from amniotic sacs of E10.5 embryos.

DOI: http://dx.doi.org/10.7554/eLife.07780.008

Heterozygous Nf1 mice appeared normal and were able to breed, but heterozygous intercrosses failed to produce any viable homozygous Nf1 pups (Table 1). One out of 61 embryos genotyped at E12.5 was Nf1, and 11of 63 (17.5%) were Nf1 at E11.5 (Table 1). Hence, homozygous R1276P mutation of Nf1 causes midgestation embryonic lethality with most embryos succumbing by E12.5.

Table 1.

Genotypes from Nf1intercrosses.

DOI: http://dx.doi.org/10.7554/eLife.07780.006

Age	Total	+/+	Nf1GRD/+	Nf1GRD/GRD
E11.5	63	13	39	11
E12.5	61	19	41	1
P0	62	23	39	0

Total cell lysates from Nf1and Nf1 embryos exhibited similar levels of neurofibromin protein of expected apparent molecular weight of 250–280 kDa (Figure 4A). The relative neurofibromin protein expression was similar to that of wild-type embryos and was increased relative to Nf1 embryos (Figure 4B).

Figure 4.

Neurofibromin protein expression and activity from the Nf1 alleles.

(A) Total cell lysates from E10.5 Nf1, and Nf1embryos were analyzed by SDS-PAGE followed by immunoblotting with either anti-neurofibromin (top panel) or anti-beta tubulin (bottom panel) antibodies as indicated. (B) Band intensities from 5 immunoblots as in (A) were quantified by ImageJ. The relative neurofibromin expression for each genotype compared to wild-type is indicated. All data are represented as the mean ± S.E. **, p<0.05; ***, p<0.001; NS = not significant (p<0.001, one-way ANOVA between groups, post hoc multiple comparisons, Tukey’s test). (C) A cross-section of a peripheral nerve (demarcated in white and indicated by an arrow) from each of Nf1 and Wnt1-Cre; Nf1 P0 animals shows elevated expression of pERK, a downstream indicator of Ras activity, in Wnt1-Cre; Nf1 animals (right panel). An adjacent blood vessel (BV) is indicated. (D) Adrenal medullary tissue within an adrenal gland from either a Nf1 or Wnt1-Cre; Nf1 animal shows increased pERK expression in a hyperplastic area from the Wnt1-Cre; Nf1 animal (right panel). pERK-positive cells are marked by arrowheads. Background fluorescence from non-neural-crest-derived adrenal cortical and blood cells is evident in the Nf1 sample. (E) Cardiac cushions from E11.5 embryos show elevated pERK staining in Nf1 embryos compared to Nf1 animals as indicated within the dashed oval. Scale bars = 50 μm.

DOI: http://dx.doi.org/10.7554/eLife.07780.007

(A) Schematic diagram outlining the targeting strategy to develop the Nf1GRD mouse line by modifying the endogenous mouse Nf1 locus with a mutation corresponding to the human NF1 R1276P missense allele. This mutation abrogates neurofibromin GAP activity without impairing secondary or tertiary protein structure or reducing cellular levels of neurofibromin (Klose, et al., 1998). (B) Strategy to develop the Nf1knock-in 'control' mouse by targeting the endogenous mouse Nf1 locus with a construct identical to that used to target the NF1 R1276P mutation in (A) with the exception that no mutation is introduced. Knock-in Nf1mice generated from this construct are a stringent control for Nf1animals. For both (A) and (B) asterisks denote regions where additional DNA sequences are identically introduced into introns as part of the targeting process. The 'an' cassette imparts G418 resistance and is self-excised in the male germ line. N = NcoI restriction endonuclease site. (C) Southern blots of genomic DNA from embryonic stem (ES) cell clones targeted with either the Nf1or Nf1allele display a 13 kb wild type (WT) band as well as a 5.5 kb mutant (MT) band. Five and three positive clones were isolated with genotype Nf1or Nf1, respectively, as shown. (D) DNA products from PCR reactions performed with primers specific for the Nf1 wildtype, Nf1 knockout (KO), or Nf1 GRD alleles using template DNA isolated from amniotic sacs of E10.5 embryos.

