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Hum Mol Genet. Apr 15, 2010; 19(8): 1577–1592.
Published online Jan 27, 2010. doi:  10.1093/hmg/ddq030
PMCID: PMC2846163

A primary cilia-dependent etiology for midline facial disorders

Abstract

Human faces exhibit enormous variation. When pathological conditions are superimposed on normal variation, a nearly unbroken series of facial morphologies is produced. When viewed in full, this spectrum ranges from cyclopia and hypotelorism to hypertelorism and facial duplications. Decreased Hedgehog pathway activity causes holoprosencephaly and hypotelorism. Here, we show that excessive Hedgehog activity, caused by truncating the primary cilia on cranial neural crest cells, causes hypertelorism and frontonasal dysplasia (FND). Elimination of the intraflagellar transport protein Kif3a leads to excessive Hedgehog responsiveness in facial mesenchyme, which is accompanied by broader expression domains of Gli1, Ptc and Shh, and reduced expression domains of Gli3. Furthermore, broader domains of Gli1 expression correspond to areas of enhanced neural crest cell proliferation in the facial prominences of Kif3a conditional knockouts. Avian Talpid embryos that lack primary cilia exhibit similar molecular changes and similar facial phenotypes. Collectively, these data support our hypothesis that a severe narrowing of the facial midline and excessive expansion of the facial midline are both attributable to disruptions in Hedgehog pathway activity. These data also raise the possibility that genes encoding ciliary proteins are candidates for human conditions of hypertelorism and FNDs.

INTRODUCTION

Craniofacial abnormalities comprise approximately one-third of all birth defects and of those developmental defects of the forebrain and midface, such as holoprosencephaly (HPE), are the most common (1,2). Although the phenotypic presentation of HPE is variable, HPE is associated with a distinct facial ‘gestalt’, a reduced facial midline. The most extreme cases of HPE are characterized by a complete collapse of the facial midline, such as cyclopia and the congenital absence of a mature nose. Less severe forms of HPE feature close-set eyes (hypotelorism), defects of the upper lip and nose, and variable central nervous system (CNS) defects (reviewed in 36).

In contrast to defects associated with a reduced facial midline, there is a second class of midline disorders that are associated with an expanded facial midline. The most extreme cases of midline expansion result in craniofacial duplication, or diprosopus. The phenotype comprises a wide spectrum and ranges from partial duplication of a few facial structures to complete dicephalus (7). Less severe forms of midline expansion are the hallmark of syndromes like frontonasal dysplasia (FND) in which a broad nasal root, medial clefting and extreme ocular hypertelorism are common (8). Although the phenotypic presentation of syndromes such as FND is variable, hypertelorism and an expanded facial midline are the characteristic features.

The molecular basis for midline disorders has been the subject of intense scrutiny. For syndromes associated with midline collapse loss-of-function mutations in the Hedgehog signaling pathway are common, but no direct correlations have been uncovered between genotype and phenotype (9). Some evidence suggests a relationship between the timing of Hedgehog disruption, and the severity of the HPE phenotype (10,11), and other genetic studies indicate a dose-dependent relationship between pathway activity and the extent to which the facial midline is reduced (12,13).

Understanding the genetic basis for midline expansions has not been as straightforward. Numerous syndromes characterized by midline expansion including, frontorhiny, craniofrontonasal syndrome and FND have been attributed to loss of Alx3 (14), mutations in Ephrin-B1 (15) and defects in Six2 (16), respectively. Loss of the Hedgehog repressor Gli3 has been linked to Greig cephalopolysyndactyly (a syndrome characterized by hypertelorism) (17), and introduction of ectopic Hedgehog has been shown to increase the width of the midline facial prominence (frontonasal prominence) in an avian model (18). Our own studies have demonstrated that Wnt pathway activity is important for midline patterning (19); however, it was the loss of Wnt dependent proliferation in the maxillary prominence that permitted the expansion of the midline.

To gain insight into the mechanisms that produce midline expansion, we opted for an approach that, rather than disrupting any member of a signaling pathway such as Wnt or Hh, disrupted the ability of the cell to respond to signals in their environment. For this, we exploited an ubiquitous organelle, the primary cilia. Primary cilia have been reported to be required for both Hedgehog and Wnt signal transduction (2023). Loss of the intraflagellar transport protein (IFT) Kif3a results in non-functional primary cilia (24). We used a conditional knockout approach to eliminate Kif3a and thus disrupt the function of primary cilia in the cells that give rise to the facial skeleton, the neural crest cells. Our data demonstrate that Kif3a-mediated truncation of primary cilia causes a gain of Hedgehog function, and aberrant neural crest cell proliferation in the facial midline. The resulting embryos exhibit a form of FND, characterized by hypertelorism and diprosopus. Our data support a common molecular etiology linking loss and gain of Hedgehog function with a spectrum of midline facial anomalies.

RESULTS

Cranial neural crest cells extend primary cilia

We began our study by demonstrating that neural crest cells extend primary cilia in vivo. We identified primary cilia using immunostaining for acetylated tubulin (25) and Arl13b, a small GTPase of the Arf/Arl family (26). Cranial neural crest cells in the frontonasal prominence showed robust immunostaining for both proteins (Fig. 1A and C). We next tested the consequences of deleting Kif3a from cranial neural crest cells. A complete inactivation of Kif3a results in embryonic lethality at early stages of development (27); we circumvented this difficulty by crossing floxed Kif3a mice (28) with the neural crest deleter Wnt1-Cre (29) to produce Wnt1-Cre::Kif3afl/fl embryos (referred to as Kif3a CKO).

