- Top of page
- MATERIALS AND METHODS
- LITERATURE CITED
- Supporting Information
Normal patterning and morphogenesis of the complex skeletal structures of the skull requires an exquisite, reciprocal cross-talk between the embryonic cephalic epithelia and mesenchyme. The mesenchyme associated with the jaws and the optic and olfactory capsules is derived from a Hox-negative cranial neural crest (CNC) population that acts much as an equivalence group in its interactions with specific local cephalic epithelial signals. Craniofacial pattern and morphogenesis is therefore controlled in large part through the regulation of these local cephalic epithelial signals. Here, we demonstrate that Pax6 is essential to the formation and maturation of the complex cephalic ectodermal patterning centers that govern the development and morphogenesis of the upper jaws and associated nasal capsules. Previous examinations of the craniofacial skeletal defects associated with Pax6 mutations have suggested that they arise from an optic-associated blockage in the migration of a specific subpopulation of midbrain CNC to the lateral frontonasal processes. We have addressed an alternative explanation for the craniofacial skeletal defects. We show that in Pax6SeyN/SeyN mutants regional CNC is present by E9.25 while there is already specific disruption in the early ontogenetic elaboration of cephalic ectodermal expression, associated with the nascent lambdoidal junction, of secreted signaling factors (including Fgf8 and Bmp4) and transcription factors (including Six1 and Dlx5) essential for upper jaw and/or nasal capsular development. Pax6 therefore regulates craniofacial form, at stages when CNC has just arrived in the frontonasal region, through its control of surface cephalic ectodermal competence to form an essential craniofacial patterning center. genesis 49:307-325, 2011. © 2011 Wiley-Liss, Inc.
Although remarkable for their stunning array of transitional and end-point phenotypic forms, gnathostome skulls are equally well characterized by their possession of conserved regions of structure (Barghausen and Hopson,1979; de Beer,1985; Goodrich,1958). This applies to both the components of the chondrocranium (the jaw-associated branchial arch (BA) derived splanchnocranium and the neurocranium consisting of the midline trabecular and parachordal basal plates and the otic, optic, and olfactory capsules) and the skeletal elements of the dermatocranium (the dermal skeleton) that forms in association with the chondrocranium and is well conserved along macrotaxanomic lines. Fate-mapping studies in gnathostome vertebrates have demonstrated that the craniofacial complex—including the splanchnocranium, the trabecular basal plate, and the olfactory and optic capsules and their associated dermal skeletons—is derived from a rostral Hox-negative, or “trigeminal,” cranial neural crest (CNC) cell population (Chai et al.,2000; Couly et al.,1993,1998; Kuratani,2005a,b; Le Douarin et al.,2004; Osumi-Yamashita et al.,1994; Santagati and Rijli,2003).
Understanding both the conserved and divergent patterns of structural designs in the gnathostome skull requires explaining how this Hox-negative CNC population is informed to manifest structure within and between taxa. A “Hinge and Caps” (H&C) model has previously been proposed that places CNC competence to respond to localized epithelial signals at the root of much of craniofacial development and evolution (Fig. 1; Depew and Compagnucci,2008; Depew et al.,2002a,b, 2005; Depew and Simpson,2006). Although the Hox-negative CNC carries with it taxa-specific interpretive information, there is ever accumulating evidence that both cephalic endodermal and ectodermal epithelial populations supply patterning information to which the CNC component responds to form the jaws, trabecular basal plate and olfactory and optic capsules (Couly et al.,2002; Creuzet et al.,2005; Depew et al.,2002b; Depew and Simpson,2006; Graham et al.,2008; Hu et al.,2003; Marcucio et al.,2005; Song et al.,2004; Trumpp et al.,1999). Significant among the H&C model's posited localized epithelial patterning centers is that at the lambdoidal junction (or λ-junction), so called for the “λ” shape found at the confluence of the maxillary process of the first BA (mxBA1) and the medial (mFNP) and lateral (lFNP) frontonasal processes (Fig. 1; Depew and Compagnucci,2008; Depew and Simpson,2006; Tamarin and Boyde,1977). The epithelial component of the mature λ-junction, as typically seen in mice around embryonic day 10.5 (E10.5), stems from the surface cephalic ectoderm (SCE) that gives rise to the optic, olfactory, and hypophyseal placodes as well as covers the distal end of mxBA1 (see Fig. 1), and disruption of positioning and strength of signaling factors in this region (as with the application of growth factor-soaked beads) is well known to disrupt skeletal patterning of the region (Barlow and Francis-West,1997; Foppiano and Marcucio,2007; Lee et al.,2001). This position in mammals is particularly developmentally and functionally complex and critical as it outlines the positions of the nostrils (choanae), the formation of the upper lips, the nasal capsules, and the primary and secondary palates (Depew and Compagnucci,2008; Gaare and Langman,1977; Pourtois,1972; Tamarin,1982; Tamarin and Boyde,1976,1977; Warbrick,1960; Waterman and Meller,1973).
