Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Genesis. Author manuscript; available in PMC 2012 June 13.
Published in final edited form as:
Published online 2011 April 1. doi:  10.1002/dvg.20727
PMCID: PMC3374396

A Phenotype-Driven ENU Mutagenesis Screen Identifies Novel Alleles With Functional Roles in Early Mouse Craniofacial Development


Proper craniofacial development begins during gastrulation and requires the coordinated integration of each germ layer tissue (ectoderm, mesoderm, and endoderm) and its derivatives in concert with the precise regulation of cell proliferation, migration, and differentiation. Neural crest cells, which are derived from ectoderm, are a migratory progenitor cell population that generates most of the cartilage, bone, and connective tissue of the head and face. Neural crest cell development is regulated by a combination of intrinsic cell autonomous signals acquired during their formation, balanced with extrinsic signals from tissues with which the neural crest cells interact during their migration and differentiation. Although craniofacial anomalies are typically attributed to defects in neural crest cell development, the cause may be intrinsic or extrinsic. Therefore, we performed a phenotype-driven ENU mutagenesis screen in mice with the aim of identifying novel alleles in an unbiased manner, that are critically required for early craniofacial development. Here we describe 10 new mutant lines, which exhibit phenotypes affecting frontonasal and pharyngeal arch patterning, neural and vascular development as well as sensory organ morphogenesis. Interestingly, our data imply that neural crest cells and endothelial cells may employ similar developmental programs and be interdependent during early embryogenesis, which collectively is critical for normal craniofacial morphogenesis. Furthermore our novel mutants that model human conditions such as exencephaly, craniorachischisis, DiGeorge, and Velocardiofacial sydnromes could be very useful in furthering our understanding of the complexities of specific human diseases.

Keywords: ENU mutagenesis, craniofacial development, neural crest, pharyngeal arch, vasculature, mouse


The vertebrate head is a complex assemblage of cranial specializations including the central and peripheral nervous systems, viscero- and neurocraniums, musculature, vasculature, and connective tissue. In all higher vertebrates the facial prominences from which the head and face are derived, share a common basic plan or blueprint. With respect to the pharyngeal arches for example, the central core region of each arch is composed of cranial mesoderm, which will ultimately generate the branchiomeric musculature and vasculature (Noden, 1982, 1983b; Trainor et al., 1994). The mesodermal cores are enveloped by neural crest cells, which are a specialized migratory population of progenitor (Trainor and Tam, 1995) cells that generates most of the bone, cartilage and connective tissue in the head and face (LeDouarin and Kalcheim, 1999). The pharyngeal arches are then lined internally by the endoderm and externally by the ectoderm. There is a general axial registration that exists between neural crest cells and mesoderm and ectoderm that persists during their migration and differentiation (Noden, 1991; Trainor and Tam, 1995). These relationships and the tissue boundaries they create are maintained throughout development (Kontges and Lumsden, 1996). Moreover, the neural crest-derived connective tissue mesenchyme provides the cues necessary to direct the distribution and alignment of the mesoderm-derived myoblasts (Noden, 1983a). This congruence and axial registration also includes the cranial motor nerves and precursors of epi-pharyngeal placodes, (Baker and Bronner-Fraser, 2001; D’Amico-Martel and Noden, 1983) which will innervate specific craniofacial muscles. These co-ordinated interactions are essential for generating a fully functioning jaw and indicate that the registration between different tissues in the head during early embryogenesis is critical for establishing the blueprint or foundations of vertebrate craniofacial development.

Craniofacial abnormalities account for approximately one third of all birth defects (Gorlin et al., 1990) and are typically recognized as problems in the underlying structure of the face, that is anomalies in bone and cartilage development. These tissues are derived predominantly from neural crest cells. It is not surprising therefore that many craniofacial abnormalities are attributed to problems in neural crest cell development. Neural crest cells are however only transiently generated hence it is critical that the embryo produces and maintains a sufficient pool of neural crest progenitors that survive, proliferate, migrate and differentiate appropriately as deficiencies in these processes underlie a number of congenital craniofacial malformation disorders. In fact, depending on which phase of neural crest cell development is disrupted (i.e., formation versus differentiation), very different craniofacial anomalies can arise. For example if neural crest cell formation or migration is perturbed such that too few neural crest cells are produced or they fail to migrate to their final destinations, this can result in babies with small noses and jaws, defects in ear development and cleft palate. These phenotypes are characteristic of Treacher Collins syndrome (Dixon et al., 2006; Jones et al., 2008). In contrast, if differentiation of the neural crest cell-derived suture mesenchyme is perturbed, craniosynostosis, which is characterized by dysmorphic cranial shape, midface hypoplasia, seizures and mental retardation can occur (Morriss-Kay and Wilkie, 2005).

Craniofacial anomalies, however, are not always the consequence of defects autonomous or intrinsic to the neural crest. Abnormal neural crest cell patterning can also arise secondarily as a consequence of cell non-autonomous or extrinsic defects in the mesoderm, ectoderm, and endoderm tissues with which the neural crest cells interact (Trainor and Krumlauf, 2001). In fact, each of the ectoderm, endoderm, and mesoderm tissues play critical roles in regulating neural crest cell patterning and reciprocal interactions between each of these tissues are absolutely critical for normal craniofacial development (Trainor and Krumlauf, 2000). Current analyses of craniofacial development therefore must be cognizant of the fact that not all craniofacial anomalies arise through defects intrinsic to the neural crest. Furthermore, it is vital to investigate the molecular and cellular nature of reciprocal interactions between all of these tissues during embryonic development to better understand the etiology and pathogenesis of congenital craniofacial malformations.

With these principles in mind, we performed a forward genetic screen in mice to identify novel alleles with functional roles in early craniofacial development using N-ethyl-N-nitrosourea (ENU), which generates point mutations in mouse spermatogonial cells. Point mutations induced by N-ethyl-N-nitrosourea (ENU) provide a unique mutant resource because they: (i) reflect the consequences of single gene changes independent of position effects; (ii) provide a fine-structure dissection of protein function; (iii) display a range of mutant effects from complete or partial loss of function to exaggerated function (Justice et al., 1999). ENU has been widely used in mutagenesis screens to uncover novel alleles with specific developmental roles and one of the major advantages of the ENU approach is that it is unbiased. No preference is given to genes of a specific type or family, nor expression status in a particular tissue, which is critical because as described above, intrinsic as well as extrinsic regulators of neural crest cell patterning contribute to craniofacial disease. What is important is to identify interesting, consistent, and reproducible craniofacial malformation phenotypes.

Here we describe an ENU screen that has successfully identified a number of novel alleles that are critically required for early craniofacial development and embryo survival in mice. The data we have obtained from our mutants to date imply that endothelial cells and neural crest cells may employ similar developmental programs and be interdependent during early embryogenesis which collectively is critical for normal craniofacial morphogenesis. Furthermore, we have identified mutations eliciting phenotypes that model human syndrome which will be of value to the craniofacial community. Our mutant series demonstrates that phenotype-driven ENU screens can be very powerful in furthering our understanding of the complexities of human disease.


Screen for Early Craniofacial Morphogenesis Mutants

We generated 50 founder (F1) males which were heterozygous for ENU-induced mutations. Each founder male was outcrossed to FVB/NJ females and at least six daughters were then backcrossed to each founder male to identify lines that reproducibly generated autosomal recessive mutant phenotypes in early craniofacial morphogenesis. Embryos were initially examined using bright field dissection microscopy. We focused our screen on morphological abnormalities readily identifiable at E9.5–10.5 that were consistent and reproducible within litters and between litters. Consequently, we identified 10 recessive mutant lines that exhibited phenotypes including defects in embryo size, frontonasal, and pharyngeal arch patterning, neural and vascular development, eye, ear, and limb morphogenesis (see Fig. 1). The names ascribed to each mutant reflect their distinctive individual features; trex -short forelimbs typical of dinosaur T.rex (Fig. 1b); wiggable—excessive leaf-like laminae or folia in the brain resembling a wig (Fig. 1c); grimace—contorted facial expression (Fig. 1d); palloncino—Italian for balloon, reflecting inflated vasculature (Fig. 1e); mullet—exencephaly and craniorachischisis resembling embryo with 80s hair style (Fig. 1f); arco piccolo—Italian for small (pharyngeal) arch (Fig. 1g); sottile tubo—Italian for thin (neural) tube (Fig. 1h); pacman—open gaping mouth (Fig. 1i); orvieto—Italian city known for dry white wine, reflecting appearance of embryos due to absence of blood cells and vasculature (Fig. 1j); snouty—shortened frontonasal region (Fig. 1k). A full description of the phenotypes and their significance are described below.