Figure 1.
Cranial neural crest cells do not extend primary cilia in Kif3a CKO embryos. (A) Acetylated tubulin staining (green) in WT frontonasal neural crest cells. (B) Punctate acetylated tubulin staining in Kif3a CKO frontonasal neural crest cells. (C) Arl13b ...

Kif3a CKO embryos specifically lost primary cilia in neural crest derived facial mesenchyme. Acetylated tubulin immunostaining identified long, finger-like primary cilia in the e10.5 wild-type frontonasal prominence (Fig. 1A). The punctate pattern of immunostaining demonstrated that primary cilia were truncated in the Kif3a CKO frontonasal prominence (Fig. 1B). Arl13b immunostaining confirmed the loss of primary cilia [compare wild-type (Fig. 1C) with Kif3a CKO (Fig. 1D)].

We next verified the tissue specificity of our Kif3a CKO. The neuroepithelium lies just dorsal to the neural crest-derived facial mesenchyme, and is derived from ectoderm. Arl13b immunostaining confirmed the extension of primary cilia from the neuroectoderm into the ventricle in both the hindbrain (Fig. 1E) and forebrain (Fig. 1F). Kif3a CKO mutant hindbrain and forebrain neuroectodermal cells were also immunopositive for Arl13b (Fig. 1G and H). Facial ectoderm, which surrounds the neural crest mesenchyme, is derived from surface ectoderm. Although immunopositive cells were difficult to detect at e10.5, we nonetheless saw an equivalent distribution of Arl13b-positive cells in wild-type and Kif3a CKO facial ectoderm (arrows, Fig. 1I and J). Collectively, these data confirmed that the loss of primary cilia was confined to neural crest cells in Kif3a CKO embryos. We next analyzed the facial phenotype of Kif3a CKO embryos.

Kif3a is required for normal craniofacial patterning

At e10.5, when neural crest migration into the facial prominences is complete (30), wild-type and Kif3a CKO embryos have very similar appearances (n = 17 wild-type, n = 15 mutant, data not shown), but shortly thereafter, subtle alterations in facial phenotype became evident. Compared to wild-type littermates, e11.5 Kif3a CKO faces were wider. Using infra-nasal measurements, we ascertained that e11.5 Kif3a CKO embryos had a 19% increase in frontonasal width (wild-type n = 15, mutant n = 7; Fig. 2A, B and I). After e11.5, the Kif3a CKO midline defect was exacerbated. Compared to wild-type littermates, the Kif3a CKO infra-nasal width was increased by 97% at e12.5 (wild-type n = 9, mutant n = 3; dotted yellow lines, Fig. 2C, D and I).

Figure 2.
Truncation of primary cilia in cranial neural crest cells causes facial dysmorphology. (A and B) Frontal view of e11.5 wild-type and Kif3a CKO embryos. White lines measure distance between nasal pits. (C and D) Frontal view of e12.5 wild-type and Kif3a ...

By e14.5, the Kif3a CKO frontonasal prominence was almost 100% wider than normal (wild-type n = 12, mutant n = 4; dotted yellow lines, Fig. 2E, F and I). At e17.5, the Kif3a CKO infra-nasal distance was 120% wider than the wild-type (wild-type n = 5, mutant n = 3; dotted yellow lines, Fig. 2G, H and I). These measurements demonstrated that the increased mid-facial width of Kif3a CKOs was significant and maintained relative to wild-type embryos throughout craniofacial development (Fig. 2I).

Clefting of the secondary palate accompanied the widened frontonasal prominence in Kif3a CKO embryos (Fig. 2J and K). Examining the palate in frontal section revealed that the wild-type medial edge epithelium fused, and the underlying palatine bones nearly merged in the midline (Fig. 2L, dotted lines), whereas in the Kif3a CKO, the palatine bones were dysmorphic. The bones did not extend towards the midline (Fig. 2M). Some small ectopic ossifications were detectable in the midline (Fig. 2M, dotted black lines), but whether these represented fragments of the palatine bones was unclear.

Kif3a CKO embryos phenocopy human frontonasal dysplasia

Deletion of Kif3a from cranial neural crest cells resulted in an extreme and significant expansion of the facial midline. In humans, abnormal expansion of the facial midline can manifest as FND. Defining features of FNDs include ocular hypertelorism; a flat broad nose; in more severe cases, the nose may separate vertically into two parts (bifid nasal septum), and cleft lip and/or palate. In addition, an abnormal skin-covered gap in the front of the head (anterior cranium occultum) and CNS defects are frequently present (31,32). We next examined Kif3a CKO embryos for defects analogous to human FND.

At e12.5, the wild-type nasal septum begins as a single mesenchymal condensation in the frontonasal prominence which becomes continuous with condensations of the nasal capsule (Fig. 3A). In Kif3a CKO littermates, the nasal septum was evident as a bifid condensation (Fig. 3B). By e16.5, the wild-type nasal septum condensation has matured into a midline cartilaginous rod (Fig. 3C). In Kif3a CKO littermates, the bifid condensation had matured into a duplicated nasal septum (Fig. 3D). Given previously published reports (33), we were surprised to find that maturation of the craniofacial skeleton was unaffected by loss of Kif3a. For example, Kif3a CKO chondrocytes were similar to wild-type chondrocytes in morphology (Fig. 3C and D; insets), distribution and intensity of alkaline phosphatase activity (data not shown). The primary defect with the Kif3a CKO skeleton appeared not to be in the differentiation of chondrogenic or osteogenic tissues, but rather in their patterning: the wild-type nasal septum is located in the midline and is surrounded by the pre-maxillary bones and the nasal bones (dorsal view, Fig. 3E; frontal view, Fig. 3G), but in Kif3a CKO embryos, the nasal septum was duplicated, which displaced both the nasal and pre-maxillary bones laterally (dorsal view, Fig. 3F; frontal view, view,33H).