Figure 1. Expression of Pax6 and its relationship to the development of the lambdoidal junction (λJ). (a) Expression of Pax6 in the SCE at E8.5 and E8.75. The dashed red line represents the line of section for center figure. (b) SEM micrographs of E9 and E9.5 embryos with the regional λJ-associated SCE pseudocolored blue. (c) Diagram of the relationship of the λJ to the “Hinge and Caps” model of jaw development. In the model, positional information for the jaw “Hinge” (purple disc) is driven by factors at the junction of the mxBA1 and mdBA1 and the first pharyngeal plate (at PC1). Patterning the forming jaw regions functionally furthest from the hinge involves positional signals from the distal-most BA1 midline (dml), denoted the lower “Caps” (lower blue disc), and those associated with the λJ (denoted λmc), where the mxBA1 meets the olfactory placode associated frontonasal prominences (mFNP and lFNP), denoted the upper “Caps” (upper blue discs). (d) Diagram of the components of the mature λJ, a complex, significant ontogenetically dynamic organizing region formed from the coalescence of a number of cell populations from which emanate striking patterns of signaling molecule and transcription factor gene expression that rim the olfactory pits, FNP, nasolacrimal grooves, mxBA1, and extensions toward Rathke's pouch, and underlying the optic stalk. Diagrammed as blue rami, the elements of the mature λJ include: 1, maxillary; 2, odontogenic; 3, inner medial pit; 3′, outer medial pit; 4, inner lateral pit; 4′, outer lateral pit; 5, lateral prominence; 6, nasolacrimal; 7, substalk oral; 8, Rathke's; 9, commissural; and 10, central/median body. (e) Schemae of late fetal and neonatal mouse skulls (1, 2) and chondrocrania (3–5). The darker blue portions indicate skeletal structures associated, in whole or in part, with λJ patterning (the epicenters of which are indicated by the blue discs). Abbreviations: cmp, commissural plate; cp; cribriform plate; fr, frontal; hyp, hypophyseal axis; MC, Meckel's cartilage; mdBA1, mandibular first arch; mx, maxillae; mxBA1, maxillary first arch; na, nasal; nc, nasal capsule; ne, neuroepithelium; nld, course of the nasolacrimal duct; ns, nasal septum; oe, oral ectoderm; olf, olfactory ectoderm; opc, optic capsule; pal, paltine; PC1, first pharyngeal cleft; pmx, premaxillae; ps, presphenoid; psc, paraseptal cartilage; RP, Rathke's pouch; tbp, trabecular basal plate; λmc, centre of mature λJ.