DOI: http://dx.doi.org/10.7554/eLife.07780.008

(A) pERK staining was observed in neural crest-derived enteric ganglia within the intestinal wall that was more evident in Wnt1-Cre; Nf1 animals (right panel). Arrowheads indicate cells exhibiting positive staining. (B) Both axons (arrows) and nerve cell bodies (arrowheads) were readily visualized in Wnt1-Cre; Nf1 newborns but not in control animals (data not shown).

DOI: http://dx.doi.org/10.7554/eLife.07780.009

To assess whether the introduced R1276P mutation within the GAP domain of neurofibromin disrupts Ras-GAP activity in vivo, tissues were examined for elevated phosphorylated extracellular-signal regulated kinase (pERK), a downstream effector of Ras, as evidence of up-regulated Ras pathway activity. Nf1newborns in which Nf1 was deleted by Wnt1-Cre, displayed elevated pERK staining in neural crest-derived tissues such as peripheral nerves (Figure 4C), within hyperplastic adrenal medullary tissue (Figure 4D), and in enteric ganglia (Figure 4—figure supplement 2). Wnt1-Cre; Nf1newborns showed prominent pERK staining in the axons and cell bodies of peripheral nerves (Figure 4—figure supplement 2) that was not observed in control animals. Elevated pERK staining was also seen in the enlarged cardiac cushions of Nf1embryos, indicating the R1276 mutation is sufficient to elevate pERK levels (Figure 4E). Multiple reports showed that mutation of the conserved 'arginine finger' within the GAP domain decreases neurofibromin GAP function while leaving the domain structurally intact (Ahmadian et al., 1997; Scheffzek et al., 1997; Klose et al., 1998; Hiatt et al., 2004). These observations indicate that inactivation of neurofibromin GAP activity elevates the phosphorylation of the Ras pathway effector ERK in vivo.

Figure 4—figure supplement 2.

Increased pERK staining in neural crest derivatives of Nf1 newborn animals following deletion by Wnt1-Cre.

(A) pERK staining was observed in neural crest-derived enteric ganglia within the intestinal wall that was more evident in Wnt1-Cre; Nf1 animals (right panel). Arrowheads indicate cells exhibiting positive staining. (B) Both axons (arrows) and nerve cell bodies (arrowheads) were readily visualized in Wnt1-Cre; Nf1 newborns but not in control animals (data not shown).

DOI: http://dx.doi.org/10.7554/eLife.07780.009

Nf1 mice either heterozygous or homozygous for the control allele in which arginine 1276 was left intact, appeared normal. Intercrosses of Nf1 mice produced 6 of 26 Nf1 offspring (23%). These control mice were not examined further, and we conclude that embryonic lethality observed in Nf1 embryos is due specifically to the R1276P mutation.

Nf1 embryos exhibit cardiac endocardial cushion and neural crest defects

Histologic analysis of E11.5 Nf1 embryos revealed abnormal cardiac outflow tract morphology and enlarged endocardial cushions, similar to those seen in Nf1 embryos (Figure 5A), which have been previously described in detail (Brannan et al., 1994; Jacks et al., 1994; Lakkis and Epstein, 1998). Atrioventricular endocardial cushions were also enlarged and ventricular septal defects were present, similar to the phenotype seen in Nf1 embryos (Figure 5B). Sympathetic ganglia, derived from neural crest, were enlarged in both Nf1and Nf1 embryos (Figure 5C). Enlargement of sympathetic ganglia in Nf1 mutants was confirmed by immunofluorescence staining for neurofilament and tyrosine hydroxylase (Figure 6A,B).

Figure 5.

Inactivation of Nf1 GRD function affects heart and sympathetic ganglia development.

(A) Sections of hearts from E12.5-13.5 Nf1, and Nf1embryos. The enlarged endocardial cushions in hearts from Nf1embryos (arrowheads) are similar to the oversized cushions of Nf1 embryos. (B) Enlarged atrioventricular endocardial cushions (arrowheads) and ventricular septa defects (arrows) in Nf1and Nf1embryos. (C) Sympathetic ganglia (arrowheads) are similarly enlarged in Nf1and Nf1 embryos. Scale bars = 500 μm.

DOI: http://dx.doi.org/10.7554/eLife.07780.010

Figure 6.