Figure 3.
Skeletal analyses of Kif3a CKO embryos. (A and B) Pentachrome stain on transverse sections of e12.5 WT and Kif3a CKO embryos. The nasal septum condensation (dotted white line, ns) is a single skeletal element in wild-type embryos. The nasal septum of ...

We continued our examination of the Kif3a CKO craniofacial skeleton by examining the palate, the skull and the mandible. In Kif3a CKO embryos, the bones of the palate and ventral cranial midline, including the maxilla, trabecular basal plate, palatine and basisphenoid were either laterally displaced, or if they were midline elements, reduced to bony nodules or absent (Fig. 3I and J). The Kif3a CKO cranium was also severely dysmorphic. Compared to wild-type littermates (Fig. 3K), mutant embryos had laterally displaced, underdeveloped frontal bones, which resulted in an abnormal opening in the skull (i.e. cranium occultum; Fig. 3L) that was covered by an epithelial membrane (see whole mounts in Fig. 2). In addition, we found that the Kif3a CKO mandibles were 30% shorter than their wild-type littermates, and lacked a discernable ramus and condyle (Fig. 3M versus N).

Finally, we examined Kif3a CKO embryos for the CNS defects associated with FND: namely agenesis of the corpus callosum (32). Nerve fibers, which normally connect the left and right hemispheres of the brain (Fig. 3O), failed to span the midline in Kif3a CKO mutants (Fig. 3P). Taken together, these analyses revealed that Kif3a CKO mutants shared a number of features found in human conditions that fall within the FND sequence, including bifid nasal septum, cleft palate, cranium occultum and agenesis of the corpus callosum (31). Thus, the Kif3a CKO mutants displayed the murine equivalent of a human FND.

Truncation of primary cilia causes region-specific alterations in facial Wnt signaling

We sought to clarify the molecular basis for the Kif3a CKO midline phenotype. Previous work from our lab has shown that Wnt pathway activity plays an integral role in patterning the vertebrate face (19). Although some studies indicate that primary cilia are not essential for Wnt signal transduction (34,35), other experiments suggest a key role for primary cilia in regulating β-catenin-dependent Wnt signaling (23). To address the possibility that loss of cilia disrupted the pattern of Wnt pathway activity in the face, we evaluated the expression of a transgenic reporter of canonical Wnt signaling in Kif3a CKO embryos. We crossed TOPgal reporter mice (36) into the mutant background to produce Wnt1-Cre::Kif3a f  l/f l::TOPgal embryos (referred to as Kif3a CKO::TOPgal). At e10.5, prior to obvious morphologic differences, the patterns of Xgal staining in wild-type and Kif3a CKO mutants appeared similar (n = 3; Fig. 4A and B). For example, both embryos showed evidence of Xgal staining in the telencephalon, and in the lateral nasal, maxillary and mandibular prominences (Fig. 4A and B). Careful examination from a lateral perspective showed a slight expansion of the lateral nasal and maxillary domains of Xgal staining (Fig. 4C and D).

Figure 4.
Wnt signaling is altered in a site-specific manner in Kif3a CKO embryos. Xgal staining (blue) indicates sites of Wnt responsiveness. (A and B) Frontal view of e10.5 TOPgal and Kif3a CKO::TOPgal embryos. (C and D) Lateral view of e10.5 TOPgal and Kif3a ...

We previously showed that domains of Wnt responsiveness in the face coincide with localized regions of proliferation, and that these regions are ultimately responsible for shaping the vertebrate face (19). We reasoned that the expanded midline in the Kif3a CKO mutant could be the consequence of ectopic Wnt responsiveness (and therefore increased proliferation) in the frontonasal prominence. We were surprised to find that despite the gross morphologic variation in the width of the mutant frontonasal prominence, the boundaries and domains of Wnt responsiveness in wild-type TOPgal and Kif3a CKO::TOPgal embryos were largely maintained (Fig. 4E and F). For example, the lateral nasal and maxillary domains of Xgal staining were remarkably well conserved in both wild-type and mutants, and the frontonasal prominence, which in wild-type embryos is normally free of Xgal staining, maintained this Xgal free status in the mutant (Fig. 4E–H).

We examined other domains of Wnt responsiveness in the face. Focal domains of Wnt and Shh signaling define the sites where teeth will develop (37). In wild-type TOPgal embryos, discrete Xgal-positive domains were evident in the dental lamina (Fig. 4G), but in Kif3a CKO::TOPgal embryos, the domain was an unbroken line (Fig. 4H). The dental lamina staining pattern differed slightly between the upper and lower jaws: in the mutant mandible, Xgal domains were smaller in comparison to wild-type littermates (Fig. 4I and J), and the mutant tongue was reduced to a remnant.

Analyses of Xgal-stained tissue sections from Kif3a CKO::TOPgal embryos revealed other changes in the pattern of Wnt pathway activity. Initially, ectoderm is the first Wnt responsive tissue in the face, followed by Wnt responsiveness in underlying neural crest-derived mesenchyme (Fig. 4K and see (19)). In Kif3a CKO::TOPgal embryos, the mesenchymal Xgal domains were smaller or in some cases, absent (Fig. 4L). By e16.5, the Xgal staining pattern shifted again: whole-mount analyses showed ectopic patches of Xgal positive tissue in the mutant frontonasal prominence (n = 3; Fig. 4M wild-type compared to Fig. 4N). In tissue sections, these ectopic Xgal patches demarcated small cell aggregates (compare control, Fig. 4O with P and Q). Morphologic and histologic analyses demonstrated that these condensations were undergoing chondrogenesis and osteogenesis (Fig. 4R), and ultimately formed the ectopic islands of bone and cartilage we observed in the e17.5 mutant facial midline (Figs 2M and and33L).