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Pax6 is expressed early during embryogenesis in the anterior ectoderm, including that associated with the forming neurectoderm, eye, nose, and the λ-junction (Fig. 1; Grindley et al.,1995; Walther and Gruss,1991). Pax6 appears to be involved with optic, olfactory, brain, and pancreatic development but it is also required for proper craniofacial skeletal development (Collinson et al.,2003; Hill et al.,1991; Hogan et al.,1986; Kaufman et al.,1995; Matsuo et al.,1993; Osumi-Yamashita et al.,1997a; Quinn et al.,1996,1997). There are more than 20 known mutations involving the rodent Pax6 locus (collectively referred to as Small eye alleles for the hypoplastic eyes of the heterozygotes) and a range of cephalic phenotypes have been documented for these mutations (Hill et al.,1991; Hogan et al.,1986). While the optic, olfactory, and forebrain nervous system defects in a small number of these Pax6 mutants, in particular the rat rSey/rSey and mouse Pax6Sey/Sey and Pax6Sey-neu/Sey-neu (hereafter Pax6SeyN/SeyN) lines, have been analyzed many times, the craniofacial skeletal deficits encountered and their mechanistic etiologies have been less well characterized (Depew et al.,2002b).
With regard to skeletal development, the most extensively investigated Pax6 mutant has been the rSey/rSey mutant. Analysis of the rSey/rSey mutants demonstrated a number of regional defects in the olfactory and optic capsules and associated dermatocranium. Because Pax6 is not expressed in the mesenchymal populations that directly yield the skeletal structures that are deficient in the rSey/rSey mutants, noncell autonomous explanations for these deficiencies have previously been investigated (Fujiwara et al.,1994; Matsuo et al.,1993; Nomura et al.,2007; Osumi-Yamashita et al.,1997a). As most of the defective skeletal elements are derived from the CNC, this population of cells has been most intensively investigated in the rSey/rSey mutants. For instance, using a DiI-labeling approach, it was seen that a subpopulation of CNC cells migrating from the anterior midbrain stack up behind the malformed eye primordia rather than populate the frontonasal region; all other populations of CNC cells were seen to migrate normally (Matsuo et al.,1993; Osumi-Yamashita et al.,1997a,b). Fate-mapping evidence suggested that this population of anterior midbrain CNC migrated specifically to the lFNP, the derivatives of which were thought to be specifically lost in the mutant. Armed with this apparent correlation, investigators then sought an explanation for this stacking phenomenon, and found a specific upregulation in the HNK-1 epitope (a carbohydrate residue found on various cell adhesion molecules) in the SCE around the eye (Nagase et al.,2001). It was thus concluded that (1) loss of Pax6 resulted in upregulated, focal levels of HNK-1; (2) this traps the anterior midbrain CNC, which then fails to reach the frontonasal region; (3) deficiencies in the elaboration of the lFNP skeletal derivatives ensue.
A number of reasons, however, suggest that this is not a sufficiently complete explanation for the craniofacial skeletal defects found in the Pax6SeyN/SeyN mouse mutants. First, the murine and the rat Small eye mutants exhibit some phenotypic incongruity: for instance, the rSey/rSey mutants exhibit an ectopic cleft within the mxBA1 (Kriangkrai et al.,2006; Matsuo et al.,1993) but mouse mutant embryos do not (see Fig. 2). Second, all but a small subpopulation of Hox-negative CNC has been shown to enter the region of the optic and olfactory primordia of both the rat and murine mutant embryos (Matsuo et al.,1993; Nomura et al.,2007; Osumi-Yamashita et al.,1997a,b). This is significant because, as suggested by various neural fold rotation, regeneration, and ablation studies, the Hox-negative CNC—which includes the freely mixing forebrain crest as well as the posterior and anterior midbrain crest—appears to act as an “equivalence group” with regard to translating patterning information into morphology (Creuzet et al.,2002,2005; Kuratani,2005a,b; Le Douarin et al.,2004). Third, Pax6Sey/+ embryos exhibit a similar stacking of CNC behind the eye to the one that is seen in the rSey/rSey mutants (Kanakubo et al.,2006) and yet do not exhibit severe skeletal defects. And fourth, a long history of experimental evidence had suggested a more direct, primal role for the SCE in regional skeletal development, as exemplified, for instance, by evidence that the control of nasal capsular chondrogenesis is mediated by the regional placodal epithelium (Bell,1907; Burr,1916; Corsin,1971; LaMantia et al.2000; Reiss,1998; Toerien and Roussouw,1977; Zwilling,1940).
