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First published online 30 November 2005
doi: 10.1242/dev.02171

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The homeoprotein engrailed 1 has pleiotropic functions in calvarial intramembranous bone formation and remodeling

¹ Department of Cell Biology, New York University School of Medicine, MSB room 614, 550 1st Avenue, New York, NY 10016, USA.
² Faculty of Medicine, Centre for Bone and Periodontal Research, McGill University, Room 2203, 740 Avenue Dr Penfield, Montreal, Québec H3A 1A4, Canada.
³ Departments of Pathology and Dermatology, New York University School of Medicine, MSB room 614, 550 1st Avenue, New York, NY 10016, USA.

View larger version (110K): [in a new window]   Fig. 1. Analysis of En1 expression during calvarial bone development between E11.5 and P1, as recapitulated by X-gal staining of En1lki/+ mice. (A,B) Calvarial En1 expression within the craniofacial mesenchyme (Aa-Ac, white arrows) at E11.5 (Aa,Ba), E12.5 (Ab) and E13.5 (Ac,Bb). Expression of En1 during overt calvarial bone formation (E15.5-P1) is prominent along the osteogenic fronts (arrowhead) and developing sutures (black arrows) (Ad-Af); it is expressed by ectocranial periosteal osteoblasts (Ac, Af, asterisks; Bc,Bd, arrow) as well as by the endosteal osteoblasts and osteocytes of the frontal bone trabeculae (Bc,Bd, arrowheads). In the interfrontal suture, En1 is expressed in the mesenchyme (Bc,Be, arrow; Be'), but not in osteoprogenitors populating the frontal bone margins (Be, arrowheads). By contrast, En1 is expressed by osteoprogenitors within the coronal suture (Bf, arrowheads; Bf'). p, parietal bone; f, frontal bone; e, eye. Scale bars: 2 mm in A; 1 mm in Ba-Bc; 0.1 mm in insets in Ba,Bb, and in Bd-Bf.

View larger version (61K): [in a new window]   Fig. 2. Gross calvarial malformations in En1-/- mice. (A) Schematic diagram and staining at P1. (Aa) Schematic depicting the organization of the murine skull vault consisting of: the paired frontal bones (F), the paired parietal bones (P) and the interparietal bone (IP). These are intervened by the lambdoid, coronal, sagittal and interfrontal (metopic) sutures. (Ab,Ac) Alizarin Red and Alcian Blue staining of mineralized bone and cartilage, demonstrating a generalized calvarial hypoplasia, coronal and lambdoid suture gaping (arrow, double-headed arrow), and frontal foramina (asterisk) in the skull vault of En1-/- mice at P1. (B) At P35, En1 mutants (Bb,Bd) exhibit abnormal coronal, lambdoid and interfrontal suture patency (Bb, arrows, asterisk), and severe malocclusion due to maxillary and nasal bone hypoplasia (Bd, arrow, arrowhead) compared with wild type (Ba,Bc). Scale bars: 2 mm in A; 5 mm in B.

View larger version (73K): [in a new window]   Fig. 3. Osteopenia and impaired calvarial bone mineralization in En1-/- mice. (Aa,Ab) Micro-CT analysis of heterozygous and En1-/- calvariae at P1 and P5, demonstrating reduced ossification in the mutants. (B) Quantitative morphometric measurements of calvariae ultrastructure obtained by micro-CT analysis, demonstrating reduced bone volume and increased trabecular separation in En1-/- mice. (C) Histological assessment of tissue mineralization by Von Kossa staining. The parietal bone matrix of En1+/- mice is mostly mineralized at birth, whereas that of En1 mutants is heavily perforated and undermineralized (arrows). (D) En1-null calvarial osteoblasts exhibit a poor capacity to mediate extracellular marix mineralization over the course of a 21-day differentiation period. (E) ALP activity, assessed spectrometrically between 7-21 days of culture, is 2- to 3-fold higher in wild-type osteoblasts than in En1-/- cells. Scale bar: 0.1 mm in C.