FIG. 1
Fluorescent images of E9.5-10.5 DAPI stained (a) Wild-type; (b) trex; (c) wiggable; (d) grimace; (e) palloncino; (f) mullet; (g) arco piccolo; (h) sottile tubo; (i) pacman; (j) orvieto; (k) snouty embryos. Abbreviations: ba, branchial arch; lnp, lateral ...

Growth Defects in Mutant Embryos

One of the most recognizable and consistent features of mutant embryos obtained in our screen was a distinct size difference compared to wild-type littermates (Fig. 1; embryos not photographed to scale). At E9.5, grimace, palloncino, arco piccolo, sottile tubo, pacman, orvieto, and snouty mutant embryos were each considerably smaller than their wild-type littermates. Typically the mutants were only half to two-thirds the size of controls. At E9.5, each of the mutant embryos exhibited hearts with regular beating and there was little evidence of any overt developmental delay. This indicates the embryos were still alive and the size difference was likely due to alterations in cell proliferation and survival. At E9.5, mullet embryos were comparable in size to wild-type but by E10.5 were slightly smaller. However, not all the mutant embryos were smaller in size. For example, trex mutant embryos are identical in overall size at E9.5-11.5 to their wild-type littermates. In contrast, E9.5-11.5 wiggable embryos were noticeably larger than their wild-type littermates, which was suggestive of enhanced growth and cell proliferation in this particular mutant. The size differences observed for each mutant were evident not only in terms of overall embryo size, but also with respect to specific structures as described below.

Frontonasal and Pharyngeal Arch and Cleft Anomalies

The frontonasal prominences and pharyngeal arches comprise a series of bilateral outgrowths that give rise to many of the structures of the head and face (Fig. 2a). For example, the frontonasal region can be subdivided into medial and lateral prominences that line either side of the nasal placode or pit and give rise to the forehead and nose. In mammals such as mice, there are four (1–4) clearly identifiable pharyngeal arches and two arches (5 and 6), which are considered rudimentary. Each pharyngeal arch consists of a mesoderm core, which is covered externally by ectoderm and lined internally by endoderm. The ectoderm between the arches form grooves called pharyngeal clefts while the endoderm forms pharyngeal pouches. Together the clefts and pouches delineate the individual pharyngeal arches. The maxillary and mandibular prominences that constitute the first arch generate much of the upper and lower jaw, respectively.

FIG. 2
High magnification fluorescent images of the pharyngeal arch region of E9.5-10.5 DAPI stained (a) Wild-type; (b) trex; (c) wiggable; (d) grimace; (e) palloncino; (f) mullet; (g) arco piccolo; (h) sottile tubo; (i) pacman; (j) orvieto; (k) snouty embryos. ...

Hypoplasia and abnormal development of the frontonasal mesenchyme and individual pharyngeal arches was prevalent with high penetrance in a number of our ENU generated mutants. E10.5. trex embryos for example, displayed laterally displaced nasal placodes with distal maxillary and frontonasal hypoplasia (Figs. 1b and and2b).2b). With respect to the medial and lateral nasal prominences, insufficient growth and fusion leads to midfacial clefting by E12.5-13.5 (Sandell et al., 2007). In addition, the third arch was severely reduced in size leaving only a rudimentary structure, which was fused distally with the second arch (Figs. 1b and and2b;2b; arrowhead). trex embryos also exhibited complete agenesis of the 4th pharyngeal arch.

Embryos of the wiggable mutant also displayed pharyngeal arch agenesis at E10.5. Specifically, the third and fourth arches were absent and a large cleft remained in their place (Figs. 1c and and2c;2c; arrowhead). The second arch was severely hypoplastic. Interestingly, despite agenesis and hypoplasia of the caudal arches, the maxillary and mandibular prominences that comprise the first arch were massively overgrown in this mutant. The maxillary prominence in particular appeared about twice the size of controls and protruded to the same distal extent as the mandibular prominence. Similar to trex, the nasal placode in wig-gable mutant embryos was displaced and protruded laterally.

The grimace, palloncino, and orvieto embryos also exhibited agenesis of the caudal third and fourth pharyngeal arches (Fig. 2d,e; arrowhead). Although these mutants had caudal arch defects that were manifested by E9.5, each had well-defined but abnormally developed rostral pharyngeal arches with respect to maxillomandibular morphology. In grimace embryos, the proximal portion of the mandibular prominence protruded over the medial portion of the arch and there was no clearly defined maxillary prominence (Figs. 1d and and2d;2d; asterisk). Consequently there was no evidence of the maxillomandibular cleft or pharyngeal arch hinge, which is critical for patterning the upper and lower jaw (Depew and Simpson, 2006). Palloncino embryos similarly displayed abnormal pharyngeal hinge morphology in the form of a modest cleft due to hypoplasia of both the maxillary and mandibular prominences (Figs. 1e and and2e;2e; asterisk). Abnormal maxillomandibular hinge morphology is also evident in orvieto embryos. Overall, the mandibular prominence is reduced in size, particularly at its proximal end, which is extremely narrow, often giving the impression that the mandibular prominence has physically separated from the maxillary prominence (Figs. 1j and and2j;2j; arrowhead). A similar phenomenon is observed with respect to the hypoplastic second pharyngeal arch in orvieto embryos, which also displays a proximal constriction and apparent ventrolateral separation from the face.

Embryos of the arco piccolo mutant are readily identifiable at E9.5 due to their severely hypoplastic second arch, which abnormally resembles the third pharyngeal arch in size (Figs. 1g and and2g;2g; arrowhead). By E10.5 all of the pharyngeal arches in arco piccolo embryos are noticeably reduced in size compared to wild-type control littermates. Mutant embryos of the pacman line, at E9.5, display rostral and caudal pharyngeal arches that are distinct from each other but exhibit abnormally expanded mesoderm derived arch arteries (Figs. 1i and and2i;2i; arrowhead). The expanded arch artery is particularly evident within the first pharyngeal arch giving it the appearance of being massively enlarged and also hollow.

Frontonasal and pharyngeal arch morphology in snouty mutant embryos was also affected (Fig. 1k; asterisk). In this mutant the lateral and medial frontonasal mesenchyme was considerably reduced giving rise to frontonasal foreshortening. The second arch in snouty mutants is almost always hypoplastic and is occasionally absent in very severe mutants (Figs. 1k and and2k;2k; asterisk).

In contrast, pharyngeal arch formation in sotille tubo and mullet embryos was relatively normal. In sotille tubo embryos the pharyngeal arches are present and clearly delineated by clefts/pouches, but are reduced in size compared to their wild-type littermates, consistent with the smaller nature of sotille tubo embryos overall (Fig. 2h). Only minor or subtle reductions in pharynegeal arch size were observed in E10.5 mullet embryos (Fig. 2f).

Neural Tube Defects

Neurulation is a fundamental process that occurs during embryogenesis culminating in formation of the neural tube, which is the precursor of the brain and spinal cord. During neurulation, specialized regions of the neural plate, the bilateral dorsal neural folds, elevate, appose each other and fuse to create the neural tube. Neurulation is typically divided into primary and secondary phases. Primary neurulation creates the brain and most of the spinal cord, whereas secondary neurulation occurs at the anterior sacral level and is associated with formation of the caudal-most region of the spinal cord. We identified two mutants in our screen with defects in primary neurulation, however, none of the mutants in our screen exhibited secondary neurulation anomalies. At E10.5 wiggable embryos exhibit an excess of laminae or folia within the neural plate, particularly at the level of the forebrain, midbrain, and anterior hindbrain (Fig. 1c; asterisks). The cranial neural folds therefore are persistently open and have an everted and enlarged appearance. Consequently, this results in exencephaly and rostral craniorachischisis in wig-gable embryos. Similarly, E10.5 mullet embryos also display exencephaly and rostral craniorachischisis (Fig. 1f; asterisk). However, although the neural folds are open and everted, mullet embryos do not display the same excessive neural folia as is observed in wiggable embryos. This suggests that despite exhibiting a similar open neural tube defect, the mechanisms underpinning the pathogenesis of exencephaly and craniorachischisis in wiggable and mullet embryos are probably different.

