Mesodermal expression of Fgfr2S252W is necessary and sufficient to induce craniosynostosis in a mouse model of Apert syndrome

Abstract

Coordinated growth of the skull and brain are vital to normal human development. Craniosynostosis, the premature fusion of the calvarial bones of the skull, is a relatively common pediatric disease, occurring in 1 in 2500 births, and requires significant surgical management, especially in syndromic cases. Syndromic craniosynostosis is caused by a variety of genetic lesions, most commonly by activating mutations of FGFRs 1-3, and inactivating mutations of TWIST1. In a mouse model of TWIST1 haploinsufficiency, cell mixing between the neural crest-derived frontal bone and mesoderm-derived parietal bone accompanies coronal suture fusion during embryonic development. However, the relevance of lineage mixing in craniosynostosis induced by activating FGFR mutations is unknown. Here, we demonstrate a novel mechanism of suture fusion in the Apert Fgfr2(S252W) mouse model. Using Cre/lox recombination we simultaneously induce expression of Fgfr2(S252W) and β-galactosidase in either the neural crest or mesoderm of the skull. We show that mutation of the mesoderm alone is necessary and sufficient to cause craniosynostosis, while mutation of the neural crest is neither. The lineage border is not disrupted by aberrant cell migration during fusion. Instead, the suture mesenchyme itself remains intact and is induced to undergo osteogenesis. We eliminate postulated roles for dura mater or skull base changes in craniosynostosis. The viability of conditionally mutant mice also allows post-natal assessment of other aspects of Apert syndrome.

Fig. 1

Mesodermal expression of Fgfr2^S252W causes CS. (A) Heads of 1-year old wild-type (WT; upper panel) and Meso^S252W/+ (MesoAp; lower panel) mice. (B) Dorsal view of skulls from mice in (A). Vertical bars mark the suture position between interparietal (ip), parietal (p), frontal (f) and nasal (n) bones. The short vertical bar marks the jugum limitans of the frontal bone. The dotted line along the MesoAp internasal suture indicates the curvature of the snout. (C) Dorsal view of coronal sutures showing full (middle panel) or partial fusion (lower panel) in MesoAp skulls. The interfrontal suture is at left. In the lower panel, lateral fusion is interrupted by patent regions (arrowheads). The upper and lower panels are from the same skulls as in (B). (D) Ventral view of palatal extensions of the premaxilla (pm) and maxilla (m) of skulls in (B). (E) Dorso-lateral view of basisphenoid (bs), intersphenoidal synchondrosis (iss), and presphenoid (ps) in a second pair of WT and MesoAp mice. (F) Dorsal view of the ethmoid (e) and anterior frontal (f) bones of the MesoAp mouse in (E). Black arrowhead indicates partial fusion of the frontoethmoidal suture.

Fig. 2

Neural crest expression of Fgfr2^S252W causes facial shortening but not CS. (A) Heads of 3-week old WT (upper panel) and mediumly affected (middle panel) or strongly affected (lower panel) NC^S252W/+(NCAp) mice. (B) Lower incisors of mice in (A). (C) Dorsal view of skulls from mice in (A). The left parietal is removed in the lower panel. The NCAp skull in the middle panel shows zygomatic arch fusion (arrowhead) and a deviated snout (dotted line). (D) Ventral view of the palate in WT (left panel) and NCAp (right panel) skulls. Shortening of the maxillary (m) and palatine (p) bones and fusion of the maxillary–palatine (arrowhead) and interpalatine (arrow) sutures occurs in NCAp mice. (Same mice as in A, upper and lower panels). (E) Ventral view showing curvature of the snout and palate in an NCAp mouse at 3 weeks. (F) Dorsal view of the ethmoid plate in an NCAp mouse shows extensive fusion of the dorsal frontoethmoidal suture between the two arrows. (G) PCR to detect the wild-type Fgfr2 (black arrow) and Cre-recombined Fgfr2^S252W (blue arrow) alleles in genomic DNA prepared from individual frontal (F) and parietal (P) bones of two WT (Fgfr2^NeoS252W/+) and two NC^S252W/+ (Wnt1Cre/Fgfr2^NeoS252W//+) newborn mice. The larger PCR band contains the additional LoxP sequence resulting from Neo excision. The schematic of frontal and parietal bones below indicates the region of expected Wnt1Cre activity in blue.