View larger version (84K): [in a new window]   Fig. 4. Impaired calvarial osteogenesis in En1 mutant mice. (A) Whole-mount in situ hybridization analysis of calvarial Opn expression, showing that En1-/- embryos exhibit a delay in the commencement of osteogenesis (E14.5), followed by deficient osteoblast differentiation (arrows, E16.5-E18.5). (B) Section ISH analysis demonstrating the absence of Osx expression in the mesenchyme of En1-/- calvariae at E13.5. At E16.5, Osx is expressed by wild-type osteoblasts of the frontal bone, but is reduced in En1-/- osteoblasts (arrowheads). (C) Analysis of osteopontin (Opn) and osteocalcin (Ocn) on sagittal sections of wild-type and En1-/- calvariae at P1. Although Opn normalizes to wild-type levels in En1-/- calvariae postnatally, it is aberrantly expressed by terminally differentiated osteocytes of the mutant frontal bones (inset, arrowheads). By comparison, normal Ocn expression in ectoperiosteal osteoblasts is nearly abolished in En1-/- calvariae. (D) Northern blot analysis of Opn, Bsp, Ocn and Osx expression by calvarial osteoblasts over a 21-day culture period. p, parietal bone; f, frontal bone. Scale bars: 1.5 mm in A; 0.1 mm in B; 0.2 mm in C.

View larger version (59K): [in a new window]   Fig. 5. Osteoprogenitor proliferation at the interfrontal and coronal sutures of wild-type and En1-/- embryos between E12.5-E18.5. (A) Immunohistochemical detection of BrdU-labeled cells within the interfrontal sutures (dashed outline). (B) Proliferation was numerically assessed as the fraction of labeled to non-labeled cells. A specific reduction in osteoprogenitor proliferation was observed in En1-/- calvariae at E18.5. (C) Enhanced osteoprogenitor proliferation within the coronal suture (arrowheads) of En1 mutants at E18.5. p, parietal bone; f, frontal bone. Scale bar: 0.2 mm.

View larger version (92K): [in a new window]   Fig. 6. Reduced FGF signaling in En1 mutant calvariae. (A) Expression of Fgfr1, Fgfr2 and Fgf18 in the coronal suture, and the parietal and frontal bones, at P1. Fgfr1, Fgfr2 and Fgf18 are co-expressed by ectocranial periosteal osteoblasts in the parietal and frontal bones. Fgfr1 is absent from wild-type sutural osteprogenitors, whereas Fgfr2 and Fgf18 are expressed at these locations. In En1 mutants, Fgfr1 and Fgfr2 are upregulated in the sutural mesenchyme (arrowheads indicate the parietal and frontal bone margins). (B) Northern blot analysis of Fgfr1 and Fgfr2 expression during the differentiation of cultured primary calvarial osteoblasts. Relative to Gapdh, no significant differences in Fgfr1 or Fgfr2 levels were observed in wild-type and En1-/- osteoblasts. (C) Loss of Spry2 expression in the calvarial osteoblasts (arrow) and osteoprogenitors (arrowheads) of En1 mutants. (D) Expression of pERK in calvarial bones of wild-type and En1-/- mice at P1. pERK displays strong activity in wild-type endosteal osteoblasts lining the frontal bone trabeculae, but is only weakly present at the osteogenic fronts (arrowheads), and is absent from ectoperiosteal osteoblasts (arrow). En1 mutants display severely reduced pERK in endosteal osteoblasts. p, parietal bone; f, frontal bone. (E) Proposed model for the interactions between EN1 and FGF signaling events during calvarial osteogenesis. The calvarial bone is postulated to divide into spatial subdomains (sutural osteoprogenitors, ectoperiosteal osteoblasts, endosteal osteoblasts) that respond differentially to FGF signaling. Scale bars: 0.2 mm in A; 0.1 mm in C; 0.1 mm in D.

View larger version (59K): [in a new window]   Fig. 7. Increased remodeling and osteoclast recruitment in En1-/- calvariae. (A) Alizarin Red stained wild-type and En1-/- calvariae at P1 showing multiple perforations in the mutants (arrows). (B) Whole-mount detection of osteoclast-specific TRAP activity in calvariae at P5, depicting increased staining in En1 mutants (compare with wild type, arrow). (C) Histological detection of TRAP+ osteoclasts located along the endocranial (arrows) and trabecular (arrowheads) bone surfaces of the frontal bones at P1. (D) Quantitative histomorphometric assessment of osteoclast number in sections of wild-type and En1-/- frontal bones. (E,F) Northern blot and semi-quantitative analysis of OPG and Rankl expression in cultured calvarial osteoblasts. At day 7 of postconfluent growth, wild-type and En1-/- cells express similar levels of OPG, whereas expression of Rankl is strongly upregulated in mutant osteoblasts. F, frontal bone; P, parietal bone. Scale bars: 2 mm in A,B; 0.1 mm in C.