Enlarged sympathetic ganglia in E11.5 Nf1embryos.

(A) Transverse sections of E11.5 Nf1, and Nf1 embryos stained with antibodies against neurofilament. Arrowheads indicate sympathetic ganglia. (B) Transverse sections of E11.5 Nf1, and Nf1embryos stained with antibodies against tyrosine hydroxylase. Scale bars = 100 μm.

DOI: http://dx.doi.org/10.7554/eLife.07780.011

Neural crest-specific loss of Nf1 Ras-GAP function leads to tissue overgrowth

Tissue-specific loss of Nf1 in neural crest results in late gestation lethality, bypassing the midgestation cardiac defects seen in Nf1 null mutants (Gitler et al., 2003). In order to examine in more detail if the Ras-GAP function of neurofibromin is necessary in developing neural crest in embryos surviving past midgestation, we crossed Wnt1-Cre; Nf1mice with Nf1 mice to generate Wnt1-Cre; Nf1 offspring. At E18.5-P0, no viable Wnt1-Cre; Nf1 pups were identified out of 80 genotyped, although 12 non-viable pups (15%) were stillborn or died shortly after birth (Table 2). Live Wnt1-Cre; Nf1 embryos were recovered between E12.5 and 16.5 at the expected frequency (Table 2).

Table 2.

Genotypes from Wnt1-Cre; Nf1X Nf1crosses.

DOI: http://dx.doi.org/10.7554/eLife.07780.012

Age	Total	Nf1flox/+	Nf1GRD/flox	Wnt1-Cre;Nf1flox/+	Wnt1-Cre;Nf1GRD/flox
E12.5-–16.5	27	10	5	3	7 *
E18.5-P0	80	32	18	18	0 **

*2 non-viable Wnt1-Cre; Nf1 embryos were recovered at E12.5-–16.5.

**12 non-viable Wnt1-Cre; Nf1 pups were recovered at E18.5-P.

Histologic examination of Wnt1-Cre; Nf1 embryos revealed overgrowth of the adrenal medulla when compared to control Nf1 embryos that phenocopied adrenal medullary defects seen in Wnt1-Cre; Nf1 embryos (Figure 7A), described previously (Gitler and Epstein, 2003). Massive enlargement of paraspinal neural crest-derived ganglia was also noted in both Wnt1-Cre; Nf1 and Wnt1-Cre; Nf1 embryos (Figure 7B,C). These findings suggest that loss of neurofibromin Ras-GAP function in neural crest is sufficient to reproduce the late-gestation lethality and tissue overgrowth that results from by tissue-specific deletion of Nf1 in neural crest.

Figure 7.

Hyperplasia of neural crest derivatives is similar in Nf1and Nf1newborn animals in which Nf1 is deleted in neural crest cells with Wnt1-Cre.

(A) Adrenal medullary tissue (demarcated in white and indicated with an arrowhead) contained within an adrenal gland of P0 wild-type, P0 Wnt1-Cre; Nf1, or E16.5 Wnt1-Cre; Nf1animals. The tissue is similarly overgrown in Wnt1-Cre; Nf1and Wnt1-Cre; Nf1newborns/fetuses. Scale bars = 100 μm. The arrow indicates a tumor-like medullary protrusion. (B) Sagittal sections showing peripheral ganglia (arrowheads) in Nf1newborn pups, and abnormally enlarged ganglia and tumor-like overgrowth of nerve tissue adjacent to the lumbar spine in a Wnt1-Cre; Nf1newborn pup and an E16.5 Wnt1-Cre; Nf1fetuse. Scale bars = 500 μm. (C) Magnifications of images in (B), with hyperplastic tissue demarcated in black and marked by arrowheads. Lu, lung; Li, liver; Scale bars = 500 μm.