Sensory Organ Defects of the Eyes and Ears

The head houses the primary sensory organs including the eyes and ears, the precursors of which are the optic vesicle and otocyst, respectively. These structures are readily recognized morphologically in E9.5 embryos. In contrast the nasal placode is not morphologically recognizable until invagination of the placode commences around E10.5-11.0. Since all of our ENU-induced mutants except trex and mullet are lethal by E11.0, we have limited our analyses of sensory organ morphology primarily to the optic and otic vesicles. Interestingly trex and palloncino embryos exhibit very similar anomalies in otic development (Fig. 1b,e). Instead of the otocyst lying at the axial level adjacent to the junction of pharyngeal arches 2 and 3 as is the case in wild-type embryos, the otocyst in trex and palloncino embryos is displaced rostrally and the otic placode is often observed to have given rise to multiple supernumerary otocysts adjacent to the caudal hindbrain and rostral spinal cord (Fig. 1b; asterisk). trex and palloncino embryos differ however with respect to eye development. Morphologically the initiation and morphogenesis of optic vesicle formation in palloncino embryos is normal, but in the case of trex embryos, the optic vesicle is smaller than in wild-type controls. This optic vesicle phenotype becomes more pronounced with age such that by E12.5, it is clear that the small eye is due in large part to agenesis of the ventral half of the eye.

E10.5 wiggable embryos also exhibit ear and eye anomalies (Fig. 1c). The otocysts in wiggable embryos are only mildy affected and to the extent that they are smaller in overall size than in wild-type controls. In contrast the eyes are dramatically perturbed in wiggable embryos which exhibit complete ocular agenesis. Since the optic vesicles are derived from the neuroepithelium, ocular agenesis in wiggable embryos is likely to be a manifestation of abnormal growth and patterning of the neural primordium. However, the absence of optic vesicles in these embryos may also be secondary to the massive overgrowth of the maxillary prominence, which is a significant part of the wiggable phenotype.

Neural Crest Cell Formation and Migration

Cranial neural crest cells comprise a migratory progenitor cell population that generates the majority of bone, cartilage, and connective tissue in the head and face and also contributes to sensory organ and peripheral nervous system development (Le Douarin and Kalcheim, 1999). During early embryogenesis, neural crest cells are induced to form within the dorsal region of the neural plate, after which they delaminate and migrate, populating the distal regions of the head and face. Cranial neural crest cells that colonize the facial prominences and pharyngeal arches give rise to the visceroskeleton of the face. Cranial neural crest cells that coalesce with ectodemal placode cells establish and pattern the sensory organs and peripheral nervous system. Neural crest cells also contribute smooth muscle cells and pericytes, which are integral to vascular development. We hypothesized that many of the craniofacial anomalies observed in our ENU induced mutants were associated with abnormal neural crest cell development. Therefore to begin documenting any anomalies in neural crest cell formation, migration and early differentiation in mutant lines, we performed RNA in situ hybridization with the SoxE family member, Sox10, mutations in which are associated with neural crest cell abnormalities and congenital disease (Pingault et al., 1998; Southard-Smith et al., 1998). The Sox10 gene is a useful marker for neural crest development owing to its expression in migrating cranial neural crest cells in wild-type embryos at E8.5-9.0. Later Sox10 expression becomes refined primarily to neurogenic neural crest (E9.5) and then to glial cells within the peripheral nervous system (E10.5; Fig. 3a).

FIG. 3
Bright field images of Sox10 in situ hybridization stained E9.5-10.5 (a) Wild-type; (b) trex; (c) wiggable; (d) grimace; (e) palloncino; (f) mullet; (g) arco piccolo; (h) sottile tubo; (i) pacman; (j) orvieto; (k) snouty embryos. Arrowheads and arrows, ...

Sox10 in situ hybridization revealed considerable neural crest cell patterning anomalies in each of the ENU-induced mutants between E9.5-10.5 (see Fig. 3). For example, abnormal Sox10 staining in E10.5 trex embryos revealed perturbations in the neural crest populations that contribute to cranial ganglia. With respect to the trigeminal ganglion, the neural crest that normally gives rise to dorsal half or proximal root of this ganglion is missing indicating a perturbation of neural crest cell contribution to the trigeminal in trex embryos. A similar defect is apparent in the neural crest population that gives rise to the dorsal root of the facial ganglion (Fig. 3b; asterisk). The caudal ganglia in trex embryos are even more severely affected as evidenced by absence of Sox10-positive neural crest populations in the cervical region (Fig. 3b; arrowhead). This is indicative of complete agenesis of the facioacoustic/vestibulocochlear, glossopharyngeal and vagal ganglia in these embryos.

Severe perturbations in cranial sensory ganglia formation were also observed in E10.5 wiggable embryos through Sox10 staining. Similar to trex, it appears that the trigeminal ganglion is lacking its neural crest cell derived proximal root in wiggable embryos (Fig. 3c; asterisk). Anterior to the trigeminal, multiple ectopic Sox10-positive ganglion-like condensations can be observed, one of which is connected to the ophthalmic axon branch of the trigeminal (Fig. 3c; arrow). In this mutant the facioacoustic/vestibulocochlear ganglia are abnormally shaped and persist as round hyoplastic ganglia instead of forming their usual finger like projections (Fig. 3c; asterisk). Caudal to the otocyst, the cranial ganglia are hypoplastic or absent (Fig. 3c; arrowhead). Rudimentary condensations corresponding to the glossopharyngeal and vagal may exist, but there is little evidence of formation of the caudal cranial ganglia as revealed by the limited domains of Sox10 staining.

Grimace mutant embryos exhibit defects in cranial ganglia development that are even more severe than trex and wiggable. The anterior-most domain of Sox10 in E9.5 grimace embryos, which lies proximal to the first pharyngeal arch, correlates with the expected positioning of the trigeminal ganglion (Fig. 3d; arrowhead). However, the trigeminal is considerably hypoplastic. Moreover, the spatiotemporal reduction in Sox10 staining more caudally is consistent with agenesis of the facioacoustic/vestibulochochlear, glossopharyngeal, and vagal ganglia. The circular domains of Sox10 staining that remain coincided with small ectopic supernumerary otocysts. Thus, grimace embryos display severe defects in cranial ganglia patterning.

Palloncino mutant embryos exhibited the most profound reduction in Sox10 staining of all of the mutants identified in our screen. Apart from labeling of the otocyst and a small number of cells proximal to the first pharyngeal arch, the cranial region was largely devoid of Sox10 positive cells (Fig. 3e; arrowhead). The absence of Sox10 staining is consistent with perturbation of cranial neural crest cell development and extreme hypoplasia or agenesis of the trigeminal, facioacoustic/vestibulocochlear, glossopharyngeal, and vagal ganglia.

Embryos of the orvieto mutant are distinctive for their particularly small second arch, and based on Sox10 RNA in situ hybridization at E8.5-9.5, exhibit a failure of cranial neural crest cell colonization of the second pharyngeal arch (Fig. 3j; arrowhead). In fact, rhombomere 3–5 derived neural crest cells failed to migrate beyond the proximal extent of the second pharyngeal arch. Furthermore caudal to the otic vesicle there is an apparent loss of migrating neural crest cells as evidenced by the absence of Sox10 positive cells.

In contrast to the mutants described above, defects in neural crest cell development E9.5 arco piccolo, sottile tubo, and pacman mutant embryos were relatively mild (Fig. 3g–i). Sox10 RNA in situ hybridization in E8.5-9.5 embryos revealed the presence of three discrete segregated streams of neural crest cells populating the rostral pharyngeal arches and contributing to formation of the trigeminal, facioacoustic/vestibulocochlear, glossopharyngeal, and vagal ganglia. Although the formation and migration of cranial neural crest appears relatively normal in each of these mutants the trigeminal ganglion is abnormally shaped and the Sox10 positive neural crest cell population within the forming ganglion is disorganized and dispersed rather than forming a tight coalescence as it does in wild-type embryos (Fig. 3g–i; arrowhead).

