Interaction between Foxc1 and Fgf8 during Mammalian Jaw Patterning and in the Pathogenesis of Syngnathia

Syngnathia (bony fusion of the upper and lower jaw) is a rare human congenital condition, with fewer than sixty cases reported in the literature. Syngnathia typically presents as part of a complex syndrome comprising widespread oral and maxillofacial anomalies, but it can also occur in isolation. Most cartilage, bone, and connective tissue of the head and face is derived from neural crest cells. Hence, congenital craniofacial anomalies are often attributed to defects in neural crest cell formation, survival, migration, or differentiation. The etiology and pathogenesis of syngnathia however remains unknown. Here, we report that Foxc1 null embryos display bony syngnathia together with defects in maxillary and mandibular structures, and agenesis of the temporomandibular joint (TMJ). In the absence of Foxc1, neural crest cell derived osteogenic patterning is affected, as osteoblasts develop ectopically in the maxillary prominence and fuse with the dentary bone. Furthermore, we observed that the craniofacial musculature is also perturbed in Foxc1 null mice, which highlights the complex tissue interactions required for proper jaw development. We present evidence that Foxc1 and Fgf8 genetically interact and that Fgf8 dosage is associated with variation in the syngnathic phenotype. Together our data demonstrates that Foxc1 – Fgf8 signaling regulates mammalian jaw patterning and provides a mechanistic basis for the pathogenesis of syngnathia. Furthermore, our work provides a framework for understanding jaw patterning and the etiology of other congenital craniofacial anomalies, including temporomandibular joint agenesis

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

Foxc1^−/− neonates exhibit syngnathia and TMJ agenesis.

<p>Alizarin red (bone) and alcian blue (cartilage) stained skeletal preparations of <i>Foxc1^+/+</i> (A, C, E, F) and <i>Foxc1^−/−</i> (B, D, G) P0 neonates. (A,B) Intact view of cranial skeleton showing relative position of upper and lower jaw elements. Syngnathia (syn*) is evident in <i>Foxc1^−/−</i> neonates. Circles highlight the articulating joint. Arrows in A and B highlight lack of ossification of malleus, incus, and stapes in mutant middle ear. (C) Dissected wild-type maxilla (mx) and jugal (jg). (D) The mutant maxilla is fused in the zygomatic region to the dentary (dnt) which displays hypoplastic coronoid (crp), condylar (cdp), and angular (agp) processes compared to controls (E). The <i>Foxc1^−/−</i> condyle is bifurcated (arrowhead in D). (F, G) The mutant squamosal (sq) and alisphenoid (als) are hypoplastic, and the squamosal lacks a zygomatic process (asterisk). Scale bars: 500 µm Abbreviations: cps, caudal process of squamosal; fmx, frontal process of maxilla; iof, infraorbital foramen; li, lower incisor; rps, retrotympanic process of squamosal; zmx, zygomatic process of maxilla; zps, zygomatic process of squamosal.</p

Early morphologic abnormalities of <i>Foxc1−/−</i> PA1 detected despite no significant alteration of cell cycle dynamics or apoptosis.

<p>(A) Confocal z-stack projections of DAPI stained, whole mount <i>Foxc1^+/+</i> and <i>Foxc1^−/−</i> embryos showing gross PA1 morphology. A red asterisk marks the maxillary prominence in all panels. A white arrowhead indicates the maxilla-mandibular junction (Mx-Md). At E9.0-9.5, the mutant maxillary prominence is smaller than wild-type, and the Mx-Md is shallower than in controls. By E10.5, the lambdoidal junction (λ) and nasal prominence epithelium has formed in <i>Foxc1^+/+</i> and <i>Foxc1^−/−</i> embryos. The <i>Foxc1^−/−</i> maxillary prominence continues to develop between E10.5-11.5, but remains smaller compared to wild-type embryos. The distance from the λ to Mx-Md junction is reduced in <i>Foxc1^−/−</i> embryos. (B) Representative sections of IdU and BrdU immunostained embryos. (C) Average S-phase and cell cycle lengths were determined based upon incorporation of IdU and BrdU. A trend toward longer S-phase and cell cycle length was noted in the cranial mesenchyme at 5–7-s. (D) Projections of confocal Z-stacks showing whole mount TUNEL staining to detect apoptotic cells in <i>Foxc1^+/+</i> and <i>Foxc1^−/−</i> embryos at E8.75 and E9.0. Although change in morphology of PA1 is evident, no abnormal level or localization of apoptosis is detected in the mutants. Scale bars: 100 µm.</p

Dynamic expression of <i>Foxc1</i> in PA1.