DOI: http://dx.doi.org/10.7554/eLife.07780.013

Ectopic cardiac blood island formation is due to loss of NF1 Ras-GAP activity

We examined E11.5 Nf1 embryos for evidence of cardiac blood island formation to determine if this results from the loss of Ras-GAP activity. Nf1 embryos appeared grossly normal at E11.5 (Figure 8A), but blood-filled protrusions were often visible on the ventricles (Figure 8B, arrowheads). Whole-mount immunofluorescence revealed that the blood-filled protrusions were phenotypically identical to the ectopic cardiac blood islands that formed on the ventricles of Nf1 embryos (Figure 8C, arrowheads). Furthermore, Nf1 embryos had enlarged fetal livers populated with Runx1+ CD41+ hematopoietic cells, similar to Nf1 embryos (Figure 8C). Ventricular blood islands were evident in histologic sections after hematoxylin and eosin (H and E) staining of Nf1 and Nf1embryos and had similar structural characteristics (Figure 8D, arrowheads). Single optical projections through Nf1 blood islands confirm that they were associated with CD31+ CD41+ Runx1+ phenotypic hematopoietic cells (Figure 8E). An average of 26.3 ± 9.2 blood islands could be identified via confocal microscopy on the ventricles of E11.5 Nf1 embryos, whereas Nf1and Nf1ventricles contained no cardiac blood islands (Figure 8F). However, Nf1-deficient E11.5 embryos had, on average, >2 fold more morphological cardiac blood islands as compared to Nf1 embryos (compare Figure 8F and 2D, p≤0.022), suggesting that the Nf1 is a hypomorphic Nf1 allele, at least in regard to cardiac blood island formation. Flushed Nf1 ventricles contained significantly more EMPs than Nf1and Nf1littermates (Figure 8G), but there was a trend towards fewer progenitors than in Nf1 embryos.

Figure 8.

E11.5 Nf1embryos form ectopic cardiac blood islands.

(A) Gross view of E11.5 Nf1and Nf1littermates. (B) Isolated hearts from E11.5 Nf1and Nf1embryos. Black arrowheads indicate blood-filled protrusions on the ventricle of the Nf1heart. (C) Confocal Z-projections (Z interval = 5 μm) of CD31 (red), CD41 (cyan) and Runx1 (green) immunostained E11.5 Nf1and Nf1embryos. Arrowheads point to blood islands on the ventricle of the Nf1embryo. Scale bars = 500 μm. (D) Cell aggregates resembling blood islands (arrowheads) in hearts of E12.5 Nf1 and Nf1embryos. Lower panels, are magnifications of images in top panels. Scale bars = 100 μm. (E) Single optical projection through the cardiac blood islands of an E11.5 Nf1embryo immunostained for CD31 (red), Runx1 (green) and CD41 (cyan). Scale bars = 50 μm. (F) Quantification of blood islands on the ventricles of E11.5 embryos. One-way ANOVA and Bonferroni’s multiple comparison test applied to determine significance, error bars represent the SD. (G) Number of erythroid and myeloid progenitors per flushed E11.5 ventricle. One-way ANOVA and Bonferroni’s multiple comparison test applied to determine significance; error bars represent SD. Nf1 n = 21, Nf1 n = 22 and Nf1 n = 8. * indicates that p≤0.05 and *** indicates that p≤0.001. V: ventricle; A: atrium; FL: fetal liver; Mk: megakaryocyte; Mix: granulocyte-erythroid-monocyte-megakaryocyte; BFU-E: burst forming unit-erythroid; G/M: granulocyte-macrophage colonies. 

DOI: http://dx.doi.org/10.7554/eLife.07780.014

Discussion

In this study we have identifed ectopic cardiac blood island formation as a novel phenotype that arises in Nf1-deficient embryos. Furthermore, using a mouse that expresses a mutant form of neurofibromin with decreased Ras-GAP activity, we demonstrated that the phenotype is a direct result of dysregulation of the Ras signaling pathway. We also showed that some endocardial cells in the ectopic blood islands express Runx1, a master regulator of hematopoiesis and a marker of HE. This, in addition to the enrichment of both phenotypic and functional hematopoietic progenitors in the ventricles of E11.5 Nf1-deficient embryos, suggests that the endocardial cells are producing hematopoietic cells de novo.