In contrast to the mutants described above, mullet and snouty exhibited largely normal patterns of neural crest cell formation and migration as well as initial cranial ganglia patterning in E9.5-10.5 embryos based on Sox10 in situ hybridization (Fig. 3f,k).

Neural Crest Cell Differentiation

Neural crest cells give rise to a variety of neural and non-neural cell types located throughout the entire vertebrate body, which raises interesting questions as to how progenitor cell populations generate multiple differentiated cell types in the correct spatiotemporal manner. The expression of Sox10 in migrating neural crest cells and then its refinement to neurogenic neural crest and ultimately glia is indicative of the fact that segregation between neurogenic and non-neurogenic potential is a differentiation decision that occurs during early neural crest cell development. To characterize our mutants with respect to defects in early neural crest cell differentiation and their neurogenic contributions to the peripheral nervous system we have performed immunostaining for neurofilament (2H3) and neural specific β-tubulin (Tuj1), which broadly label the sensory ganglia and their branches as well as axons and dendrites throughout the central and peripheral nervous systems (Fig. 4a).

FIG. 4
Bright and fluorescent images of Neurofilament or β-tubulin immunostained E9.5-10.5 (a) Wild-type; (b) trex; (c) wiggable; (d) grimace; (e) palloncino; (f) mullet; (g) arco piccolo; (h) sottile tubo; (i) pacman; (j) orvieto; (k) snouty embryos. ...

E10.5 trex embryos exhibit hypoplastic trigeminal ganglia with truncated maxillary and mandibular branches (Fig. 4b; arrowhead). Tuj1 immunostaining also revealed considerable disorganization and agenesis of the facioacoustic/vestibulocochlear, glossopharyngeal, and vagal branches, reflecting the anomalies in neural crest and cranial ganglia patterning observed through Sox10 staining (Fig. 4b; arrow).

E10.5 wiggable mutants display excessive neuronal differentiation in the hindbrain of the central nervous system compared to wild-type control and considerable hypoplasia of the peripheral nervous system including defects in cranial ganglia branching (Fig. 4c; asterisks and arrowheads). Although the trigeminal, facial, and glossopharyngeal ganglia form, each appears smaller than normal and disconnected from the neural tube. The small ganglia resemble the similarly small domains of Sox10-positive neural crest cells, highlighting the correlation between neural crest cell and cranial ganglia defects in wiggable embryos.

E9.5 grimace and palloncino embryos exhibit even more severe defects in neural patterning (Fig. 4d,e). For both mutants 2H3 immunostaining revealed near complete nervous system agenesis. Except for a small amount of neurofilament staining at the isthmus (midbrain–hindbrain junction) there was no evidence of the neuronal network or plexus that typically develops in the midbrain (Fig. 4d,e; arrowhead). Defects in peripheral nervous system development were similarly extreme. Except for a couple of very discrete localized domains of neurofilament staining adjacent to the hind-brain, there was little evidence of any overt neural differentiation in association with the cranial ganglia (Fig. 4d,e; arrows). Again, the defects in neuronal development are consistent with the very limited expression of Sox10, suggesting that the failure to form the cranial sensory ganglia stems from neural crest cell defects in grimace and palloncino embryos.

Mullet embryos displayed fairly normal patterns of neural crest cell formation, migration and contribution to the cranial ganglia (Fig. 3f). However, neurofilament immunostaining in this mutant revealed defects in peripheral nervous system patterning including reduction and hypoplasia of the maxillary branch of the trigeminal ganglion (Fig. 4f; arrowhead) and discontinuity between the glosssopharyngeal ganglia and hindbrain/spinal cord (Fig. 4f; arrow).

Central and peripheral nervous system development appeared to be similarly affected in arco piccolo, sottile tubo, pacman, and orvieto mutant embryos. Each mutant exhibits only rudimentary neuronal differentiation within the cranial ganglia and a complete lack of neuronal differentiation within the midbrain (Fig. 4g–j; arrowheads). Interestingly, arco piccolo, sottile tubo and pacman each had very similar and relatively normal domains of Sox10 neural crest cell patterning (Fig. 3g–i), suggesting some commonality to the neuronal phenotype occurring at the level of neural crest cell differentiation. The ganglia in arco piccolo appear reduced and axonal branching of the trigeminal and facial ganglia is lacking. The glossopharyngeal ganglion is abnormally fused with the vagal and nerves from this combined ganglion projects ectopically towards the facial ganglion (Fig. 4g; arrows).

E10.5 orvieto and snouty mutants exhibit a pattern of neuronal differentiation almost identical to grimace and palloncino with only a single domain of neurofilament or β-tubulin staining localized to the proximal end of the first pharyngeal arch probably representing a rudimentary trigeminal ganglion (Fig. 4j,k; arrowhead). There is little evidence of neuronal differentiation caudal to the trigeminal ganglion and no evidence of any axonal branching throughout the cranial region (Fig. 4j,k arrows). Plenty of Sox10 positive neural crest cells are observed in the cranial territory prior to ganglia maturation in orvieto and snouty mutants suggesting that their defects occur at the level of neural crest cell differentiation. In the central nervous system the midbrain neuronal plexus formed in snouty embryos but is conspicuously absent in orvieto mutants.

Overall, defects in peripheral nervous system formation and maturation with respect to the cranial sensory ganglia were observed in all of the ENU-induced mutants. In many cases cranial ganglia abnormalities were reflective of earlier defects in neural crest cell development. This was not always the case, however, and it must be noted that the peripheral nervous system is derived from a combination of neural crest cells and cells of the ectodermal placodes. Whereas the hypoplasia and agenesis of the cranial ganglia observed in some of our mutants likely results from defects in neural crest cell development, in other mutants it may occur due to deficiencies in ectodermal placode patterning.

Vascular Differentiation

As described earlier, one of the most recognizable and consistent features of our ENU-generated mutant embryos was their distinct size difference compared to wild-type littermates. In addition, the majority of our ENU generated mutants are embryonic lethal by E11. The combination of small size and early embryonic lethality pointed toward cardiovascular anomalies as a mechanistic basis underpinning these phenotypes. Within the head, the circulatory system consists of a distinctive series of primary blood vessels, including the dorsal aorta (arrow) and pharyngeal arch arteries (arrowhead) together with the vascular plexus that initially forms over the midbrain and subsequently expands over other parts of the central nervous system (Fig. 5a). The vasculature is generated via two distinct processes; vasculogenesis, which is the de novo formation of new blood vessels, and angiogenesis, which describes the remodeling or branching of pre-existing blood vessels. Each process depends upon primary contributions from endothelial cells. Therefore we characterized the establishment and remodeling of the vasculature in our mutant series via immunostaining for platelet endothelial cell adhesion molecule (PECAM-1; Fig. 5a), a glycoprotein component of intercellular junctions that is expressed on the surface of endoethelial cells (Vecchi et al., 1994).

FIG. 5
Bright and fluorescent images of PECAM-1 immunostained E9.5-10.5 (a) Wild-type; (b) trex; (c) wiggable; (d) grimace; (e) palloncino; (f) mullet; (g) arco piccolo; (h) sottile tubo; (i) pacman; (j) orvieto; (k) snouty embryos. Arrowheads and arrows indicate ...

No overt vascular patterning anomalies were detected in E10.5 mullet embryos based on PECAM-1 immunostaining (Fig. 5f). Perhaps this is not surprising as mullet embryos are initially equivalent in size to their wild-type littermates at E9.5 and remain viable late into gestation (E18.5). While this is not uncommon for embryos with exencephaly and craniorachischisis, it is considerably longer than all the other mutants in our series, which are typically lethal midgestation.

For example, trex embryos, survive only until E12.5-13.5. PECAM-1 immunostaining revealed an absence of the third-sixth pharyngeal arteries in trex mutant embryos, which is likely a consequence of agenesis of the third through sixth pharyngeal arches (Fig. 5b; arrow). Compared to wild-type littermates, E10.5 trex embryos exhibit a persistent connection between the second arch artery and endocardium of the outflow tract of the heart (Fig. 5b; arrowhead). In addition, the dorsal aorta, which connects to the pharyngeal arch arteries, is considerably narrower in trex mutants compared to controls. This constellation of cardiovascular anomalies likely underpins the midgestation lethality of trex embryos.