<p>Whole mount <i>in situ</i> hybridization (A–F) and β-galactosidase (β-gal) (G, H) staining showing a timecourse of expression of <i>Foxc1</i> in the developing first pharyngeal arch. (A–C) At E8.5, <i>Foxc1</i> is broadly expressed in the cranial mesenchyme, PA1 mesenchyme and strongly expressed in the oral ectoderm. (D, E) By E9.5, expression is diffuse within PA1 mesenchyme, and is no longer detected in the PA1 oral ectoderm. (F–H) By E10.5, a discrete domain of <i>Foxc1</i> is detected in the mandibular mesenchyme, which is more easily seen in β-gal stained specimens. Scale bars: A, B, D, F–H, 200 µm; C, E, 100 µm.</p

Time-course of PA1-derived skeletal abnormalities in <i>Foxc1^−/−</i> mice.

<p>(A, D) Gross view of fixed <i>Foxc1^+/+</i> (A) and <i>Foxc1^−/−</i> (D) embryos at E13.5. Cerebral hemispheres are enlarged and the snout is foreshortened in the mutant. (B, E) Whole mount alcian blue staining to detect cartilage differentiation in the wild type (B) and mutant (E) embryos pictured in (A, D). A single, normal Meckel's cartilage (arrows in B, E) is seen in both control and <i>Foxc1^−/−</i>. (C, F) Bissected E13.5 heads of <i>Foxc1^+/+</i> (C) and <i>Foxc1^−/−</i> (F) embryos stained for endogenous alkaline phosphatase (AP) activity to detect early osteoblast differentiation. In the absence of <i>Foxc1</i>, early osteoblasts of the dentary (dnt) and maxillary (mx) region initially differentiate in a fused, syngnathic pattern (*). (G–L) Whole mount alcian blue (cartilage) and alizarin red (bone) staining of <i>Foxc1^+/+</i> (G, I, K) and <i>Foxc1^−/−</i> (H, J, L) embryos. (G, H) At E15.0, ossification of the wild type maxilla (mx) with a wispy frontal process (fmx) and dentary can be seen. In the mutant, wispy projections are seen in the maxillary region similar to controls. However, this ossification is connected to a larger ossified region that is fused (syn*) directly to the dentary. (I–L) In <i>Foxc1^+/+</i> embryos at E16.5 (I) and E18.5 (K), the zygomatic process of the maxilla (zmx), jugal (jg), zygomatic process of the squamosal (zps), and alisphenoid (als) are clearly identified as separately ossifying elements. By contrast, in the <i>Foxc1^−/−</i> specimens (J, L), syngnathia is observed in the zygomatic region, rather than the distinct elements found in wild type. The alisphenoid and the squamosal (sq) are hypoplastic compared to controls. The zygomatic process of the squamosal does not form (arrowheads). Scale bars: 500 µm Abbreviations: agp, angular process; cdp, condylar process; crp, coronoid process; mx, premaxilla; tongue, tg.</p

<i>Foxc1</i> is required to maintain <i>Fgf8</i> signaling and genetically interacts with <i>Fgf8</i>.

<p>(A) <i>Fgf8</i> expression is maintained in the frontonasal prominence and midbrain-hindbrain boundary regions of <i>Foxc1^−/−</i> embryos, but it is reduced in the PA1 oral ectoderm (red asterisks). (B) Quantification of <i>Fgf8</i> mRNA in <i>Foxc1</i>, <i>Fgf8^Null/+</i>, and <i>Fgf8^Null/Neo</i> embryos. (C) <i>Foxc1</i> is normally localized in <i>Fgf8^Null/Neo</i> embryos. (D) Quantification of <i>Foxc1</i> mRNA in <i>Foxc1</i>, <i>Fgf8^Null/+</i>, and <i>Fgf8^Null/Neo</i> embryos. Gross appearance (E–H) and skeletal preparations (I–P) of E17.5 embryos comparing <i>Fgf8^Null/+</i> (E, I, M), <i>Foxc1^−/−</i> (F, J, N), <i>Fgf8^Null/Neo</i> (G, K, O), <i>Foxc1^+/−</i>; <i>Fgf8^Null/+</i> (L, inset), and <i>Foxc1^−/−;Fgf8^Null/+</i> (H, L, P) phenotypes. In both gross view (E) and skeletal preparations (I, M), <i>Fgf8^Null/+</i> are indistinguishable from wild-type embryos. (F) <i>Foxc1^−/−</i> embryos have shortened frontonasal regions, open eyelids, abnormal and shifted external ears, and enlarged, hydrocephalic cerebral hemispheres. (G) <i>Fgf8^Null/Neo</i> embryos have a more rounded frontonasal region, small lower jaw, and abnormal, shifted external ears. (H) Compound <i>Foxc1^−/−;Fgf8^Null/+</i> embryos resemble <i>Foxc1^−/−</i> specimens, but have more severe frontonasal shortening and no externally visible oral opening/lower jaw (black arrowhead). (J, N) Hypoplastic squamosal (sq), syngnathia (syn*), and abnormal condyle formation in the absence of <i>Foxc1</i>. This specimen shows fusion in alveolar region of dentary and absence of the coronoid process (arrow in N). (K, O) Severe hypoplasia and malformation of the squamosal (sq*) was observed in <i>Fgf^Null/Neo</i> specimens. The frontal process of the maxilla with a characteristic infraorbital foramen (iof) formed, and the maxilla fused to the dentary in the alveolar region, more distally than seen in <i>Foxc1^−/−</i>. The proximal processes of the dentary are absent (asterisk in O), but distal incisors form. (L, P) In <i>Foxc1^−/−;Fgf8^Null/+</i> embryos, the syngnathic phenotype is further exacerbated. No squamosal formed and a small frontal process of the maxilla is attached to the hypoplastic maxilla. This region is fused to the dentary just proximal to the lower incisors resulting in flattening of the normally curved dentary. The proximal dentary is severely truncated and lacks all processes (asterisk in P). (inset in L) <i>Foxc1^+/−;Fgf8^Null/+</i> compound heterozygote (2/12) in which calvaria had developed normally. This specimen also displayed a syngnathic jaw with TMJ abnormalities grossly identical to that of the <i>Foxc1</i> null. Scale bars: (A,C) 200 µm; (G–H) 1000 µm; (M–P) 500 µm.</p