We also observed dysregulation of de novo hematopoietic progenitor formation in Nf1-deficient embryos in normal sites of hematopoiesis. A previous study in zebrafish embryos found that the downstream effector of the Ras signaling pathway, pERK, has a biphasic role in blood cell formation from endothelium (Zhang et al., 2014). When zebrafish embryos were treated with an ERK signaling inhibitor prior to artery-—vein specification, runx1 and myb expression in the dorsal aorta decreased; however, when treated with an ERK signaling inhibitor after artery-—vein specification but before EHT, runx1 and myb expression increased (Zhang et al., 2014). Thus, early in development pERK is necessary for de novo generation of hematopoietic cells, but after artery-vein specification, pERK inhibits the specification of HE cells (Zhang et al., 2014). Consistent with a role for ERK signaling in HE specification, increased signaling through the fibroblast growth factor (FGF) receptor, which is upstream of ERK and regulated by Ras-GAP, decreases runx1 expression in the dorsal aorta of zebrafish (Pouget et al., 2014). The mechanism by which increased FGF signaling decreases runx1 expression in the HE involves inhibition of bone morphogenic protein signaling, which is required for runx1 expression (Pouget et al., 2014; Wilkinson et al., 2009; Pimanda et al., 2007). Consistent with these findings, we show that loss of Nf1, which is associated with activation of the Ras-pERK pathway, results in fewer Runx1+ HE cells in the dorsal aorta at E10.5, as well as fewer CD31+ Kit+ Runx1+ hematopoietic cluster cells. In contrast, the yolk sac of Nf1 embryos produced more EMPs when compared to littermate controls, consistent with the positive role for FGF signaling in regulating erythropoiesis and myelopoiesis at a similar earlier stage in the zebrafish embryo (Yamauchi et al., 2006; Walmsley et al., 2008). Thus the level of Ras activity must be carefully titrated, as elevating Ras signaling in the dorsal aorta limits HE specification, but enhances EMP formation in the yolk sac, and unleashes the hematopoietic potential of the endocardium.

It was previously reported that hematopoietic cells derived from the fetal livers of Nf1-deficient mice are hyperproliferative and cause a JMML-like myeloid proliferative disorder when transplanted into irradiated recipients (Birnbaum et al., 2000; Largaespada et al., 1996; Zhang et al., 2001; Zhang et al., 1998). Based on immunoflourescence, it appears that hematopoiesis is elevated as early as E11.5 in fetal livers of both Nf1 and Nf1 embryos compared to controls, thus implicating activated Ras in hyperproliferation of hematopoietic cells (at E11.5, primarily EMPs) that populate the fetal liver. Likewise, our results implicate the loss of neurofibromin Ras-GAP function within neural crest cells as sufficient to result in overgrowth of sympathetic and dorsal root ganglia and of the adrenal medulla.

The ability of the Nf1 gene product to act as a Ras GAP has been known for a quarter of a century (Ballester et al., 1990; Xu et al., 1990), but the degree to which this function accounts for some or all Nf1 phenotypes has been an ongoing topic of research with relevance for therapeutic strategies. We and others have provided evidence for the ability of neurofibromin to affect alternate signaling pathways, including PKA and cAMP (Guo et al., 1997; The et al., 1997; Hegedus et al., 2007; Brown et al., 2010; Wolman et al., 2014). Prior work has suggested that midgestation embryonic lethality resulting from loss of Nf1 can be rescued by transgenic expression of the isolated neurofibromin GRD, but this was not sufficient for rescue of neural crest overgrowth (Ismat et al., 2006). Failure to rescue neural crest overgrowth could have been the result of inadequate transgenic expression of GRD in this tissue, or because of the necessity for an additional function of neurofibromin outside of the GRD. The findings reported here for Nf1 embryos do not rule out the existence of critical non-GRD functions of neurofibromin in the neural crest or other tissues. In fact, the Nf1 has characteristics of a hypomorphic allele that could be explained by non-GRD related functions. Rather, we demonstrate the necessity of GRD function for normal embryonic development. The development of the Nf1 mouse line described here will allow researchers to determine the necessity of GRD function across the spectrum of developmental and tumor phenotypes observed in mouse models of neurofibromatosis.