Taken together, the analyses of Kif3a CKO::TOPgal embryos clearly demonstrated that β-catenin-dependent Wnt signaling was altered in certain domains that have lost functional primary cilia. An expansion in Wnt responsiveness, however, did not appear to be the direct cause the expanded Kif3a CKO frontonasal prominence as originally hypothesized. We next employed another genetic strategy to determine whether a disruption in Hedgehog signaling was the root cause for this facial malformation.

Loss of Kif3a results in an expansion of Hedgehog responsiveness in the face

Most studies suggest that a mutation in Kif3a reduces Hedgehog signal transduction (3840), and it is known that the loss of Hedgehog signaling causes hypotelorism and cyclopia (10,41,42). Since our mutant embryos have hypertelorism, we hypothesized that neural crest-specific deletion of Kif3a might cause a gain of Hedgehog pathway activity within the face. This hypothesis is not without precedent, as other investigators have reported that Hedgehog target genes are expressed in broader domains after the loss or truncation of primary cilia (33,43). The mechanism(s) behind this contradictory effect have not been elucidated, but our conditional genetic knock-out offered a unique opportunity to explore this problem in more detail.

To directly assess the effect of Kif3a deletion on Hedgehog signal transduction, we crossed Ptc-LacZ reporter mice (44) into the mutant background to produce Wnt1-Cre::Kif3afl/fl::Ptc-LacZ embryos (referred to as Kif3a CKO::Ptc-LacZ). We began analyses at e10.5, a time-point prior to any obvious differences between wild-type and Kif3a CKO facial morphology. In Ptc-LacZ controls, Xgal staining was detectable in all known sites of Hedgehog pathway activity (n = 4; Fig. 5A). By whole-mount analysis, the staining pattern appeared similar in Kif3a CKO::Ptc-LacZ embryos (n = 6; Fig. 5B), but sagittal tissue sections revealed subtle changes in the pattern of Xgal staining. For example, relative to e10.5 Ptc-LacZ controls, Kif3a CKO::Ptc-LacZ embryos showed broader domains of Xgal staining in the midline facial mesenchyme and facial ectoderm (Fig. 5C and D, white and black arrows). Most notable was an expansion of Hedgehog responsiveness within the facial ectoderm itself. Although in our conditional knockout facial ectodermal cells maintain their primary cilia (Fig. 1I and J), the ectodermal Xgal domain was clearly expanded (Fig. 5D). Broader domains of Hedgehog activity were also observed in e14.5 Kif3a CKO::Ptc-LacZ embryos relative to Ptc-LacZ littermates (Fig. 5E and F). In particular, the regions of Xgal staining in the Kif3a CKO::Ptc-LacZ facial mesenchyme were markedly expanded (Fig. 5G and H). The most dramatic increase in Xgal staining was observed in the mesenchyme of the primary palate (Fig. 5I and J). The molecular basis and cellular consequences for this effect on Hedgehog pathway activity within the facial prominences became the focus of our next series of experiments.

Figure 5.
Hedgehog pathway activity is expanded in Kif3a CKO embryos. (A and B) Frontal view of whole mount Xgal stained e10.5 Ptc-LacZ and Kif3a CKO::Ptc-LacZ embryos. (C and D) Sagittal sections through e10.5 Xgal stained Ptc-LacZ and Kif3a CKO::Ptc-LacZ embryos. ...

Shh and Gli1 expression domains are expanded in Kif3a CKO facial prominences

Hedgehog signals from forebrain neuroectoderm, facial ectoderm and pharyngeal endoderm are required for proper development of the craniofacial complex (10,45,46). We first examined tissue sections to determine whether the pattern of Shh expression in these epithelial domains was altered. At e11.0, the domains of Shh in the telencephalon, diencephalon, ventral hindbrain and oral ectoderm are equivalent between wild-type and mutant tissues (Fig. 6A–D), indicating that Kif3a deletion in cranial neural crest cells does not affect the initial Shh domains in epithelia established during neurogenesis (18). The targets of Shh signaling, however, are ectopically expressed. The Gli transcription factors are direct targets of Hedgehog signaling (47). In the presence of a Hedgehog ligand, Gli1 is transcriptionally activated and functions to extend the duration and strength of a Hedgehog signal (47,48). At e11.0, Gli1 expression domains were dramatically altered in the Kif3a CKO facial prominences (Fig. 6E–H). In the proximal frontonasal prominence, the mutant Gli1 domain was smaller, but in the distal frontonasal prominence and lateral nasal prominences the Gli1 domains were larger (Fig. 6E and F). Examination of Gli1 expression in sagittal section reveals an expansion of the expression domain posteriorly into the palatal mesenchyme and within the nasal epithelium (Fig. 6G and H, dotted yellow line).

Figure 6.
Expression of Hedgehog ligand and targets are perturbed in Kif3a CKO embryos. (AD) In situ hybridization for Shh in e11.0 WT and Kif3a CKO in sagittal and transverse sections. (EH) In situ hybridization for Gli1 in e11.0 WT and Kif3a ...