An extreme vascular phenotype is seen in orvieto embryos, which form no blood vessels (Fig. 5j). PECAM-1 immunostaining revealed the total absence of endothelial cells, primary vessels and secondary capillaries in E8.5-10.0 embryos in orvieto mutants along with failure in chorioallantoic fusion. These defects no doubt contribute to the small size of orvieto embryos which are typically only half the size of their E9.5 wild-type littermates. Collectively these major anomalies in vasculogenesis are terminal for orvieto embryo development during early gestation.

In contrast to orvieto embryos, which are devoid of any vasculature, grimace, palloncino, arco piccolo, sottile tubo, pacman, and snouty mutants each do generate a circulatory system (Fig. 5d,e,g–i,k) but the vessels formed exhibit varying anomalies in vascular remodeling suggestive of distinct alterations in the balance between vasculogenesis and angiogenesis. For example, the pharyngeal arch arteries in arco piccolo, palloncino, and pacman embryos are variable in size compared to controls (Fig. 5e,g,i; arrowhead). In arco piccolo each of the arch arteries appear thinner or constricted as opposed to palloncino and pacman, which exhibit dilated rostral arch arteries. Mutant grimace and palloncino embryos also lack caudal pharyngeal arch arteries, consistent with the pharyngeal arch agenesis observed in these lines (Fig. 5d,e; arrows). Additionally, they display diminished cranial vascular plexi. Embryos of the pacman line exhibit a reduction in microvasculature and arco piccolo and sottile tubo embryos display more constricted capillaries or microvascular networks (Fig. 5g–i arrows). The extent of vascular remodeling anomalies in snouty embryos is intermediate between those described above (Fig. 5k). Clearly anomalies in vascular remodeling significantly affect cardiovascular function and dramatically impacts upon embryo growth and viability. This is the likely cause of the migestational lethality observed in many mutants of our series.

Since remodeling of the vasculature of the arch arteries is integral to the normal structure and morphogenesis of the pharyngeal arches, it is perhaps not surprising to find an association between anomalies in vascular remodeling and defects in pharyngeal arch and consequently craniofacial development. This is exemplified in wiggable mutant embryos, which are typically larger than their wild-type littermates. E9.5-10.5 wig-gable embryos exhibit enhanced PECAM-1 staining compared to wild-type controls, indicative of a marked increase in endothelial cells and vasculature formation (Fig. 6c; arrow). This likely provides an early growth advantage to wiggable embryos and can account for their enlarged size and alterations in pharyngeal arch development. However, wiggable embryos do not survive beyond E11.5 likely because of abnormalities in cardiovascular function. Thus embryo morphogenesis and viability is compromised by insufficient as well as excessive vasculature together with abnormal vascular remodeling.

FIG. 6
Schematic representation of the spatial distribution of 140 microsatellite markers (red) that are distinct between C57BL/6;129S1/Sv and FVB/NJ and were used to map the gross chromosomal location of mutations in each of the ENU generated mouse lines. The ...

Mutation Identification and Affected Signaling Pathways

The goal of our screen was to identify new genes that play critical roles in craniofacial development. trex embryos, which exhibit orofacial clefts, abnormal neural crest cell development and pharyngeal and vascular anomalies serve as an example illustrating the success of our unbiased ENU mutagenesis screen in identifying novel alleles that regulate craniofacial development. Using a panel of microsatellite markers (see Fig. 6) polymorphic between the predominantly C57BL/6 and residual 129/Sv mixed background of our mutagenised males versus the FVB/NJ strain to which they were bred, we mapped the genomic interval associated with the trex phenotype initially to the proximal region of chromosome 1 (see Fig. 7). Refining this region using single nucleotide polymorphisms followed by sequencing of candidate genes, we determined that the trex phenotype was caused by a T to C mutation at position 251 in the retinol dehydrogenase 10 (Rdh10) gene (Sandell et al., 2007). Rdh10 is an enzyme of the short chain dehydrogenase/reductase (SDR) superfamily and we have previously demonstrated that Rdh10 is a critical regulator of vitamin A metabolism and plays an important role in the spatiotemporal specificity of retinoic acid signaling (Sandell et al., 2007).

FIG. 7
Definitive chromosomal position of mutations identified in individual ENU mutant lines or the refined chromosomal region to which they have been currently been mapped. Arrowheads indicate the precise chromosome position of the mutation identified in ...

Retinoic acid plays essential roles in patterning and morphogenesis of the developing embryo. Insufficient or excessive retinoic acid levels are known to cause abnormalities in pharyngeal, craniofacial, and vascular development (Clagett-Dame and DeLuca, 2002; Lai et al., 2003). Our discovery of the trex mutant uncovered Rdh10 as a new nodal point in vitamin A metabolism and retinoid synthesis during embryogenesis. Importantly trex mutants also demonstrate the integral relationship that exists between neural crest, pharyngeal arch and vascular development during embryogenesis and the importance of retinoid signaling in each of these processes.

A number of our ENU induced mutants exhibit similar pharyngeal arch and vascular anomalies suggesting we may have uncovered multiple alleles that affect the same signaling pathway and processes. This is true with respect to retinoid signaling. Using the same mapping strategy as for identification of the Rdh10trex mutation, we determined that the causal defect in grimace was a mutation in Aldh1a2 (see Fig. 7), a gene encoding an enzyme required for the final oxidation step in metabolism of Vitamin A into retinoic acid (Zhao et al., 1996). Specifically we identified a T to C base pair change in intron 4 of Aldh1a2 which disrupts splicing and leads to premature termination of Aldh1a2 expression and thereby to loss of retinoid signaling. Perhaps not surprisingly grimace and trex embryos share considerable similarities in pharyngeal arch and arch artery agenesis as well as defects in vascular remodeling. Identification of mutations in the two enzymes responsible for the oxidation of Vitamin A to retinoic acid highlights the importance of Vitamin A metabolism in craniofacial development and now allows for analyses of the metabolic regulation of Vitamin A oxidation and retinoid signaling.

The identification of mutations in retinoid signaling that impact neural crest, pharyngeal arch, craniofacial, and vascular development implies there is a developmental interdependency between these processes in craniofacial development. Notch signaling is another pathway that is known to regulate both neural crest development and vasculogenesis (Krebs et al., 2000). Consistent with the known importance of Notch signaling we identified sottile tubo as a mutation in Notch1 (see Fig. 7). Specifically we found a non-sense T to A base change which creates an early stop codon and is predicted to truncate the normally 2531 amino acid Notch1 protein to 84 amino acids.

Mutant sottile tubo embryos exhibit fairly normal primary vessel formation, however, there is a considerable deficiency in microvasculature or capillary formation. Notch1 is expressed in endothelial cells and arteries, but not in veins (Villa et al., 2001) suggesting the sottile tubo mutants have a defect in angiogenesis but not vasculogenesis, consistent with the phenotype observed in Notch1 knockout mice (Krebs et al., 2000). Notch signaling has been shown to influence neural crest cell differentiation into smooth muscle cells (High et al., 2007), a process that is critical for normal cardiovascular function. This again highlights the importance of interactions between neural crest cells and endothelial cells during normal vascular development and also that these processes are regulated by common signaling pathways, in this case Notch.

To date, our screen for novel alleles impacting early craniofacial morphogenesis has identified mutations that affect key signaling pathways such as retinoic acid and Notch. Both of these pathways regulate neural crest and vascular development suggesting an unexplored interdependency between neural crest cells and endothelial cell-derived vasculature during craniofacial morphogenesis. Further mapping and identification of the causal mutations in our novel mouse lines may reveal other signaling pathways required for both neural and vascular development. Thus the mapping of all other mutants in our ENU screen remains ongoing and to date, wiggable has been linked to chromosome 13qB2-qB3; palloncino maps to the medial region of chromosome 2, mullet lies between 88.44 and 91.06 Mb on chromosome 5; arco piccolo maps to the proximal region of chromosome 12; pacman has not yet been linked to a single chromosome; orvieto lies between 28.4 Mb (D5Mit201.1) and 54.6 Mb (D5Mit425.1) on chromosome 5 and snouty maps to the proximal region of chromosome 10 (see Fig. 7). The application of next generation sequencing and other genomic approaches should accelerate identification of the precise lesion underlying each of the mutant lines that remain to be mapped.


