Serial coronal sections o f norm al hum an em bryo — foetal crania at 14 weeks indicate that cranial m em branous ossification begins in the basicranium and extends to the vertex. Cranial m esenchym e consists o f cells in an early stage o f pre-osseous differentiation. As these m esenchym al cells differentiate, they form aggregate m esenchym al condensations, and, as osteoblasts, becom e enveloped in m atrix osteoid (Hah and Miyake, 1992;HaU and Miyake, 1995). F G FR7 m R N A is expressed in the norm al 14 - w eek parietal osteoid, periosteum and dura m ater at the cranial vertex (Fig. 3.2-1). F G F R 2 expression is ihustrated in the basicranial parietal osteoid (Fig. 3.2-1 ; ii, iii). F G F R 2 is expressed as B E K transcript in the parietal osteoid and a wide area o f surrounding cranial m esenchym e in coronal section; whereas the
K G F R transcript is weakly expressed in the pericranial aspect o f the parietal osteoid only. T h e differential expression o f the transcript isoform s o f F G F R 2 is m aintained at 10 — m onths o f norm al hum an parietal bo n e developm ent. B E K is expressed in the osteoblasts lining the parietal trabecular bone spaces, and in the ostecyte ceU bodies and dendrites perm eating the trabecular bone m atrix (Fig. 3.2-1 ; iii-vi). KG FR, how ever, is weakly expressed in the osteoblasts Hning the trabecular bone spaces, and is n o t expressed in the osteocyte ceU b o d ies/d en d rites that populate the m atrix bone.
T o investigate w hether the A p ert F G F R 2 - P253R m utation has an effect u p o n the expression o f F G F R 2 in — vivo, in — situ hybridisation for the B E K transcript has been undertaken in 14 - week A pert parietal bone developm ent and com pared w ith age- and site- m atched norm al tissue. A pert basicranial parietal osteoid is show n in transverse section (Fig 3.2-2; i-vt). T h e B E K transcript is n o t expressed in the cranial m esenchym e; and is only weakly expressed in the osteoblasts closely applied to parietal osteoid, despite the strong co — expression o f the osteonectin gene (Fig 3.2-2; i<ù^iv; ii<ù°v). This is in contrast w ith the strong expression o f B E K in the age- and site- m atched norm al hum an parietal osteoblasts and adjacent cranial m esenchym e (Fig 3.2-1 ; ii, in coronal section). F G F R l m R N A is expressed by the osteoblasts o f the A p ert basicranial parietal osteoid and the cells o f the cranial m esenchym e (Fig. 3.2-2; iii(Z^vi). T he differential density o f the F G F R l signal in the A p ert basicranial m esenchym e com pared to that o f the cranial vertex in the age — m atched norm al fetus (Fig 3.2-1 ; i) probably reflects a site - specific difference in developm ental m aturity.
Age- and site- m atched A pert and norm al foetal parietal calvariae w ere therefore investigated for differences in the expression o f F G F R 2 as protein. F G F R l, 2, and 3 proteins are
consistently and differentially detected in norm al basicranial parietal developm ent at 14 weeks in coronal section 3.2-3). F G F R l protein is detected in the osteoblasts lining the parietal osteoid and in the m esenchym al condensations, b u t the signal density is w eaker in the cells o f the wider cranial m esenchym e. F G F R 2 protein is strongly detected in aU three calvarial dom ains, including a high signal density in the cells o f the cranial m esenchym e; thus corroborating the B E K transcript data (Fi^ 3.2-1, it). F G F R 3 protein is detected in all
osteoblast and cranial m esenchym al dom ains. This norm ative co n tro l protein data is in keeping w ith previously published in — situ hybridisation studies (D elezoide et al, 1998;Chan and T h o ro g o o d , 1999). T he 14 - w eek A pert m em branous parietal basicranium is
characterised in transverse section by ‘islands’ o f osteoid m atrix w ithin the cranial m esenchym e (Fig 3.2-3; v-viit). T he parietal bone form s from the aggregation o f cranial m esenchym al cells into m esenchym al condensations, w hich establish an ‘osteogenic fro n t’. T he cells o f the osteogenic fro n t further differentiate into osteoblasts th at eventually generate parietal osteoid matrix. F G F R l and FG F R 3 are expressed as p rotein in the osteoblasts o f the parietal osteoid, the osteogenic front, and cells o f the condensing cranial m esenchym e (F g 3.2-3). F G F R 2 protein is maximally detected in the osteoblasts closely applied to the parietal osteoid. T he F G F R 2 — signal intensity is w eak in the cells o f the A pert m esenchym al condensations, how ever, w hen com pared to that o f the neighbouring A pert parietal osteoblasts; and the F G F R l - signal in com parative A pert dom ains. This is a reversal o f the com parative F G F R l/F G F R 2 expression in the m esenchym al dom ains o f norm al basicranial parietal osteogenesis, w here F G F R 2 has a w ider expression dom ain than F G F R l
(Fig. 3.2-3; ii, it). T hus, the A pert P253R m utation is associated w ith a relative contraction o f the expression dom ain o f F G F R 2 as protein at 14 — weeks o f h um an basicranial parietal ossification.
T h e two principal A p ert m utations (S252W and P253R in FG FR 2) have been show n to confer increased ligand - binding affinity for F G F 2 b u t n o t F G F 4 u p o n the m u ta n t receptor (A nderson et al, 1998c). A pert 14 — week foetal cranial tissue expresses the ligand F G F 2 strongly in b o th m esenchym al condensations and m atrix osteoblasts, b u t F G F 4 and F G F 7 are n o t expressed at 14 weeks, com pared to positive controls in to o th germ and cranial dermis respectively (Fig. 3,2A , i — vi).