Materials and methods

Hematopoietic progenitor assay

Embryos were removed from the uterus and dissected in phosphate buffered saline (PBS) with 20% fetal bovine serum and antibiotics. The yolk sacs were removed and the hearts were dissected, the atrium was cut away and the ventricles were flushed with PBS using an insulin needle and syringe to remove circulating blood cells. The ventricles and yolk sacs were then dissociated in 0.125% collagenase Type I (Sigma, St Louis, MO) for 20–30 min at 37°C, triturated, washed, and filtered to obtain a single cell suspension. Single cell solutions of embryonic ventricles or yolk sacs were plated in methylcellulose (MethocultM3434; Stem Cell Technologies, Vancouver, BC) and colonies were counted 7–8 days after plating.

Whole-mount immunofluorescence and confocal microscopy

Embryos were prepared as described previously (Yokomizo et al., 2012). The following primary antibodies were used; rat anti-mouse CD31 (Mec 13.3, BD Pharmingen, San Diego, CA), rat anti-mouse CD117 (2B8, eBiosciences, San Diego, CA), rat anti-mouse CD41 (MWReg30, BDBiosciences, Franklin Lakes, NJ) and rabbit anti-human/mouse Runx (EPR3099, Abcam, Cambridge, MA). Secondary antibodies used were goat anti-rat Alexa Fluor 647 (Invitrogen, Carlsbad, CA), goat-anti rat Alexa Fluor 555 (Abcam) and goat anti-rabbit Alexa Fluor 488 (Invitrogen). Images were acquired on a Zeiss LSM 710 AxioObserver inverted microscope with ZEN 2011 software and processed with Fiji software (Schindelin, et al., 2012). Hematopoietic cells in the dorsal aorta were counted using the cell counter plugin (version February 29, 2008, Kurt De Vos; http://rsb.info.nih.gov/ij/plugins/cell-counter.html).

Immunoblotting

E10.5 embryos were dissected free of the amniotic sac, frozen in liquid nitrogen, thawed, and disrupted by pipetting in Hank’s Balanced Salt Solution containing 5 mM ethylenediaminetetraacetic acid (EDTA). Total cell lysates were prepared by heating samples in boiling Laemli buffer (66 mM Tris–HCl, pH 6.8, 2% (w/v) SDS, 10 mM EDTA). The samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis using anti-neurofibromin antibody ab17963 (Abcam). Immunoreactive bands were visualized by chemiluminescence. Quantification of individual band intensities was performed using ImageJ. One-way analysis of variance (ANOVA) was used to assess statistical differences between band intensities. Significant ANOVA results were analyzed post hoc by the Tukey-Kramer multiple comparisons test.

Histology and immunofluorescence analyses

Whole mouse embryos or dissected hearts were fixed in 2 or 4% paraformaldehyde, dehydrated in ethanol, and embedded in paraffin for sectioning. Tissues were visualized with H and E stain or by immunofluorescent detection of marker proteins according to standard practices. Detailed protocols are available at http://www.pennmedicine.org/heart/. Antibodies used for immunofluorescence include rabbit polyclonal anti-tyrosine hydroxylase (AB152, EMD Millipore/Chemicon, Billerica, MA), rabbit polyclonal anti-pERK (#9101, Cell Signaling Technology, Inc., Danvers, MA) and mouse monoclonal anti-neurofilament (2H3, Developmental Studies Hybridoma Bank, Department of Biology, University of Iowa, Iowa City, IA). Images were adjusted using Adobe Photoshop using settings applied across the entirety of each image.

Mice

All mouse manipulations were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania following guidelines described in the US National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Nf1 and Wnt1-Cre mice have been described previously (Brannan et al., 1994; Jacks et al., 1994; Zhu et al., 2001; Danielian et al., 1998; Jiang et al., 2000). Nf1 and Nf1 mice were produced by targeting C57BL/6 ES cells (Genoway, Lyon, France) with a targeting vector designed to replace arginine 1276 with proline (R1276P) or, in the case of Nf1to leave arginine 1276 as arginine. The selection strategy (Figure 4—figure supplement 1) included a self-excising floxed neomycin resistance cassette that, after excision, leaves a single loxP site within intron 27. The Nf1 mice were created in order to control for possible unpredicted effects related to the introduction of small changes in genomic sequence, other than those encoding the R1276P missense mutation, necessitated by the targeting strategy. Nf1 and Nf1 mice were genotyped using polymerase chain reaction primers listed below, which produce a 175 bp wild-type band and a 248 bp mutant band (Figure 4—figure supplement 1). All mice were maintained on a C57BL/6 background.