ENU-induced mutagenesis provides an unbiased forward genetic approach for identifying novel alleles important for embryogenesis. We performed a morphology-based screen aimed at uncovering new alleles playing functionally important roles in early craniofacial development. We successfully recovered 10 mutant lines that exhibited craniofacial anomalies such as exencephaly and craniorachischisis; midfacial clefting; hypoplasia, hyperplasia, and agenesis of the frontonasal prominences and pharyngeal arches; anophthalmia and ventral eye defects; supernumerary otocysts; defects in vasculogenesis, angiogenesis, and vascular remodeling; cranial ganglia agenesis and hypoplasia together with defective branching or outgrowth; and aberrant neural crest cell formation, migration and differentiation. Phenotype-driven ENU screens can also generate mutations in genes that model loss-of-function human syndromes and are thus extremely powerful in helping to understand the complexities of congenital disease. We identified trex as a mutation in Rdh10. trex mutant mice display midfacial clefting, pharyngeal arch agenesis, defects in pharyngeal arch artery remodeling, and vasculogenesis as well as anomalies in organ development. This combination of anomalies mimics those observed in patients with Velocardiofacial and DiGeorge syndromes (Stevens et al., 1990). Subsequently, we have demonstrated that Rdh10 is a critical regulator of vitamin A metabolism and retinoid signaling (Sandell et al., 2007). Thus trex mice can serve as a useful model for understanding both the role of Vitamin A signaling and the importance of neural crest and endothelial interactions in the pathogenesis of Velocardiofacial and DiGeorge syndromes.

Similarly, mutations in genes that affect Notch signaling are associated with congenital disorders, including Alagille syndrome (Li et al., 1997; Oda et al., 1997), which is characterized by anomalies in craniofacial shape, skeletogenesis and organ development and CADASIL (Joutel et al., 1996) which is a product of the progressive degeneration of smooth muscle cells. sottile tubo embryos exhibit defects in angiogenesis and neural and cranial ganglia differentiation. Therefore as a new mutant allele of Notch1, sottile tubo could be used to model aspects of the etiology and pathogenesis of Alagille syndrome and CADASIL. Even mutants for which we have not yet identified the causative gene can still provide new models of congenital anomalies. For example mullet embryos exhibit exencephaly and craniorachischisis and the region of chromosome 5 to which we have putatively linked the mullet mutation has not been previously associated with open neural tube defects. Thus, the mullet mutation occurs in a novel gene involved in neural tube closure and skull development.

Most of our ENU-induced craniofacial mutants were embryonic lethal during early to midgestation and were generally small in size compared to controls. They also exhibited defects in neural crest cell patterning, pharyngeal arch development, and vasculogenesis. Despite their different embryonic origins, both neural crest cells and endothelial cell-derived vasculature (in the form of arch arteries) contribute structurally to the proper formation and patterning of the pharyngeal arches, structures which subsequently give rise to major components of the craniofacial complex. Consistent with the idea that craniofacial anomalies do not always arise as a consequence of primary defects within the neural crest, two of the mutations we have identified—Rdh10trex and Notch1sottile tubo—are in genes not expressed in neural crest cells. Interestingly both Rdh10 and Notch1 are expressed in mesoderm cells that give rise to endothelial cells and vasculature. These mutations underscore the critical role of vasculature development in patterning early craniofacial morphogenesis. Moreover, they reinforce the idea that defects in cranial mesoderm play an important role in the pathogenesis of congenital craniofacial malformations and provide models for exploring interactions between endothelial cells and neural crest cells in patterning neural crest, pharyngeal arch and craniofacial development.

Although, interdependency of neural crest and endothelial progenitor cells has yet to be explored, it is well established that their derivative neural and vascular networks are functionally interdependent. Arteries supply neurons with oxygenated blood and nerves control vessel dilation and contraction. Furthermore, blood vessels and nerves often run in parallel (Carmeliet and Tessier-Lavigne, 2005). Recently it has become clear that neuronal and vascular morphogenesis is not just tightly interwoven at the tissue level but also at the molecular level. At least four major axon guidance molecule families (Eph/Ephrin, Neuropilin/Sempaphorins, Slit/Robo and Netrin), which are widely expressed in multiple cell types have been implicated in angiogenesis (Freitas et al., 2008; Legg et al., 2008; Schwarz et al., 2008; Suchting et al., 2005; Wang et al., 1998). Conversely, some of the same signaling pathways used during vasculogenesis have recently been implicated in patterning the nervous system. For example, VEGF signaling is well known as a promoter of angiogenesis. Endothelial tip cells migrate toward higher concentrations of VEGF, and stalk cells proliferate in response to high VEGF concentrations (Gerhardt et al., 2003). However, in vitro studies have indicated that VEGF signaling can also mediate cortical neuron proliferation (Jin et al., 2002). Thus neural crest and endothelial progenitor cell interdependency combined with shared signaling cascades could facilitate functional integration between the neuronal and vascular systems both of which are essential for craniofacial development and organism survival.

In support of this idea, endothelial cells have previously been shown to provide neurotrophic factors that promote the recruitment, growth and survival of neural precursors (Leventhal et al., 1999). Furthermore, endothelial cells are known to modulate astrocyte proliferation and differentiation in brain capillaries (Estrada et al., 1990; Mi et al., 2001). Endothelial cells also maintain a microenvironment that allows for active neurogenesis in the hippocampus throughout adult life (Palmer et al., 2000). Thus there are critical requirements for endothelial cell signaling in neural development during both early and late neurogenesis. However, it is not currently known whether endothelial cells and the vascular network influence neural crest development by playing roles in regulating neural crest cell formation, migration and/or differentiation during craniofacial development. Nonetheless, a recent screen for novel factors involved in neural crest cell induction, uncovered at least eight genes that had previously been implicated in endothelial cell development (Gammill and Bronner-Fraser, 2002). These included factors involved in VEGF production and signaling (e.g., ORP150 or neuropilin 2a1) as well as proteins important for endothelial cell migration (such as laminin α5 and γ1). Interestingly, VEGF mutant embryos exhibit poorly developed branchial arches (Ferrara et al., 1996). VEGF itself is expressed in tissues that could affect neural crest development, including the head-folds, neural tube and cephalic mesenchyme of E8.5-9.0 mouse embryos (Miquerol et al., 1999). The orvieto mouse mutant isolated in our screen also points toward interdependent neural crest-endothelial cell interactions. orvieto embryos exhibit complete vascular agenesis and a loss of migrating neural crest cells caudal to the otocyst. Additionally, cranial neural crest cells in orvieto embryos fail to colonize the second arch. Consistent with these gross neural crest anomalies only a rudimentary trigeminal ganglion forms which fails to undergo axon branching or outgrowth.

In summary, our ENU screen has successfully identified a number of novel alleles that are critically required for early craniofacial development and embryo survival which will be of value to the craniofacial community. Furthermore, the data we have obtained from our mutants to date, provocatively implies that endothelial cells and neural crest cells may employ similar developmental programs and be interdependent during early embryogenesis which collectively is critical for normal craniofacial morphogenesis.


ENU Mutagenesis

Our screen for recessive mutations was modeled after standard three generation screens performed in other laboratories (Justice et al., 1999; Kasarskis et al., 1998). Mice were housed in the Laboratory Animal Services Facility at the Stowers Institute for Medical Research according to IACUC animal welfare guidelines. To induce mutations, essentially congenic C57BL/6 (but with potentially miniscule 129S1/Sv background remnants) male mice of 8–12 weeks of age were injected intraperitoneally once per week for 3 weeks with a 100 mg kg−1 dose of ENU as previously described (Justice et al., 2000). These mutagenized males were designated generation 0 (G0) and 90 days later were bred to wild-type FVB/NJ female mice. The G0 males that regained fertility produced animals heterozygous for the induced mutations. Individual males of this first intercross generation were considered founder animals of individual mutant lines (F1). Founder males were crossed with FVB/NJ females to produce generation 2 (G2). G2 females were backcrossed to their fathers, and their litters were examined at E9.5-10.5 for morphological defects to identify lines that yielded reproducible craniofacial abnormalities such as hypoplastic or absent facial prominences and pharyngeal arches. For embryo collection the day of plug observation was noted as 0.5 days post coitum (dpc). Embryos were fixed and yolk sacs were collected for genotyping. From 50 founder (F1) males, 10 recessive mutant lines were generated that exhibited consistent and reproducible (within litters and between litters) abnormal craniofacial morphology. Individual mutant lines were maintained by backcrossing heterozygous carrier animals to FVB/NJ.


