Morphometric study of the primary ossification center of the frontal squama in the human fetus.

PURPOSES: Detailed morphometric data on the development of ossification centers in human fetuses is useful in the early detection of skeletal dysplasias associated with a delayed development of ossification centers and their mineralization. Quantitative analysis of primary ossification centers of cranial bones is sporadic due to limited availability of fetal material. MATERIAL AND METHODS: The size of the primary ossification center of the frontal squama in 37 human (16 males and 21 females) spontaneously aborted human fetuses aged 18-30 weeks was studied by means of CT, digital-image analysis and statistics. RESULTS: With neither sex nor laterality differences, the best-fit growth dynamics for the primary ossification center of the frontal squama was modelled by the following functions: y = 13.756 + 0.021 × (age)2 ± 0.024 for its vertical diameter, y = 0.956 + 0.956 × age ± 0.823 for its transverse diameter, y = 38.285 + 0.889 × (age)2 ± 0.034 for its projection surface area, and y = 90.020 + 1.375 × (age)2 ± 11.441 for its volume. CONCLUSIONS: Our findings for the primary ossification center of the frontal squama may be conducive in monitoring normal fetal growth and screening for inherited faults and anomalies of the skull in human fetuses.

Prickle1 regulates differentiation of frontal bone osteoblasts.

Enlarged fontanelles and smaller frontal bones result in a mechanically compromised skull. Both phenotypes could develop from defective migration and differentiation of osteoblasts in the skull bone primordia. The Wnt/Planar cell polarity (Wnt/PCP) signaling pathway regulates cell migration and movement in other tissues and led us to test the role of Prickle1, a core component of the Wnt/PCP pathway, in the skull. For these studies, we used the missense allele of Prickle1 named Prickle1Beetlejuice (Prickle1Bj). The Prickle1Bj/Bj mutants are microcephalic and develop enlarged fontanelles between insufficient frontal bones, while the parietal bones are normal. Prickle1Bj/Bj mutants have several other craniofacial defects including a midline cleft lip, incompletely penetrant cleft palate, and decreased proximal-distal growth of the head. We observed decreased Wnt/ß-catenin and Hedgehog signaling in the frontal bone condensations of the Prickle1Bj/Bj mutants. Surprisingly, the smaller frontal bones do not result from defects in cell proliferation or death, but rather significantly delayed differentiation and decreased expression of migratory markers in the frontal bone osteoblast precursors. Our data suggests that Prickle1 protein function contributes to both the migration and differentiation of osteoblast precursors in the frontal bone.

A novel ciliopathic skull defect arising from excess neural crest.

The skull is essential for protecting the brain from damage, and birth defects involving disorganization of skull bones are common. However, the developmental trajectories and molecular etiologies by which many craniofacial phenotypes arise remain poorly understood. Here, we report a novel skull defect in ciliopathic Fuz mutant mice in which only a single bone pair encases the forebrain, instead of the usual paired frontal and parietal bones. Through genetic lineage analysis, we show that this defect stems from a massive expansion of the neural crest-derived frontal bone. This expansion occurs at the expense of the mesodermally-derived parietal bones, which are either severely reduced or absent. A similar, though less severe, phenotype was observed in Gli3 mutant mice, consistent with a role for Gli3 in cilia-mediated signaling. Excess crest has also been shown to drive defective palate morphogenesis in ciliopathic mice, and that defect is ameliorated by reduction of Fgf8 gene dosage. Strikingly, skull defects in Fuz mutant mice are also rescued by loss of one allele of fgf8, suggesting a potential route to therapy. In sum, this work is significant for revealing a novel skull defect with a previously un-described developmental etiology and for suggesting a common developmental origin for skull and palate defects in ciliopathies.

Sphenofrontal distance on three-dimensional ultrasound in euploid and trisomy-21 fetuses at 16-24 weeks' gestation.

OBJECTIVE: To compare the distance between the sphenoid and frontal bones on three-dimensional (3D) ultrasound in euploid and trisomy-21 fetuses at 16-24 weeks' gestation. METHODS: We acquired 3D volumes of the fetal profile from 80 normal and 30 trisomy-21 fetuses at 16-24 weeks' gestation. We used the multiplanar mode to obtain the mid-sagittal plane and measured the sphenofrontal distance as the shortest distance between the most anterior edge of the sphenoid bone and the lowest edge of the frontal bone. RESULTS: In normal fetuses, the sphenofrontal distance increased linearly with gestational age, from 15.1 mm at 16 weeks to 18.2 mm at 24 weeks. In fetuses with trisomy 21, the mean sphenofrontal distance delta value was significantly smaller than in normal cases (-3.447 mm (95% CI, -5.684 to -1.211 mm); P < 0.01). The sphenofrontal distance was below the 5(th) and 1(st) percentiles of the normal range in 29 (96.7%) and 27 (90.0%) trisomy-21 fetuses, respectively. CONCLUSIONS: The sphenofrontal distance is shorter at 16-24 weeks' gestation in fetuses with trisomy 21 than in normal fetuses. A reduction in the growth of the anterior cranial base contributes to the mid-facial hypoplasia observed in fetuses with trisomy 21. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Frontal Bone Insufficiency in Gsk3ß Mutant Mice.