GRDF: 5’- GAGGGGAGATGTCAAAGATGTATTGTGTAACTAC-3’

GRDR: 5’- CAACCTTCAAACAGTACTAAAGTCCATCATGG-3’

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for submitting your work entitled "Loss of Ras-GAP activity of neurofibromin enhances formation of cardiac blood islands in murine embryos" for peer review at eLife. Your submission has been favorably evaluated by Sean Morrison (Senior editor) and three reviewers, one of whom served as a guest Reviewing editor.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.

This paper shows that germline inactivation of the Nf1 tumor suppressor gene leads to aberrant formation of cardiac blood islands, which are observed during a discrete developmental window. This study also describes a novel knock in Nf1 mutant allele containing an amino acid substitution in the catalytic arginine residue that interacts with the P loop of Ras proteins. This conditional "GRD" knock in allele failed to rescue developmental phenotypes associated with Nf1 inactivation including aberrant blood island formation. The analyses of blood islands and of hematopoietic cluster cells that appear earlier in development extend our knowledge regarding the role of Nf1 in hematopoietic development. These observations are interesting and relevant given the association of NF1 with JMML and the finding of somatic NF1 gene mutations in other blood cancers. The GRD knock in allele is a novel and valuable resource that will facilitate additional studies on the role of NF1 in development, neuro-cognitive abnormalities, and tumorigenesis.

Essential revisions:

1) The finding that disruption of NF1 increases blood island formation and blood progenitor development highlights the hemogenic capacity in the endocardium. Given this, a more complete analysis of endocardial hematopoiesis in these embryos is warranted. The assays reported in the manuscript are limited to immunofluorescence for hemogenic endothelial markers and colony assays, which suggest that NF1 deficient endocardial cells are at least capable of erythro-myeloid (EMP) progenitor production, which is similar to the second wave of hematopoiesis in the yolk sac. A general FACS assessment for the key cell types of embryonic hematopoietic progenitor/stem cell hierarchy in flushed hearts (e.g. primitive erythroid, EMPs and HSCs) should be well within the expertise of the team.

2) A critical missing link in the paper is that yolk sac hematopoiesis was not assessed. Is the increased EMP production limited to the heart, or also observed in the yolk sac, which is the classical site of EMP production? This would be important conceptually as it would reveal whether the increased endocardial hematopoiesis upon NF1 inactivation reflects unique regulation in the heart, or indicates a broader requirement of NF1 in hemogenic vessels that generate EMPs. FACS quantification of hematopoietic stem and progenitor cells in the key hematopoietic organs should be performed to serve as a point of reference to compare hematopoiesis in the hearts of NF1 mutant embryos.

3) There is no biochemical proof shown that the knock in mutation results in Ras hyperactivation. Some data assessing evidence of biochemical activation of Ras – perhaps in fetal liver cells from GRD/GRD mice – should be provided.

4) Quantitative data comparing blood island formation in Nf1 WT versus GRD/ + versus GRD/GRD for multiple embryos of each genotype should be provided (similar to panel 2D). The question here (and in #5 below) is whether the GRD allele is a true null or a hypomorph.

5) The immunoblot data shown in panel 4D are central to the conclusions of the paper as they address the nature of the GRD allele. In particular, it appears that neurofibromin expression in the GRD/ + and GRD/GRD lanes are about equal to the Nf1 lane and less that the wild-type. This panel is somewhat problematic as a loading control is not included. This is a key point as the data as presented do not exclude the possibility that wild-type expression levels of the GRD mutant might retain some biologic activity. It is essential that the authors address this point carefully through orthogonal approaches that might include quantitative RT PCR to assess GRDand GRDCTL mRNA expression and more quantitative Western blotting with appropriate loading controls to assess the expression levels of the respective GRD proteins relative to wild-type Nf1.