Since mutant lines were maintained by backcrossing to FVB/NJ, the chromosomal position of each mutation could be mapped by identification of the region of C57BL/6 or 129 genome that co-segregated with the mutant phenotype. To that end we utilized a panel of microsatellite markers that were polymorphic between the mixed C57BL/6 × 129 background and FVB/NJ strains (see Fig. 6). For each microsatellite marker, an FVB/NJ allele could be unequivocally distinguished from a C57BL/6 or 129 allele. Each chromosome of the mouse genome with the exception of the Y chromosome was represented by several microsatellite markers. PCR reactions were multiplexed such that 140 microsatellite markers could be analyzed in 48 individual reactions. Fluorescently labeled primers for PCR amplification of microsatellite markers were purchased as a set from ABI (Mouse Mapping Primers version 1.0). Of the 314 markers available, 140 were used for mapping polymorphisms between the C57BL6 mutagenized strain background and the FVB backcross breeding strain. PCR products were analyzed with a 3730 DNA Analyzer and GeneMapper software. Yolk sac DNA samples from phenotypically mutant and control embryos and ear biopsy DNA samples from carrier parent animals were screened with the microsatellite panel to determine the region of the C57BL/6 genome that was genetically linked to the mutant phenotype. Once a genomic interval associated with the mutant phenotype was mapped to a single chromosomal location, the region was then refined using selected single-nucleotide polymorphisms and individuals were genotyped using a procedure known as derived cleaved amplified polymorphic sequences (Neff et al., 2002). When the genomic interval was narrowed to the extent it spanned a manageable number of genes, transcripts from prioritized candidate genes were amplified and sequenced in order to identify alterations from wild type.


For wholemount immunostaining with the neuronal markers Neurofilament (2H3; Developmental Studies Hybridoma Bank) and Neuronal Class III β-tubulin (Tuj1; Covance) embryos were fixed in 4% PFA overnight at 4°C. For whole-mount immunostaining with the endothelial marker PECAM-1 (platelet endothelial cell adhesion molecule; BD Biosciences), embryos were fixed in 4% PFA for only 1–2 h. Post fixation, embryos were dehydrated in a graded methanol series. Dehydrated embryos were then bleached in methanol:DMSO:30% H2O2 (4:1:1; Dent’s Bleach) for 2–5 h at RT and rehydrated in 75%methanol:PBS, 50% methanol:PBS, 15% methanol:PBS and PBS for 30 min each. Embryos were then blocked in PBSMT (2% milk powder, 0.1% Triton X-100 in PBS) or 3%BSA in PBS twice for 1 h at RT, prior to incubation in primary antibodies (2H3 1:250; Tuj1 1:1,000; PECAM 1:500) overnight at 4°C in PBSMT. Embryos were then repeatedly rinsed in PBSMT, (5 × 1 h) and incubated in either an HRP-coupled secondary (1:200 dilution in PBSMT) antibody or a secondary Alexafluor antibody (1:300–500 dilution in PBSMT or 3% BSA in PBS) and kept in the dark. For HRP color development, embryos were rinsed in PBSMT and PBT (0.2% BSA, Sigma, 0.1% Triton X-100) three times for 20 min each at room temperature and washed in 3,3-diaminobenzidine (0.3 mg ml−1) (Sigma, St. Louis, MO) in PBT for 20 min. H2O2 (0.03%) was added and the color was developed to the desired intensity and examined via bright field microscopy. For fluorescence development, embryos were washed three times in PBSMT for 5 min each, followed by three 1 h washes in PBSMT. Embryos were counterstained directly with DAPI (1:1,000) or by immersion in Vecta-shield with DAPI to label the nucleus of individual cells. Embryos were cleared in BABB (1:1 benzyl acetate: benzyl benzoate) or glycerol and then visualized via fluorescence and confocal microscopy.

RNA In Situ Hybridization

Embryos were collected between E9.5-11.5 as described above, fixed overnight in 4% paraformaldehyde (PFA) at 4°C, dehydrated in methanol, and stored at −20°C until used in the staining protocol. Anti-sense digoxigenin-labeled mRNA riboprobes were synthesized for Sox10 and in situ hybridizations were performed following a previously described standard protocol (Nagy et al., 2003).


Contract grant sponsor: National Institute of Dental and Craniofacial Research Grant, Contract grant number: R01 DE 016082, Contract grant sponsor: Stowers Institute for Medical Research

The authors are indebted to Monica Justice and Andy Salinger for their generous help and advice in initiating the screen and are immensely grateful to Teresa Terrel, Rodney McKay, Michael Morgan, Lacey Ellington, and Melissa Childers for their technical expertise and help in caring for and maintaining our mouse lines. They also thank Karen Staehling and Brian Sanderson for assistance with microsatellite and SNP mapping as well as sequencing of candidate genes. The neurofilament (2H3) antibody developed by Jessell and Dodd was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242.