Figure 2. Hypotheses correlating the loss of Pax6 with subsequent craniofacial skeletal defects. (a) Recently migrated (green arrows) Hox-negative CNC engage in reciprocal epithelial–mesenchymal cross-talk with the SCE (depicted on pseudocolored scanning electron micrographs of E9 (1), E9.5 (2), and E10.5 (3) and E11 (4) wild type embryos), resulting in a mature λ-junction (λJ). Epithelial subregions: red, eye; blue, lFNP; green, mFNP; yellow, mxBA1; lavender, mdBA1. (b) The “Inhibited Midbrain CNC Migration Hypothesis.” A subpopulation of Hox-negative CNC (red T-bars) migrates as far as the caudal eye but is inhibited (signified by the X) from further migration in to the frontonasal region (1). Migration of other Hox-negative CNC (green arrows) proceeds to the medial frontonasal region (green) (2). This foreordained CNC specifically generates medial FNP primordia (green) but no lateral FNP structures (red Xs) as this crest has failed to migrate into place (3-4). (c) The proposed “Dysfunctional λ-Junction Hypothesis.” (1) In this scenario, a subpopulation of Hox-negative CNC (red T-bars) migrates as far as the caudal eye but does not continue to the frontonasal region. The rest of the Hox-negative CNC migrates as usual (green arrows) but does so in an environment (blue) that is molecularly deficient (white Xs). (2) This deficiency inhibits placodogenesis and the formation of the λJ (X), and subsequently vitiates (blue Xs, 3-4) the formation and functionality of mature λJs.
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We therefore hypothesized that the cranial skeletal phenotype of the Pax6SeyN/SeyN mutant is not simply due to CNC migration defects but to an inability of the SCE to elaborate a fully functioning λ-junction (see Fig. 2). We posited that Pax6 is required for the competence of the SCE to manifest a mature λ-junction, and its absence vitiates the patterning information that the λ-junction usually imparts to the underlying mesenchyme: thus, the CNC receives insufficient information to yield the normal craniofacial skeletal structures associated with the junction.
To explore this hypothesis, we addressed the following issues. First, to clarify whether early embryonic phenotypic incongruity correlates with later skeletal incongruities, we describe herein the Pax6SeyN/SeyN mutant skull and compare it with that reported for the rSey/rSey. We then determine whether Pax6SeyN/SeyN embryos exhibit molecular patterning characteristics of a normal frontonasal region at late pharyngeal stages when the craniofacial primordia are patent. Finally, we directly investigate evidence of a disruption in the ontogenetic maturation of the cellular and molecular characteristics of the λ-junction due to the loss of Pax6. We show that there is a specific disruption in the ontogenetic elaboration (spatial and temporal) of SCE gene expression, before and during the formation of the mature λ-junction, of essential secreted signaling factors or their effectors (such as Fgf8, Bmp4, and Raldh3) and of transcription factors (such as Six1 and Dlx5) variably implicated in upper jaw and nasal capsular development. Thus, we demonstrate that the epithelial environment that the Pax6 mutant Hox-negative CNC encounters in the frontonasal region outside of the eye is molecularly compromised before the CNC begins to execute its morphogenetic programs. This supports a model wherein early Pax6 expression in the SCE acts as an epithelial competence factor essential for the ectoderm to engage in regional cross-talk with the CNC to establish a proper λ-junction.
- Top of page
- MATERIALS AND METHODS
- LITERATURE CITED
- Supporting Information
Craniofacial patterning and morphogenesis requires an exquisitely timed and positioned reciprocal cross-talk between the pertinent embryonic epithelia and mesenchyme. This is particularly true of the development associated with the morphologically and molecularly complex λ-junction, whose organization reflects the future positions of the internal and external choanae and the formation of the upper lips, nasal capsules, upper incisors, and primary and secondary palates. Elaboration of this cross-talk is manifested in the induction and maintenance of intricate patterns of gene expression. The spatial and temporal details of these patterns underlie the precise translation of patterning processes and information into appropriate skeletal morphologies.