Although early domains of Shh were normal (Fig. 6A–D), between e11.0 and e12.5 the Hedgehog signaling was disrupted in Kif3a CKO embryos. By e12.5, the neuroectodermal domains of Shh were laterally displaced from their normal midline position, and the oral ectodermal domain of Shh was expanded in Kif3a CKO embryos (Fig. 6I and J). Although the tongue, a midline structure, was absent in Kif3a CKO embryos, the Shh domain that marks the lingual ectoderm persists, and was expanded (Fig. 6K and L). The Shh-positive incisor dental lamina was also expanded in Kif3a CKO oral ectoderm (Fig. 6M and N).

Expression domains of the Gli transcription factors were also altered at this later time point. The Gli1 domain was expanded in the oral ectoderm (Fig. 6O and P). Expression of the transcriptional repressor, Gli3, on the other hand, was reduced in Kif3a CKO oral ectoderm (Fig. 6Q and R). Thus, molecular data support a model whereby loss of primary cilia in neural crest cells results in ectopic Hedgehog signaling in the face.

Loss of primary cilia results in a Hedgehog-dependent increase in neural crest cell proliferation

We next sought to understand the cellular consequence of the loss of primary cilia. Neural crest cells undergo four distinct cellular behaviors; epithelial to mesenchymal transformation, migration, proliferation and differentiation. Previous studies in zebrafish proposed that a loss of cilia adversely affected the migration of neural crest cells (49). To rule out a migration defect and to confirm that neural crest cells were able to populate the facial prominences in the Kif3a CKO mutants, we crossed the Kif3a CKO mutant into the Wnt1-Cre::R26R reporter line (Supplementary Material, Fig. S1). At e10.5, Xgal staining in Wnt1-Cre::R26R and Wnt1-Cre::R26R::Kif3a CKO was similarly localized to areas populated by neural crest cells (Supplementary Material, Fig. S1A–D). To verify Xgal staining was marking neural crest cells, we sectioned these embryos and found that the mesenchyme of the frontonasal, maxillary and mandibular prominence of Wnt1-Cre::R26R and Wnt1-Cre::R26R::Kif3a CKO were both Xgal positive, whereas the forebrain and surface ectoderm were Xgal negative (Supplementary Material, Fig. S1E–H). Furthermore, we confirmed that it was the neural crest mesenchyme that lacked primary cilia in the Kif3a CKO by performing double immunostaining for the neural crest marker, AP2α, and primary cilia marker, Arl13b. We found double positive cells in the wild-type mandibular prominence, but no co-localization of AP2α and Arl13b in the Kif3a CKO mandibular prominence (Supplementary Material, Fig. S1I and J). These results allowed us to confirm that although Kif3a CKO neural crest cells lacked primary cilia, they were still able to migrate out of the neural tube and populate the facial prominences.

Since neural crest cells were able to exit the neural tube, arrived in the facial prominences and underwent skeletal differentiation, we assayed neural crest cell proliferation. Using BrdU immunostaining (Fig. 7A–F), we found exuberant proliferation throughout the Kif3a CKO facial prominences, in comparison to the localized areas of proliferation seen in wild-type facial prominences. The enhanced cell proliferation was most notable in the Kif3a CKO facial midline. Analysis of BrdU incorporation was performed through various section planes including the nasal capsule (Fig. 7A and B) and palatal mesenchyme (Fig. 7C–F). The most robust increase in proliferation was observed in the ventral midline of the frontonasal prominence (future primary palate) in the Kif3a CKO (Fig. 7C–F). To confirm this observed increase in proliferation, we quantified BrdU incorporation in various planes of section via ImageJ software. Quantification of BrdU incorporation showed an over 5-fold increase in neural crest proliferation in Kif3a CKO facial prominences compared with wild-type control (Fig. 7G, P = 0.002). We next explored the molecular basis for this increased proliferation.

Figure 7.
Loss of Kif3a results in a Hedgehog-dependent increase in neural crest cell proliferation. (AF) BrdU incorporation in e12.5 WT and Kif3a CKO embryos. (A and B) BrdU incorporation in the dorsal nasal capsule of e12.5 WT and Kif3a CKO embryos. ...

The most notable molecular difference in Kif3a CKO embryos was the expansion of the expression domain of the Hedgehog target Gli1. We hypothesized that the increased proliferation in the Kif3a CKO mutant was a Hedgehog dependent process. To test this hypothesis, we first sought to determine whether the increased proliferation and increased Gli1 expression were co-localized. Using equivalent planes of section, we found that the expanded Gli1 domains (Fig. 7H and I) in the Kif3a CKO coincided with regions of increased BrdU incorporation (Fig. 7J and K) in the medial mesenchyme. Co-localization of Gli1 expression and BrdU incorporation was also found in lateral maxillary mesenchyme within the Kif3a CKO mutants (Fig. 7L–O).

To support a causal relationship between Hedgehog signaling and proliferation in the facial mesenchyme, frontonasal neural crest cells were isolated and exposed in vitro to exogenous N-terminal Sonic Hedgehog (Shh-N). First, we confirmed that the concentration of Shh-N was sufficient to induce the downstream target, Gli1 (Fig. 7P). Next, we assayed the amount of proliferation by measuring BrdU incorporation in wild-type and Kif3a CKO neural crest cells in the presence of increasing doses of Shh-N. Relative to wild-type cells, which showed no significant increase in proliferation in response to varying doses of Shh-N, the Kif3a CKO facial neural crest cells showed a significant, dose-dependent increase in BrdU incorporation (Fig. 7Q, P = 0.0028). Therefore, we hypothesize that Kif3a deletion altered the response of neural crest cells to a Hedgehog signal and led to their exuberant proliferation in the frontonasal prominence.