The development of the mammalian skull is a complex process that requires multiple tissue interactions and a balance of growth and differentiation. Disrupting this balance can lead to changes in the shape and size of skull bones, which can have serious clinical implications. For example, insufficient ossification of the bony elements leads to enlarged anterior fontanelles and reduced mechanical protection of the brain. In this report, we find that loss of Gsk3ß leads to a fully penetrant reduction of frontal bone size and subsequent enlarged frontal fontanelle. In the absence of Gsk3ß the frontal bone primordium undergoes increased cell death and reduced proliferation with a concomitant increase in Fgfr2-IIIc and Twist1 expression. This leads to a smaller condensation and premature differentiation. This phenotype appears to be Wnt-independent and is not rescued by decreasing the genetic dose of ß-catenin/Ctnnb1. Taken together, our work defines a novel role for Gsk3ß in skull development.

Difference in apical and basal growth of the frontal bone primordium in Foxc1ch/ch mice.

The frontal and parietal bones form the major part of the calvarium and their primordia appear at the basolateral region of the head and grow apically. A spontaneous loss of Foxc1 function mutant mouse, congenital hydrocephalus (Foxc1(ch/ch)), results in congenital hydrocephalus accompanied by defects in the apical part of the skull vault. We found that during the initiation stage of apical growth of the frontal bone primordium in the Foxc1(ch/ch) mouse, the Runx2 expression domain extended only to the basal side and bone sialoprotein (Bsp) and N-cadherin expression domains appeared only in the basal region. Fluorescent dye (DiI) labeling of the frontal primordium by ex-utero surgery confirmed that apical extension of the frontal bone primordium of the mouse was severely retarded, while extension to the basal side underneath the brain was largely unaffected. Consistent with this observation, decreased cell proliferation activity was seen at the apical tip but not the basal tip of the frontal bone primordium as determined by double detection of Runx2 transcripts and BrdU incorporation. Furthermore, expression of the osteogenic-related genes Bmp4 and-7 was observed only in the basal part of the meninges during the initiation period of primordium growth. These results suggest that a loss of Foxc1 function affects skull bone formation of the apical region and that Bmp expression in the meninges might influence the growth of the calvarial bone primordium.

Foxc1 controls the growth of the murine frontal bone rudiment by direct regulation of a Bmp response threshold of Msx2.

The mammalian skull vault consists of several intricately patterned bones that grow in close coordination. The growth of these bones depends on the precise regulation of the migration and differentiation of osteogenic cells from undifferentiated precursor cells located above the eye. Here, we demonstrate a role for Foxc1 in modulating the influence of Bmp signaling on the expression of Msx2 and the specification of these cells. Inactivation of Foxc1 results in a dramatic reduction in skull vault growth and causes an expansion of Msx2 expression and Bmp signaling into the area occupied by undifferentiated precursor cells. Foxc1 interacts directly with a Bmp responsive element in an enhancer upstream of Msx2, and acts to reduce the occupancy of P-Smad1/5/8. We propose that Foxc1 sets a threshold for the Bmp-dependent activation of Msx2, thus controlling the differentiation of osteogenic precursor cells and the rate and pattern of calvarial bone development.

The BMP ligand Gdf6 prevents differentiation of coronal suture mesenchyme in early cranial development.

Growth Differentiation Factor-6 (Gdf6) is a member of the Bone Morphogenetic Protein (BMP) family of secreted signaling molecules. Previous studies have shown that Gdf6 plays a role in formation of a diverse subset of skeletal joints. In mice, loss of Gdf6 results in fusion of the coronal suture, the intramembranous joint that separates the frontal and parietal bones. Although the role of GDFs in the development of cartilaginous limb joints has been studied, limb joints are developmentally quite distinct from cranial sutures and how Gdf6 controls suture formation has remained unclear. In this study we show that coronal suture fusion in the Gdf6-/- mouse is due to accelerated differentiation of suture mesenchyme, prior to the onset of calvarial ossification. Gdf6 is expressed in the mouse frontal bone primordia from embryonic day (E) 10.5 through 12.5. In the Gdf6-/- embryo, the coronal suture fuses prematurely and concurrently with the initiation of osteogenesis in the cranial bones. Alkaline phosphatase (ALP) activity and Runx2 expression assays both showed that the suture width is reduced in Gdf6+/- embryos and is completely absent in Gdf6-/- embryos by E12.5. ALP activity is also increased in the suture mesenchyme of Gdf6+/- embryos compared to wild-type. This suggests Gdf6 delays differentiation of the mesenchyme occupying the suture, prior to the onset of ossification. Therefore, although BMPs are known to promote bone formation, Gdf6 plays an inhibitory role to prevent the osteogenic differentiation of the coronal suture mesenchyme.