Outside of dental enamel, cephalic epithelial tissues involved in this dialogue are not known to directly yield cranial skeletal structures: their importance to skeletal form hence largely lies in their influences on the CNC mesenchyme that forms the cranial tissues. Understanding the ontogeny of such influences on the CNC, including the inception and maturation of the λ-junction from the SCE, remains a key endeavor. Evidence for the developmental importance of the SCE for cranial skeletal development has come from a number of directions, including experimental embryological studies involving ectoderm extirpation, tissue recombination, and bead implantation. For example, embryological studies manipulating portions of the nascent SCE in chicks have identified a subregion, identified as the “facial ectodermal zone,” and factors expressed therein such as Fgf8, Shh, and Bmp4, as critical for the development of subcomponents of the cranial skeleton (Hu et al.,2003; Marcucio et al.,2005).
Although generally not emphasized, phenotypic analyses of mutations of genes expressed in the ectoderm (and not the associated CNC) have also clearly demonstrated the necessity of a properly informed and competent SCE (Depew et al.,2002b). The essential issue in such studies has been how to detail and explain the nature of the noncell autonomous actions exerted by these genes on the CNC: this has particularly been true in the case of the Pax6 mutants. Previous studies of Pax6 mutations have suggested that this occurs via an upregulation in expression of HNK-1, a putative adhesion-related molecule, and that this inhibits the migration of a subpopulation of midbrain CNC to the frontonasal region which subsequently impairs the development of specific cranial skeletal elements (Matsuo et al.,1993; Osumi-Yamashita et al.,1997a,b).
An inherent premise of this “inhibited migration” hypothesis has been that the CNC reaching the lFNP is “prepatterned” before its final destination: absence of structure is due to absence of a strictly foreordained population of cells. However, accumulating evidence, including that suggesting the “equivalence” of the Hox-negative CNC and that demonstrating a specific role for the SCE in the formation of regional cranial skeletal structures, is at variance with this premise. Despite a portion of midbrain CNC failing to migrate past the eye, by E9-9.25 there are equipotent midbrain and forebrain CNC that do reach the frontonasal region (see Fig. 4): without recourse to “pre-patterned” crest, there is no reason why fungible CNC that do migrate to the frontonasal region should fail to compensate for the absent fraction. Such reasoning led us to challenge the established view that the etiology of Pax6 mutant cranial skeletal deficiencies lies essentially in a lost subpopulation of CNC. Following from the “Hinge and Caps” model, we hypothesized that the Hox-negative CNC in the Small eye mutants in general, and of the Pax6SeyN/SeyN mutants in particular, fail to generate a full complement of regional structures because the informative epithelial environment of the SCE that they have migrated into contact with has been altered to the point that it cannot support and elaborate the fully functioning λ-junction: this vitiates the complex epithelial-mesenchymal dialogue that normally regulates the formation of the nasal capsules, trabecular basal plate, and upper jaws.
To address this “dysfunctional λ-junction” hypothesis, we first compared the morphologic phenotype reported for the rSey/rSey mutants with that of the Pax6SeyN/SeyN mutants. In most significant respects, the Pax6SeyN/SeyN skull defects mirror those reported for the rSey/rSey: for instance, perinates of both mutants have similar catastrophic malformations of their nasoethmoidal structures, possessing truncated trabecular basal plates and lacking naral openings and elaborated nasal capsules. In the Pax6SeyN/SeyN mutants, however, the caudal end of the trabecular basal plates is abnormally bifurcated, giving the region a distinct triton-like appearance (see Fig. 3). Although this separation of the caudal trabecular basal plate has not been reported for the rSey/rSey mutants (Matsuo et al.,1993; Osumi-Yamashita et al.,1997a), we find that this defect is also evident in perinates of a disparate murine mutant (Pax6LacZ/LacZ; Supporting Information Fig. 1).