Primary cilia function in the cranial neural crest is conserved among species

We next examined the facial phenotype of another animal with a genetic defect that disrupts primary cilia function. Talpid chicken embryos exhibit polydactyly (50) as well as facial malformations (51). Talpid3 phenotypes arise as a consequence of mutation in a basal body protein that eliminates primary cilia (52). We found that cranial neural crest cells of Talpid2 embryos also lacked primary cilia (Fig. 8A and B) and basal bodies (Fig. 8C and D). As a consequence of the loss of functional primary cilia, Talpid2 embryos exhibited numerous facial malformations (Fig. 8E and F). For example, by St. 19, the maxillary prominences and the stomodeum were dysmorphic. Later in embryonic development, disproportionate growth of the frontonasal prominence relative to the maxillary prominences resulted in facial clefting (Fig. 8G and H). Furthermore, like the Kif3a CKO, Talpid2 embryos exhibited midline defects including the absence of a tongue (Fig. 8G and H, asterisk), duplication of the midline nasal septum (Fig. 8I and J) and expanded expression domains of Shh and Hedgehog targets in the face (Fig. 8K–N, and see what follows).

Figure 8.
The avian primary cilia mutant Talpid2 has craniofacial abnormalites. (A and B) Acetylated tubulin staining (green) in control and Talpid2 mutant neural crest cells. (C and D) Gamma tubulin staining (green) in control and Talpid2 mutant neural crest cells. ...

In Talpid2 embryos, we found alterations in gene expression extending to all craniofacial tissues. For example, Shh was reduced in the Talpid2 midbrain (Fig. 8O and P) even though Shh and Gli1 expression in the limb bud were unchanged (51). Since Talpid2 phenotypes have previously been attributed to a constitutive activation of Hedgehog signaling (53), we extended our evaluation to include the Gli genes. As expected, the Gli1 domain was expanded (n = 3; Fig. 8Q and R) and the Gli3 domain was reduced in mesenchyme (Fig. 8S and T).

DISCUSSION

The face as a barometer of Hedgehog pathway activity

In 1908, the anatomist Harris Hawthorn Wilder postulated a unifying theory to explain the relationship between extreme forms of facial dysmorphologies. On either side of a normal face, Wilder speculated, symmetrical anomalies that constituted a single malformation spectrum existed. At one extreme was cyclopia, characterized by a single median eye and a proboscis replacing the nose, and at the other extreme was a duplication of facial features (Fig. 9). In between were the conditions of hypo- and hypertelorism and their associated craniofacial and neurologic defects. In effect, Wilder's continuum ranged from a complete collapse to a duplication of the face. Wilder hypothesized that these deformities were due to ‘some modification in the germ itself, leading the organisms to develop in accordance with laws as definite and natural, though not as usual, as those governing normal development.’ Wilder sought experimental evidence to support his theory on the ‘morphology of Cosmobia’, but was unable to produce embryos exhibiting this range of phenotypes (54).

Figure 9.
A molecular model of cosmobia. (A) Schematic diagram indicating Hedgehog levels in embryonic tissue sections corresponding to various intervals of the cosmobic spectrum. (A) ‘Normal state’ has moderate Hedgehog activity in the midline ...

Accumulating evidence indicates that Wilder's theory of Cosmobia has merit. Wilder's collapse of the face is now known as HPE. The most severe form of HPE is cyclopia, with milder forms appearing as hypotelorism (Fig. 9). Cyclopia is caused by disruptions in the Hedgehog pathway that result in the elimination (41,55) or reduction (10,12,13) of Hedgehog signaling. At the other end of the Cosmobic spectrum are hypertelorism and facial duplications. Herein we provide the first evidence that these malformations are also caused by disruptions in the Hedgehog pathway, but of the kind that produce expanded or ectopic activity (Fig. 9).

Primary cilia are an essential component for Hedgehog signal transduction (21). Most studies indicate that mutations in IFT proteins, including Kif3a, result in a down-regulation of Hedgehog signaling (21,43,56). Our data show that Kif3a-mediated disruption in primary cilia function results in expanded Hedgehog pathway activity in the cranial neural crest. Other investigators have noted that Kif3a and other IFT mutations can have similar paradoxical effects (43,57), and have attributed this inconsistency to which Gli protein, Gli1 or Gli3 is acting predominantly in the tissue (40,43,58,59). What determines this Gli predominance in any given tissue is not clear. A Gli1−/− mutation does not affect craniofacial morphogenesis (60), suggesting that it is not the predominant Gli in facial tissues. Humans and mice with inactivating Gli3 mutations, however, exhibit dramatically wider faces (17,61,62) implicating Gli3 as an important regulator of craniofacial patterning. Kif3a CKO embryos show reduced Gli3 expression (Fig. 6) and they also exhibit wider faces (Fig. 2), supporting the Gli3 predominance theory.

Primary cilia may integrate signals from multiple pathways in the developing face

Hedgehog signaling directly regulates Gli3 expression, but in some tissues Gli3 expression is also directly regulated by Wnt pathway activity (63). In the facial prominences, we found both the Gli3 expression and Wnt responsiveness in facial mesenchyme. Gli3 expression levels were also reduced in the Kif3a CKO mutant, coincident with a reduction in Wnt responsiveness (Fig. 4) and an expansion of Hedgehog responsiveness (Fig. 5). Do primary cilia integrate Wnt and Hedgehog signals, thereby regulating Gli expression? Or does Kif3a have another function besides its role as an IFT motor protein? In some tissues, Kif3a regulates CKI-mediated phosphorylation of Disheveled, and that loss of Kif3a leads to increased Wnt pathway activation (23). However, we observed that the loss of Kif3a leads to a localized reduction in Wnt responsiveness (Fig. 4). While our genetic approach provides a demonstration that β-catenin-dependent Wnt signaling is affected by truncating the primary cilia on cranial neural crest cells, there is no proof that it is direct. In light of these data, more questions are raised than answered about whether primary cilia are absolutely required for Wnt pathway activity.