  • Baker CV, Bronner-Fraser M. Vertebrate cranial placodes I. Embryonic induction. Dev Biol. 2001;232:1–61. [PubMed]
  • Carmeliet P, Tessier-Lavigne M. Common mechanisms of nerve and blood vessel wiring. Nature. 2005;436:193–200. [PubMed]
  • Clagett-Dame M, DeLuca HF. The role of vitamin A in mammalian reproduction and embryonic development. Annu Rev Nutr. 2002;22:347–381. [PubMed]
  • D’Amico-Martel A, Noden D. Contributions of placodal and neural crest cells to avian cranial peripheral ganglia. Am J Anat. 1983;166:445–468. [PubMed]
  • Depew MJ, Simpson CA. 21st century neontology and the comparative development of the vertebrate skull. Dev Dyn. 2006;235:1256–1291. [PubMed]
  • Dixon J, Jones NC, Sandell LL, Jayasinghe SM, Crane J, Rey JP, Dixon MJ, Trainor PA. Tcof1/Treacle is required for neural crest cell formation and proliferation deficiencies that cause craniofacial abnormalities. Proc Natl Acad Sci USA. 2006;103:13403–13408. [PubMed]
  • Estrada C, Bready JV, Berliner JA, Pardridge WM, Cancilla PA. Astrocyte growth stimulation by a soluble factor produced by cerebral endothelial cells in vitro. J Neuropathol Exp Neurol. 1990;49:539–549. [PubMed]
  • Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, Powell-Braxton L, Hillan KJ, Moore MW. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996;380:439–442. [PubMed]
  • Freitas C, Larrivee B, Eichmann A. Netrins and UNC5 receptors in angiogenesis. Angiogenesis. 2008;11:23–29. [PubMed]
  • Gammill LS, Bronner-Fraser M. Genomic analysis of neural crest induction. Development. 2002;129:5731–5741. [PubMed]
  • Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol. 2003;161:1163–1177. [PMC free article] [PubMed]
  • Gorlin RJ, Cohen MM, Levin LS. Syndromes of the head and neck. Oxford, UK: Oxford University Press; 1990.
  • High FA, Zhang M, Proweller A, Tu L, Parmacek MS, Pear WS, Epstein JA. An essential role for Notch in neural crest during cardiovascular development and smooth muscle differentiation. J Clin Invest. 2007;117:353–363. [PMC free article] [PubMed]
  • Jin K, Zhu Y, Sun Y, Mao XO, Xie L, Greenberg DA. Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc Natl Acad Sci USA. 2002;99:11946–11950. [PubMed]
  • Jones NC, Lynn ML, Gaudenz K, Sakai D, Aoto K, Rey JP, Glynn EF, Ellington L, Du C, Dixon J, Dixon MJ, Trainor PA. Prevention of the neurocristopathy Treacher Collins syndrome through inhibition of p53 function. Nat Med. 2008;14:125–133. [PMC free article] [PubMed]
  • Joutel A, Corpechot C, Ducros A, Vahedi K, Chabriat H, Mouton P, Alamowitch S, Domenga V, Cecillion M, Marechal E, Maciazek J, Vayssiere C, Cruaud C, Cabanis EA, Ruchoux MM, Weissenbach J, Bach JF, Bousser MG, Tournier-Lasserve E. Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature. 1996;383:707–710. [PubMed]
  • Justice MJ, Carpenter DA, Favor J, Neuhauser-Klaus A, Hrabe de Angelis M, Soewarto D, Moser A, Cordes S, Miller D, Chapman V, Weber JS, Rinchik EM, Hunsicker PR, Russell WL, Bode VC. Effects of ENU dosage on mouse strains. Mamm Genome. 2000;11:484–488. [PubMed]
  • Justice MJ, Noveroske JK, Weber JS, Zheng B, Bradley A. Mouse ENU mutagenesis. Hum Mol Genet. 1999;8:1955–1963. [PubMed]
  • Kasarskis A, Manova K, Anderson KV. A phenotype-based screen for embryonic lethal mutations in the mouse. Proc Natl Acad Sci USA. 1998;95:7485–7490. [PubMed]
  • Kontges G, Lumsden A. Rhombencephalic neural crest segmentation is preserved throughout craniofacial ontogeny. Development. 1996;122:3229–3242. [PubMed]
  • Krebs LT, Xue Y, Norton CR, Shutter JR, Maguire M, Sundberg JP, Gallahan D, Closson V, Kitajewski J, Callahan R, Smith GH, Stark KL, Gridley T. Notch signaling is essential for vascular morphogenesis in mice. Genes Dev. 2000;14:1343–1352. [PubMed]
  • Lai L, Bohnsack BL, Niederreither K, Hirschi KK. Retinoic acid regulates endothelial cell proliferation during vasculogenesis. Development. 2003;130:6465–6474. [PubMed]
  • Le Douarin N, Kalcheim C. The neural crest. Cambridge University Press; 1999.
  • Legg JA, Herbert JM, Clissold P, Bicknell R. Slits and roundabouts in cancer, tumour angiogenesis and endothelial cell migration. Angiogenesis. 2008;11:13–21. [PubMed]
  • Leventhal C, Rafii S, Rafii D, Shahar A, Goldman SA. Endothelial trophic support of neuronal production and recruitment from the adult mammalian subependyma. Mol Cell Neurosci. 1999;13:450–464. [PubMed]
  • Li L, Krantz ID, Deng Y, Genin A, Banta AB, Collins CC, Qi M, Trask BJ, Kuo WL, Cochran J, Costa T, Pierpont ME, Rand EB, Piccoli DA, Hood L, Spinner NB. Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nat Genet. 1997;16:243–251. [PubMed]
  • Mi H, Haeberle H, Barres BA. Induction of astrocyte differentiation by endothelial cells. J Neurosci. 2001;21:1538–1547. [PubMed]
  • Miquerol L, Gertsenstein M, Harpal K, Rossant J, Nagy A. Multiple developmental roles of VEGF suggested by a LacZ-tagged allele. Dev Biol. 1999;212:307–322. [PubMed]
  • Morriss-Kay GM, Wilkie AO. Growth of the normal skull vault and its alteration in craniosynostosis: Insights from human genetics and experimental studies. J Anat. 2005;207:637–653. [PubMed]
  • Nagy A, Gertsenstein M, Vintersten K, Behringer RR. Manipulating the mouse embryo. Cold Spring Harbor: Cold Spring Harbor Laboratory; 2003.
  • Neff MM, Turk E, Kalishman M. Web-based primer design for single nucleotide polymorphism analysis. Trends Genet. 2002;18:613–615. [PubMed]
  • Noden DM. Patterns and organization of craniofacial skeletogenic and myogenic mesenchyme: A perspective. Prog Clin Biol Res. 1982;101:167–203. [PubMed]
  • Noden DM. The role of the neural crest in patterning of avian cranial skeletal, connective, and muscle tissues. Devl Biol. 1983a;96:144–165. [PubMed]
  • Noden DM. The embryonic origins of avian cephalic and cervical muscles and associated connective tissues. Am J Anat. 1983b;168:257–276. [PubMed]
  • Noden DM. Cell movements and control of patterned tissue assembly during craniofacial development. J Craniofac Genet Dev Biol. 1991;11:192–213. [PubMed]
  • Oda T, Elkahloun AG, Pike BL, Okajima K, Krantz ID, Genin A, Piccoli DA, Meltzer PS, Spinner NB, Collins FS, Chandrasekharappa SC. Mutations in the human Jagged1 gene are responsible for Alagille syndrome. Nat Genet. 1997;16:235–242. [PubMed]
  • Palmer TD, Willhoite AR, Gage FH. Vascular niche for adult hippocampal neurogenesis. J Comp Neurol. 2000;425:479–494. [PubMed]
  • Pingault V, Bondurand N, Kuhlbrodt K, Goerich DE, Prehu MO, Puliti A, Herbarth B, Hermans-Borgmeyer I, Legius E, Matthijs G, Amiel J, Lyonnet S, Ceccherini I, Romeo G, Smith JC, Read AP, Wegner M, Goossens M. SOX10 mutations in patients with Waardenburg-Hirschsprung disease. Nat Genet. 1998;18:171–173. [PubMed]
  • Sandell LL, Sanderson BW, Moiseyev G, Johnson T, Mushegian A, Young K, Rey JP, Ma JX, Staehling-Hampton K, Trainor PA. RDH10 is essential for synthesis of embryonic retinoic acid and is required for limb, craniofacial, and organ development. Genes Dev. 2007;21:1113–1124. [PubMed]
  • Schwarz Q, Vieira JM, Howard B, Eickholt BJ, Ruhrberg C. Neuropilin 1 and 2 control cranial gangliogenesis and axon guidance through neural crest cells. Development. 2008;135:1605–1613. [PMC free article] [PubMed]
  • Southard-Smith EM, Kos L, Pavan WJ. Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model. Nat Genet. 1998;18:60–64. [PubMed]
  • Stevens CA, Carey JC, Shigeoka AO. Di George anomaly and velocardiofacial syndrome. Pediatrics. 1990;85:526–530. [PubMed]
  • Suchting S, Heal P, Tahtis K, Stewart LM, Bicknell R. Soluble Robo4 receptor inhibits in vivo angiogenesis and endothelial cell migration. FASEB J. 2005;19:121–123. [PubMed]
  • Trainor PA, Tan SS, Tam PP. Cranial paraxial mesoderm: Regionalisation of cell fate and impact on craniofacial development in mouse embryos. Development. 1994;120:2397–2408. [PubMed]
  • Trainor PA, Tam PPL. Cranial paraxial mesoderm and neural crest of the mouse embryo-codistribution in the craniofacial mesenchyme but distinct segregation in the branchial arches. Development. 1995;121:2569–2582. [PubMed]
  • Trainor P, Krumlauf R. Patterning the cranial neural crest: Hindbrain segmentation and Hox gene plasticity. Nat Rev Neurosci. 2000;1:116–124. [PubMed]
  • Trainor PA, Krumlauf R. Hox genes, neural crest cells and branchial arch patterning. Curr Opin Cell Biol. 2001;13:698–705. [PubMed]
  • Vecchi A, Garlanda C, Lampugnani MG, Resnati M, Matteucci C, Stoppacciaro A, Schnurch H, Risau W, Ruco L, Mantovani A, Dejana E. Monoclonal antibodies specific for endothelial cells of mouse blood vessels. Their application in the identification of adult and embryonic endothelium. Eur J Cell Biol. 1994;63:247–254. [PubMed]
  • Villa N, Walker L, Lindsell CE, Gasson J, Iruela-Arispe ML, Weinmaster G. Vascular expression of Notch pathway receptors and ligands is restricted to arterial vessels. Mech Dev. 2001;108:161–164. [PubMed]
  • Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell. 1998;93:741–753. [PubMed]
  • Zhao D, McCaffery P, Ivins KJ, Neve RL, Hogan P, Chin WW, Drager UC. Molecular identification of a major retinoic-acid-synthesizing enzyme, a retinaldehyde-specific dehydrogenase. Eur J Biochem. 1996;240:15–22. [PubMed]