Fig. 3

The neural crest/mesoderm border remains intact during coronal suture fusion. (A–D) Coronal sutures of (A and B) WT(R26R/Mesp1Cre/Fgfr2^+/+) and (C and D) Meso^S252W/+ (MesoAp; R26R/Mesp1Cre/Fgfr2^NeoS252W/+) E14.5 littermates. In adjacent sections, mesoderm is stained for β-galactosidase activity (A and C; blue), and osteogenic mesenchyme of the parietal (p) and frontal (f) bones are stained for ALP activity (B and D; red). As diffuse ALP activity rises in the MesoAp suture mesenchyme, the neural crest/mesoderm border is maintained. Double-headed arrows indicate the dura mater. Arrowheads indicate mesoderm-derived endothelial cells. (E–H) Coronal sutures of (E and F) WT and (G and H) MesoAp E17.5 littermates, stained as in (A–D). After fusion, the relative positions of mesoderm and neural crest are maintained. The acellular osteoid lacks β-galactosidase activity. Scale bars=50 μm.

Fig. 4

Osteogenic induction within the Meso^S252W/+ coronal suture mesenchyme causes fusion. (A–D) Coronal sutures of WT (R26R/Mesp1Cre/Fgfr2^+/+; left panels) and Meso^S252W/+ (MesoAp; R26R/Mesp1Cre/Fgfr2^NeoS252W/+; right panels) BrdU-treated E14.5 littermates double-stained for (A) β-galactosidase activity (blue) and ALP (red). (B) Merged images of the sutures in (A) photographed under red and green fluorescence to show low ALP activity (red) and BrdU incorporation (green) extending across the suture mesenchyme in the MesoAp suture. White arrows (B,C,F,G,J,K) indicate the neural crest/mesoderm border. (C) Merged images of the sutures in (A) with mesodermal β-galactosidase rendered in false color (blue) and BrdU incorporation (green) show ectopic proliferation is within the mesoderm. (D) Schematic summary of A–C. WT (red) and mutant (striped red) osteogenic domains are shown within WT (blue) or mutant (striped blue) mesoderm and WT neural crest (gray), above the dura mater (gray). BrdU-incorporating cells are green. Endothelial cells (blue or striped blue) are shown below the dura mater. (E–H) WT and MesoAp coronal sutures at E15.5 double-stained for (I) β-galactosidase activity (blue) and ALP (red). (F) Sutures in (E) photographed under red fluorescence to show the ectopic osteoid spur extending from the WT frontal bone (arrowhead). (G) Sutures in (E) rendered in false color to show mesodermal β-galactosidase activity in green. The inset in MesoAp shows the region of the ectopic osteoid spur in (F) merged with the corresponding region in (G). (H) Schematic summary of E–G. Colors as in (D). (I–L) WT and MesoAp coronal sutures at E17.5 double-stained for (I) β-galactosidase activity (blue) and ALP (red). (J) Sutures in (I) photographed under red fluorescence to show ectopic ALP activity extending from the WT frontal bone (arrowhead). (K) Sutures in (E) rendered in false color to show mesodermal β-galactosidase activity in green. The inset in MesoAp shows the region of ectopic ALP in (L) merged with the corresponding region in (K). (L) Schematic summary of I–K. Colors as in (D). (M) Left and (N) right coronal sutures from the same WT (left panels) or MesoAp (right panels) embryos at E16.5, double-stained for ALP activity (purple) and mineralized bone (brown). Scale bars=50 μm (A–L), and 100 mm (M and N). f, frontal bone; p, parietal bone.