Molecular analysis of developing TMJ in <i>Foxc1^−/−</i> embryos.

<p><i>In situ</i> hybridization analysis on serial coronal cryosections in <i>Foxc1^+/+</i> (A, C, E, G, I, K) and <i>Foxc1^−/−</i> (B, D, F, H, J, L) mouse heads at E17.5. In (A–L) dashed lines outline the wild-type and bifurcated mutant condyles (cdp and cdp*). (A, B) <i>Scx</i> is expressed in the neural crest-derived disc (arrow) and tendon (red arrowheads) of controls. In mutants, Scx is maintained in the tendon (red arrowheads), but no disc is present. (C–F) <i>Sox9</i> is localized to the proliferating chondrocytes and <i>Acan</i> is localized to the cartilage of the condyle growth plate in wild-type and <i>Foxc1^−/−</i> condylar processes. (G, H) <i>Ihh</i> is localized to the prehypertrophic chondrocytes in wild-type and mutant condyles. (I, J) <i>ColX</i> is expressed in the zone of the hypertrophic chondrocytes of the condylar growth plate. In the absence of <i>Foxc1</i>, the hypertrophic chondrocytes make up a larger proportion of the abnormally bifurcated condyle. (K, L) <i>Coll</i> expression is localized to the osteoblasts of the condyle in control and <i>Foxc1^−/−</i> embryos; however, the <i>Coll</i> positive glenoid fossa (f) is only observed in control specimens.</p

Abnormalities of PA1 derived muscle in <i>Foxc1^−/−</i>.

<p>(A,B) Whole mount immunostaining for neonatal myosin II in P0 wild-type (A) and <i>Foxc1^−/−</i> (B) heads. The PA1 masseter (ma) and temporalis (tm) muscles are smaller in the mutant than in wild-type. (C–G) Immunostained paraffin sections of E17.5 <i>Foxc1^+/+</i> (C, E, G) and <i>Foxc1^−/−</i> (D, F, H) heads showing muscle actin localization (brown). Sagittal (C, D) and frontal (E, F) sections showing organized muscle formation in both wild type and mutant tongues (tg). The palate (pl) is more arched in <i>Foxc1^−/−</i> and shows smoother rugae (r) than in <i>Foxc1^+/+</i>. (G, H) Frontal sections in the TMJ region. The mutant temporalis muscle (tm) is shifted medially, aberrantly associates with the bifurcated condyle (cdp*), and its fibers are oriented differently than in wild type. The medial (mp) and lateral (lp) pterygoid muscles are appropriately associated with the angular (agp) and condylar processes in <i>Foxc1^−/−</i>. The mutant masseter (ma) is reduced to a small component in <i>Foxc1^−/−</i> (compare ma regions in F, H to E, G). (I, J) Ventral view of whole mount myosin II immunostained P0 wild type (I) and <i>Foxc1^−/−</i> (J) heads. The second heart field derived anterior digastric muscle (ad) is robustly detected in all specimens. Scale bars: 500 µm Abbreviations: dnt, dentary; eom, extraocular muscle; f, glenoid fossa; g, genioglossus muscle; my, mylohyoid muscle; mx, maxilla; trg, trigeminal ganglion.</p