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 ). The names ascribed to each mutant reflect their distinctive individual features; trex -short forelimbs typical of dinosaur T.rex (); wiggable—excessive leaf-like laminae or folia in the brain resembling a wig (); grimace—contorted facial expression (); palloncino—Italian for balloon, reflecting inflated vasculature (); mullet—exencephaly and craniorachischisis resembling embryo with 80s hair style (); arco piccolo—Italian for small (pharyngeal) arch (); sottile tubo—Italian for thin (neural) tube (); pacman—open gaping mouth (); orvieto—Italian city known for dry white wine, reflecting appearance of embryos due to absence of blood cells and vasculature (); snouty—shortened frontonasal region (). 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 (more ...)
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 (; 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 (). 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. (more ...)
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 ( and ). 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 ( and ; 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 ( and ; 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.
embryos also exhibited agenesis of the caudal third and fourth pharyngeal arches (; 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 ( and ; 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
embryos similarly displayed abnormal pharyngeal hinge morphology in the form of a modest cleft due to hypoplasia of both the maxillary and mandibular prominences ( and ; 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 ( and ; 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 ( and ; 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 ( and ; 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 (; 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 ( and ; 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 (). Only minor or subtle reductions in pharynegeal arch size were observed in E10.5 mullet embryos ().
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 (; 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 (; 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 (). 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 (; 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 (). 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. 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, (more ...)
Sox10 in situ hybridization revealed considerable neural crest cell patterning anomalies in each of the ENU-induced mutants between E9.5-10.5 (see ). 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 (; 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 (; 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 (; 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 (; 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 (; asterisk). Caudal to the otocyst, the cranial ganglia are hypoplastic or absent (; 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 (; 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 (; 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 (; 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 (). 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 (; 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 ().
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. 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. (more ...)
E10.5 trex embryos exhibit hypoplastic trigeminal ganglia with truncated maxillary and mandibular branches (; 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 (; 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 (; 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 (). 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 (; 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 (; 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 (). 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 (; arrowhead) and discontinuity between the glosssopharyngeal ganglia and hindbrain/spinal cord (; 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 (; arrowheads). Interestingly, arco piccolo, sottile tubo and pacman each had very similar and relatively normal domains of Sox10 neural crest cell patterning (), 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 (; 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 (; arrowhead). There is little evidence of neuronal differentiation caudal to the trigeminal ganglion and no evidence of any axonal branching throughout the cranial region ( 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.
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 (). 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; ), 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 (more ...)
No overt vascular patterning anomalies were detected in E10.5 mullet embryos based on PECAM-1 immunostaining (). 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 (; 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 (; 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 (). 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 () 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 (; 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 (; 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 ( arrows). The extent of vascular remodeling anomalies in snouty embryos is intermediate between those described above (). 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 (; 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 (more ...)
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 ) 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 ). 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
) gene (Sandell et al., 2007
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 (more ...)
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 ), 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
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 ). 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 ). 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.