1) The finding that disruption of NF1 increases blood island formation and blood progenitor development highlights the hemogenic capacity in the endocardium. Given this, a more complete analysis of endocardial hematopoiesis in these embryos is warranted. The assays reported in the manuscript are limited to immunofluorescence for hemogenic endothelial markers and colony assays, which suggest that NF1 deficient endocardial cells are at least capable of erythro-myeloid (EMP) progenitor production, which is similar to the second wave of hematopoiesis in the yolk sac. A general FACS assessment for the key cell types of embryonic hematopoietic progenitor/stem cell hierarchy in flushed hearts (e.g. primitive erythroid, EMPs and HSCs) should be well within the expertise of the team. Wild type embryonic hearts contain too few progenitors for FACS analysis. For example, Figure 2E shows that each normal embryonic heart contains approximately 25 CFU-C progenitors. At E11.5 there are approximately 3-4 HSCs per embryo, and no good markers to distinguish them from committed progenitors. Therefore, although it is well within our expertise to analyze progenitors from more abundant tissue sources, or in situations where many embryos can be pooled for analysis, we are unable to do so in this situation.

2) A critical missing link in the paper is that yolk sac hematopoiesis was not assessed. Is the increased EMP production limited to the heart, or also observed in the yolk sac, which is the classical site of EMP production? This would be important conceptually as it would reveal whether the increased endocardial hematopoiesis upon NF1 inactivation reflects unique regulation in the heart, or indicates a broader requirement of NF1 in hemogenic vessels that generate EMPs. FACS quantification of hematopoietic stem and progenitor cells in the key hematopoietic organs should be performed to serve as a point of reference to compare hematopoiesis in the hearts of NF1 mutant embryos.

We analyzed EMPs in Nf1 yolk sacs and found that they were increased relative to wild type embryos, particularly erythroid progenitors. The difference between yolk sac and heart versus dorsal aorta is quite interesting, and indicates that Ras signaling regulates aortic hematopoiesis quite differently than yolk sac or heart hematopoiesis. The new yolk sac data are included in revised Figures 1A,B.

3) There is no biochemical proof shown that the knock in mutation results in Ras hyperactivation. Some data assessing evidence of biochemical activation of Ras – perhaps in fetal liver cells from GRD/GRD mice – should be provided.

Using immunofluorescence we demonstrate that tissues from Nf1 GRD animals harboring the R1276P mutation within the GAP domain have elevated levels of the Ras effector pERK (revised Figure 4). This is consistent with multiple reports showing that mutation of the conserved “arginine finger” within the GAP domain decreases neurofibromin GAP function while leaving the domain structurally intact (Ahmadian, 1997; Scheffzek, 1997; Klose, 1998; Hiatt, K., 2001). The R1276P mutation has been utilized in multiple contexts to probe the function of neurofibromin Ras-GAP activity (Yang, FC, 2003; Hiatt, K., 2004; Hannan et al., 2006; Ho, 2007; Shannon, 2014). Just as others have found the R1276P mutation within the GAP domain a useful research tool to probe neurofibromin GAP function, we feel the Nf1 GRD mouse will be valuable in extending these earlier observations within the setting of a mouse line.

4) Quantitative data comparing blood island formation in Nf1 WT versus GRD/ + versus GRD/GRD for multiple embryos of each genotype should be provided (similar to panel 2D). The question here (and in #5 below) is whether the GRD allele is a true null or a hypomorph.

This was an excellent suggestion, and we performed the analysis requested. Indeed Nf1E11.5 embryos have fewer blood islands than Nf1 embryos. These new Nf1data are included in Figure 8F,G.

5) The immunoblot data shown in panel 4D are central to the conclusions of the paper as they address the nature of the GRD allele. In particular, it appears that neurofibromin expression in the GRD/ + and GRD/GRD lanes are about equal to the Nf1 + /- lane and less that the wild-type. This panel is somewhat problematic as a loading control is not included. This is a key point as the data as presented do not exclude the possibility that wild-type expression levels of the GRD mutant might retain some biologic activity. It is essential that the authors address this point carefully through orthogonal approaches that might include quantitative RT PCR to assess GRDand GRDCTL mRNA expression and more quantitative Western blotting with appropriate loading controls to assess the expression levels of the respective GRD proteins relative to wild-type Nf1.

We have more carefully quantified neurofibromin protein expression by performing 5 independent Westerns with loading controls. A representative Western and the averaged, normalized data is shown in revised Figure 4A,B.