Notably, the cranial skeletal defects encountered in both rodent mutants correlate with structures associated with the λ-junction. This strongly suggests that the λ-junction itself is deficient. Indeed, we argue that lack of Pax6 alters the formation and elaboration of a normal λ-junction, thereby altering skeletal morphology, for a number of reasons. First, it is clear that the epithelium of the mature λ-junction in E10.5 Pax6SeyN/SeyN mutant embryos fails to properly express critical markers of the components of the junction. This includes transcription factors, such as Dlx3, Dlx5, p63, and Six3, that have been shown to be necessary for proper cranial skeletal development and morphology.
Second, based largely on the morphological criteria of a lack of a thickened placodal epithelium, it has previously been suggested that the murine Small eye mutants fail to form olfactory placodes (Hogan et al.,1986; Grindley et al.,1997). Although the specifics of the correlation between the absence of a thickened epithelium denoting an olfactory placode and loss of nasal capsular structures has yet to be fully investigated and detailed, extirpation studies have suggested that the epithelium of the olfactory placode is essential to the formation of at least the nasal capsule. Without an olfactory placode, moreover, the morphologic events surrounding the formation of the nasal pits fail to manifest.
We have herein demonstrated that, in the absence of Pax6, gene expression patterns in the SCE of placode-essential transcription factors are disrupted in and around the presumptive olfactory placode epithelium before and during the initiation of placodogenesis. This is exemplified by the loss and/or downregulation of Six1 and Dlx5 in the SCE at E9-9.25 (see Fig. 7). Thus, Six1 and Dlx5, each a contributor to the mature λ-junction, are already mis-expressed or downregulated during placodogenesis and the initiation of λ-junction formation—a period when the crest has arrived and is ready to begin interpreting its environment. Notably, loss of function studies show that both of these genes, in addition to being essential for some aspect of olfactory placodal development, are required for the proper development of the nasal capsules (Depew et al.,1999; Laclef et al.,2003; Zou et al.,2004). This further correlates Pax6 expression in the SCE with the loss of placode development and activity, including placodal influence on the formation of the λ-junction.
Third, the ontogenetic progression of the λ-junction is perturbed by early changes in the patterns of genes encoding numerous secretory signaling molecules, and/or their effectors, known to be critical for craniofacial patterning and development. These include molecules involved in RA, Bmp, and Fgf signaling. Raldh3 mutant mice exhibit defects correlated with a disruption of the λ-junction, including the agenesis of the eye-associated Hardian glands, choanal atresia, and hypoplasia of the lateral nasal capsule (particularly the turbinals) (Dupe et al.,2003). We (and others) have failed to detect cephalic Raldh3 in Small eye mutants (Figs. 6 and 7; Enwright and Grainger,2000; Suzuki et al.,2000), placing Raldh3 at the center of previous observations that Pax6SeyN/SeyN mutant FNP exhibit severely decreased RA signaling (Anchan et al.,1997).
Bmp4 is ontogenetically dynamically expressed in the SCE epithelia, including the λ-junction, and has been shown to be involved in the epithelial–mesenchymal and epithelial–epithelial interactions that regulate gene expression patterns and overall craniofacial development (Abzhanov et al.,2004; Barlow and Francis-West,1997; Foppiano et al.,2007; Jiang et al.,2006; Lee et al.,2001; Liu et al.,2005; Wu et al.,2004). As with RA signaling, regulated levels of Bmp4 appear to be critical to proper craniofacial development: in this regard, we find clear spatial misexpression of Bmp4 in and around the mature λ-junction (E10.5) of Pax6SeyN/SeyN mutant embryos (see Fig. 6). Although increased overall (in particular in the SCE associated with the abortive eyes), Bmp4 expression appears patchier and fails to normally delineate numerous rami of the λ-junction in mutant embryos. Contrary to earlier reports (Furuta and Hogan,1998), we find that E9-9.25 mutant embryos show a clear increased level of Bmp4 expression overlying the presumptive optic lens placode. The altered ontogeny of expression of this secreted signaling factor reflects the ontogenetic alteration of the λ-junction itself, and likely contributes to the craniofacial defects observed.