Mechanistic insights into craniofacial deformities

We found that a loss of primary cilia in the facial mesenchyme resulted in expanded Hedgehog activity, including the up regulation of Gli1 and simultaneous down regulation of Gli3 (Figs 6 and and8),8), resulting in inappropriate cell proliferation in the facial mesenchyme. There are a number of human conditions where excessive Hedgehog signaling leads to uncontrolled cell proliferation. For example, Gorlin syndrome patients carry mutations in Ptc1 or Smo that lead to ectopic Hedgehog pathway activity (6466). These mutations result in the characteristic, hyper-proliferative nevi that predominantly mark the patients' faces and trunk, and which frequently transform into basal cell carcinomas (31,67). In addition to these skin lesions, Gorlin syndrome patients exhibit moderate to severe hypertelorism, a broad nasal root, large heads (macrocephaly) and an increased incidence of facial clefting (31). Our data suggest that mutations resulting in excessive Hedgehog signaling lead directly to increased proliferation of neural crest cells and that this can manifest as hypertelorism and in extreme cases, FND.

Syndromes such as FND (OMIM 136760; which include frontonasal malformation; median facial cleft syndrome and frontorhiny), acrofrontofacionasal dysostosis 1 (OMIM 201180), frontofacionasal dysplasia (OMIM 229400), oculoauriculofrontonasal syndrome (OMIM 601452) and craniofrontonasal syndrome (OMIM 304110) all present with a midline expansion accompanied by hypertelorism, a flat broad nose, and cleft lip and/or cleft palate. Although FNDs have been attributed to neural tube defects (7), abnormal cell death or aberrant patterns of vascularization in the face (31), our data and one previous report (68) argue for a different etiology. We propose that some forms of FND are due to a gain-of-Hedgehog function and Hedgehog-dependent hyperproliferation of the cranial neural crest. Finally, in view of the fact that the basis of the Kif3a CKO phenotype was ciliopathic, we put forward the possibility that genes encoding ciliary proteins could now represent a novel class of candidates for genetic analysis of disorders characterized by a hyperteloric phenotype.

MATERIALS AND METHODS

Embryo collection and preparation of tissues

Embryos were collected in 4°C PBS then fixed in 4% paraformaldehyde (PFA) overnight at 4°C, dehydrated through an ethanol series and stored in 100% ethanol. Most tissues were embedded in paraffin and cut at 8 µm using a standard microtome.

Immunohistochemistry

For acetylated and gamma tubulin antibody staining, de-paraffinized tissue sections were immersed in cold acetone and treated with 0.1% TritonX-100. Sections were incubated overnight at 4°C with a 1:1000 dilution of incubation in monoclonal anti-acetylated tubulin and anti-gamma tubulin (Sigma, St Louis, MO, USA) 1% donkey IgG overnight, and incubated for 1 h at room temperature in a 1:1000 dilution of FITC conjugated anti-mouse secondary antibody (Jackson Immunoresearch) in 1% donkey IgG. Slides were washed in 1:10 000 dilution of Hoechst in PBS. Arl13b (gift from T. Caspary) staining was performed under similar conditions with a 1:500 dilution for the primary antibody incubation and a 1:200 dilution for secondary antibody incubation. For AP2α (Developmental Studies Hybridoma Bank), similar conditions were used with a 1:25 dilution for primary antibody incubation and 1:200 dilution for secondary antibody incubation.

For BrdU antibody staining, pregnant dams were given intraperitoneal injections of BrdU labeling reagent (Zymed, Carlsbad, CA, USA) and euthanized 2 h post injection. BrdU detection was carried out as per manufacturer's instructions (Zymed, Carlsbad, CA, USA).

Whole mount and histological staining

Whole-mount bone and cartilage staining of embryos was performed as described (69). Movat's Pentachrome stain was carried out as previously described (70) and Safranin-O staining was used to detect cartilage. Briefly slides were de-waxed, washed in Weigert's hematoxylin, acid alcohol, aqueous fast green, 1% acetic acid and 0.1% aqueous Safranin-O.

Generating Kif3a CKO, Kif3a CKO::TOPgal, Kif3a CKO::Ptc-LacZ mice and Wnt1-Cre::R26R::Kif3aCKO

All mouse experiments were done in accordance with the Stanford University institutional guidelines. The Cre-lox P system was used to generate mice in which Kif3a is conditionally inactivated in the neural crest. Female mice, homozygous for the floxed Kif3a allele (Kif3afl/fl) (71), were crossed with males, heterozygous for the floxed Kif3a allele and either heterozygous or homozygous for Wnt1-Cre (Wnt1-Cre Kif3afl/+) mice, to produce Kif3a CKO mutant embryos. Genotype was confirmed by PCR for the Kif3a allele using wild-type (5′-TCTGTGAGTTTGTGACCAGCC-3′), common (5′-AGGGCAGACGGAAGGGTGG-3′) and deletion (5′-TGGCAGGTCAATGGACGCAG-3′) primers. The floxed Kif3a allele generated a 490 bp product, the wild-type allele a 360 bp product and the null allele a 200 bp product. Wnt1-Cre:R26R:Kif3afl/+ males were crossed with Kif3afl/fl females to create Wnt1-Cre::R26R::Kif3aCKO embryos.