Fig. 5

Paracrine influences from Meso^S252W/+ suture mesenchyme affect proliferation and bone deposition. (A) (Upper panel) BrdU incorporation within WT or Meso^S252W/+(MesoAp) frontal (f) and parietal (p) osteogenic fronts at E16.5. Black columns=WT (Fgfr2^+/+ or Fgfr2^NeoS252W/+) tissue; blue column=Fgfr2^S252W/+ tissue. P values are indicated. (Lower panel) Representative images of coronal sections from the corresponding sutures co-stained for ALP activity (red fluorescence) and BrdU incorporation (green fluorescence). Scale bar=70 μm. (B) (Upper panel) BrdU incorporation within WT or NC^S252W/+(NCAp) frontal (f) and parietal (p) osteogenic fronts at E16.5. P value is indicated. (Lower panel) Representative images of coronal sections from the corresponding sutures, as in (A). (C) Sagittal sections of WT (R26R/Mesp1Cre/Fgfr2^-/+) and Meso^S252W/+ (MesoAp; R26R/Mesp1Cre/Fgfr2^NeoS252W/+) coronal sutures of P0 calvaria double-stained for ALP (red) and β-galactosidase (blue) activity. (D) Sagittal sections of WT (R26R/Wnt1Cre/Fgfr2^-/+) and NC^S252W/+ (NCAp; R26R/Wnt1Cre/Fgfr2^NeoS252W/+) coronal sutures of P0 calvaria double-stained for ALP (red) andβ-galactosidase (blue) activity. Scale bar for C,D=100 μm.

Fig. 6

Ectopic midline cartilage is derived from mutant mesoderm. (A) Schematic of calvarium showing neural crest (white) and mesoderm (blue) distribution in the frontal (f), parietal (p), and interparietal (ip) bones and the sagittal suture (s). Locations of coronal plane sections in B–G are indicated. (B) Adjacent sections within the anterior sagittal suture of an E17.5 WT (Mesp1Cre/R26R) calvarium are stained for cartilage (left panel; Alcian blue, counterstained with eosin) and mesodermal β-galactosidase activity (blue, right panel; counterstained with eosin). The mesodermal parietal bone (p) expresses β-galactosidase. The unstained cartilage is derived from the neural crest. (C) As in (B), but a mid-sagittal location of a separate E17.5 WT calvarium. Cartilage is derived from both neural crest and mesoderm. (D) As in (B), from the same calvarium, but within the posterior sagittal suture. Cartilage is derived from mesoderm. Scale bar for B–D=50 μm. (E) (lower panel) Coronal section at the level of the interparietal foramen of a WT skull at E16.5, stained for cartilage. The boxed region shown enlarged in the upper panel is devoid of cartilage. (F) (lower panel) Coronal section at the level of the interparietal foramen of a Meso^S252W/+/R26R(MesoAp) skull at E16.5, stained for cartilage. The boxed region shown enlarged in the middle panel contains multiple areas of ectopic cartilage. The black arrowhead indicates the parietal bone edge. An individual cartilage island (boxed) is shown in the upper panel. (G) The section adjacent to (F), stained for β-galactosidase activity. The middle and upper panels show that the ectopic cartilages in (F) are derived from mesoderm. Scale bars for E–G=2 mm (lower panels), 400 μm (middle panels), 50 μm (upper panels).

Fig. 7

Influence of Fgfr2^S252W expression on skull base and palate formation. (A–D) Mid-sagittal sections of (A) WT, (B) Fgfr2^S252W/+ (Apert), (C) NCAp, and (D) MesoAp skulls (calvaria removed) at P0, stained for cartilage (Alcian blue, counterstained with eosin). The black line and arrow in (A) indicate the areas of enlargement of the cranial base (E–H) and palate (I–L), respectively, for all genotypes. The schematic in (A) shows a frontal view of a P0 face with the central vertical dashed line indicating the mid-sagittal plane of section for all panels. The arrow in (B) indicates the perforated palate, enlarged in (J). Scale bar=2 mm. (E–H) Enlargement of the cranial bases from (A–D). Scale bar=1 mm. (I–L) Enlargement of the anterior secondary palates from (A–D). Perforation is only present in the Apert palate (J, arrow). Scale bar=400 μm. ns, cartilage primordium of nasal septum; t, tongue; p, palatal extension of maxillary bone; bo, basioccipital; sos, spheno-occipital synchondrosis; bs, basisphenoid; iss, intersphenoidal synchondrosis; ps, presphenoid.