FGF superfamily signaling has also been implicated in numerous developmental processes, including cranial patterning and growth. Aberrant FGF signaling due to mutation in ligands or receptors, leads variously to craniosynostoses and cleft lip and palate (Dode et al.,2007; Riley et al.,2007). There is moreover strong evidence for the involvement of Fgf8 in the development of the λ-junction and the structures informed by it. In mice, for instance, Fgf8 has specifically been shown to regulate craniofacial pattern and development (reviewed in Depew et al.,2002b). Notably, mice which have either lost Fgf8 in the SCE due to conditional inactivation of a floxed allele (Kawauchi et al.,2005) or which bear hypomorphic alleles of Fgf8 (Abu-Issa et al.,2002; Frank et al.,2002) demonstrate the necessity of Fgf8 for olfactory placode and nasal capsule development. This work has been augmented by experimental embryological studies in birds where over- or under-expression of Fgf8 has been shown to affect the pattern and development of the craniofacial skeleton (Abzhanov and Tabin,2004; Szabo-Rogers et al.,2008). Significantly, the dynamic ontogenetic choreography of Fgf8 expression in the SCE is particularly out of step in Pax6SeyN/SeyN mutant embryos (see Fig. 7). In its ontogeny, as with that of Bmp4 and Raldh3, Fgf8 expression reflects a dynamic change in the signaling environment to be encountered by the Hox-negative CNC responsible for generating the structures malformed or lost in the Pax6SeyN/SeyN mutants.
A preponderance of molecular and morphologic evidence therefore indicates that Pax6 is essential for the competence of the SCE to engage in and execute the exquisitely choreographed spatiotemporal dialogue between the cephalic epithelia and mesenchyme that determines the translation of patterning information into the morphogenesis of the distinct, intricate skeletal morphologies of the skull. Its role in parsing the early SCE into appropriate placodal regions has previously been demonstrated and its interplay with Fgf8 and Dlx5 (among others) during placodogenesis has been appreciated (Bailey et al.,2006; Bhattacharyya et al.,2004). We have demonstrated, however, that Pax6 involvement in craniofacial development in general, and in the maturation of the SCE in particular, is more intricate and pervasive than perhaps its roles just in placodogenesis alone may have previously suggested. Pax6 is normally expressed early (E8-E8.5) in the SCE when the CNC is beginning its migration to the region of the future λ-junction: loss of Pax6, however, sets in motion a cascade of altered, reduced, or absent expression patterns of genes essential for normal craniofacial skeletal development. This includes, among others, early changes in the epithelial expression of genes encoding the placode-essential transcription factors Six1 and Dlx5 and the secreted signaling molecules Bmp4 and Fgf8 (as well as of that for the RA synthesizing molecule Raldh3), at embryonic stages when the CNC has just finished migrating and placodogenesis and the initiation of the λ-junction have just begun (see Fig. 8). Either directly or indirectly, this has a knock-on effect with regard to the elaboration of a normally functioning λ-junction.
We share with previous investigators the notion that loss of Pax6 leads to changes in the SCE, although perhaps not the nature and significance of all such changes. We further believe that in order for the inhibited migration hypothesis to be a valid explanation for the etiology of the craniofacial deficits seen in Small eye mutants, the anterior midbrain CNC has to either be extensively prepatterned with regard to its potential to yield distinct skeletal morphologies or else gather indispensable patterning information around the eye that it then normally passes on to the frontonasal epithelia and/or forebrain CNC—which in turn uses this information in the formation of the λ-junction. Accumulating evidence argues against the former, while the latter has yet to be conclusively demonstrated. In addition, we reconfirm here that significant CNC is present in the Pax6SeyN/SeyN mutant frontonasal region and that this neural crest maintains a degree of normal molecular patterning, including some gene expression characteristic of both the medial and lateral FNP. This suggests that cells competent to form the lFNP are present. We therefore adduce the molecular disruption of λ-junction ontogeny, due to the early loss of SCE Pax6 expression, as underling the failure of the FNP in Pax6SeyN/SeyN mutants to be fully elaborated and generative of the appropriate cranial skeletal elements.