Kif3a CKO::TOPgal mice were generated by crossing TOPgal mice, which carry a β-galactosidase transgene downstream of a c-fos minimal promoter and three consensus Tcf-binding motifs (36), to mice homozygous for the floxed Kif3a. To produce Kif3a CKO::Ptc-LacZ mice, Ptc-LacZ mice (72) were bred into Kif3afl/fl mice and the Kif3afl/fl Ptc-LacZ offspring were then crossed to Wnt1-Cre Kif3afl/+ males to generate Kif3a CKO::Ptc-LacZ.

LacZ detection

β-Galactosidase activity was visualized by Xgal staining. In brief, freshly collected tissues were fixed with 0.2% glutaraldehyde for 15 min, and stained with Xgal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside; Invitrogen, Carlsbad, CA, USA) overnight at 37°C (73). For Xgal staining of tissue sections, freshly collected embryos were fixed in 0.4% paraformaldehyde for 2 h, infused with 30% sucrose for 24 h, embedded in OCT media, frozen with dry ice in isopentane and cryosectioned. Xgal staining for cryosections followed the same protocol.

In situ hybridization

Templates for the relevant mRNAs for in situ hybridization were amplified from embryonic mouse cDNA by PCR using sequence-specific primers which included the promoter sites for T3 or T7 RNA polymerase. Antisense riboprobe for each gene was transcribed with either T3 or T7 RNA polymerase in the presence of Dig-11-UTP (Roche; Indianapolis, IN, USA). Whole-mount and section in situ hybridizations were performed by incubating tissue sections in hybridization buffer (Ambion Corporation, Austin, TX, USA) at 70°C for 12 h, followed by the addition of riboprobe (approximate concentration of 0.2–0.3 µg/ml probe per kilobase of probe complexity). Non-specifically bound probe was removed with high stringency washes (0.1× SSC, 65°C). For color detection, embryos or slides were blocked with 10% sheep serum, 1% Boehringer-Mannheim Blocking Reagent (Roche, Indianapolis, IN, USA) and levamisole, and developed using nitro blue tetrazolium chloride (NBT; Roche, Indianapolis, IN, USA) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Roche, Indianapolis, IN, USA). After developing, the slides were cover-slipped with aqueous mounting medium.

Tissue harvest and primary cell culture

Frontonasal neural crest cells were isolated from e11.5 wild-type and Kif3a CKO embryos. Embryos were collected, dissected in cold, sterile PBS and digested in 1.26 U/ml Dispase (BD Biosciences, San Jose, CA, USA). Facial ectoderm and neuroectoderm were removed from the frontonasal prominences with tungsten needles. Isolated frontonasal neural crest cells were centrifuged, resuspended and plated in standard growth medium, containing DMEM, 10% FBS and 100 IU/ml penicillin/streptomycin. Cells were expanded for a period of 2 days at 37°C, 21% O2, 5% CO2. Cells were passaged by trypsinization; only passage 1 cells were utilized for analyses.

In vitro BrdU incorporation assay

Cellular proliferation was assessed by bromodeoxyuridine (BrdU) incorporation assays (74). Briefly, e11.5 wild-type and Kif3a CKO neural crest cells were seeded in 96-well plates (1000 cells/well, n = 6), treated with 0, 100 or 250 ng/ml of N-terminal Sonic Hedgehog (Shh-N) (R&D, Minneapolis, MN, USA) or vehicle as a control (0.1% PBS). After 48 h, BrdU assays were performed according to the manufacturer's instructions (Roche Applied Science, Indianapolis, IN, USA). Means and standard deviations were calculated.

RNA isolation and quantitative real-time polymerase chain reaction

Cranial neural crest cells from the frontonasal prominence were isolated from e11.5 wild-type and Kif3a CKO embryos. Tissues were snap-frozen and homogenized by sonication, and RNA isolation was performed with the RNeasy Mini Kit (Qiagen Sciences, MD, USA). After DNase treatment, reverse transcription was performed with Taqman Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA). Quantitative real-time polymerase chain reaction was carried out using the Applied Biosystems Prism 7900HT Sequence Detection System and Power Sybr Green Master Mix (Applied Biosystems, Foster City, CA, USA). Specific primers for the genes examined were designed based on their PrimerBank (http://pga.mgh.harvard.edu/primerbank) sequence. Primers were first tested to determine optimal concentrations, and products were run on a 2% agarose gel to confirm the appropriate size and RNA integrity. The levels of gene expression were determined by normalizing to the values of GAPDH. All reactions were performed in triplicate.

Talpid2 embryo collection

Fertilized eggs were obtained from Talpid2 flocks maintained at the University of California, Davis. All eggs were incubated at 38°C in a humidified forced draft chamber and were staged as previously described (75).

FUNDING

This work was supported by the March of Dimes (FY06-335 to J.A.H.); the National Institutes of Health (NRSA-F32DE017499-01 to S.A.B., RO1-DE012462-06A1 to J.A.H.); and the Lucile Packard Foundation for Children's Health (1K99DE019853-01 to S.A.B.).

Supplementary Material

[Supplementary Data]

ACKNOWLEDGEMENTS

We would like to thank Sue McConnell (Stanford University) for kindly sharing Kif3afl/fl and Kif3afl/+::Wnt1-Cre mice, Tamara Caspary (Emory University) for kindly providing us with Arl13b antibody and Jackie Pisenti at UC Davis for supplying Talpid2 embryos.

Conflict of Interest statement. None declared.

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