Development of the dorsal circumorbital bones in the leopard gecko (Eublepharis macularius) and its bearing on the homology of these elements in the gekkota.

Five nominal elements comprise the circumorbital series of bones in gekkotans: prefrontal, postfrontal, postorbital, jugal, and lacrimal. Determination of the homology of two of these, the postfrontal and postorbital, has been particularly problematic. Two conflicting hypothesis exist relating to these: either the postorbital is lost and the postfrontal remains or they fuse during development to form a combined element, the postorbitofrontal. Such a combined element apparently occurs in at least some members of all lizard clades. There is, however, no direct developmental evidence that supports either theory. To overcome that, we investigate the sequence and pattern of ossification in the circumorbital region in a developmental series of the Leopard gecko. We posit that both the postfrontal and postorbital appear during development. Contrary to previous predictions they neither fuses to each other, nor do either degenerate. Instead, the postfrontal shifts anteriorly and fuses with the frontal to become indistinguishable from it by the time of hatching, and the postorbital persists as a robust independent element bounding the frontoparietal suture. These observations accord, in part, with both hypotheses of homology of these elements and result in the recognition of a new pattern, placing in doubt the existence of the composite postorbitofrontal. The phylogenetic implications of these findings may prove to be far reaching if similar and conserved patterns of development are encountered in other clades.

Msx1 and Dlx5 function synergistically to regulate frontal bone development.

The Msx and Dlx families of homeobox proteins are important regulators for embryogenesis. Loss of Msx1 in mice results in multiple developmental defects including craniofacial malformations. Although Dlx5 is widely expressed during embryonic development, targeted null mutation of Dlx5 mainly affects the development of craniofacial bones. Msx1 and Dlx5 show overlapping expression patterns during frontal bone development. To investigate the functional significance of Msx1/Dlx5 interaction in regulating frontal bone development, we generated Msx1 and Dlx5 double null mutant mice. In Msx1(-/-) ;Dlx5(-/-) mice, the frontal bones defect was more severe than that of either Msx1(-/-) or Dlx5(-/-) mice. This aggravated frontal bone defect suggests that Msx1 and Dlx5 function synergistically to regulate osteogenesis. This synergistic effect of Msx1 and Dlx5 on the frontal bone represents a tissue specific mode of interaction of the Msx and Dlx genes. Furthermore, Dlx5 requires Msx1 for its expression in the context of frontal bone development. Our study shows that Msx1/Dlx5 interaction is crucial for osteogenic induction during frontal bone development.

Tgfbr2 is required for development of the skull vault.

Transforming growth factor beta (TGFbeta) is known to play important roles in multiple developmental processes. One of the main functions is in skeletal development. Our previous studies demonstrated that loss of Tgfbr2 in Prx1Cre-expressing limb mesenchyme results in defects in the long bones and joints of mice. Here we show that loss of Tgfbr2 also results in defects in the development of the skull vault indicating Tgfbr2 has a critical role in intramembranous bone formation as well as endochondral bone formation. Mutant mice did not survive after birth and demonstrated an open skull. The first signs of skull defects were observed at E14.5 day. Prx1Cre(+)/Tgfbr2(f/f) embryos showed significantly reduced cell proliferation in the developing mesenchyme of the skull by E14.5 day without any detectable alteration in apoptosis suggesting that reduced cell proliferation in Prx1Cre(+)/Tgfbr2(f/f) embryos was at least partially responsible for the defects observed. Immunofluorescent staining showed a significant reduction in the expression of Runx2/Cbfa1 and Osterix/Sp7 in Prx1Cre(+)/Tgfbr2(f/f) embryos suggesting that osteoblast differentiation was also altered in Prx1Cre(+)/Tgfbr2(f/f) embryos. To distinguish between the effects of losing Tgfbr2 on mesenchymal proliferation versus osteoblast differentiation, osteoprogenitor cells from the skulls of Tgfbr2(f/f) embryos were cultured under conditions of high cell density and Tgfbr2 was deleted from the cells using Adeno-Cre virus. RT-PCR analysis showed that the mRNA level of Runx2 and Osterix as well as Dlx5 and Msx2 were down-regulated in Tgfbr2-deleted cultures compared to control cultures indicating that Tgfbr2 regulates osteoblast differentiation independent of regulating proliferation. Together, these results suggest that Tgfbr2 is required for normal development of the skull.

Requirement for Twist1 in frontonasal and skull vault development in the mouse embryo.

Using a Cre-mediated conditional deletion approach, we have dissected the function of Twist1 in the morphogenesis of the craniofacial skeleton. Loss of Twist1 in neural crest cells and their derivatives impairs skeletogenic differentiation and leads to the loss of bones of the snout, upper face and skull vault. While no anatomically recognizable maxilla is formed, a malformed mandible is present. Since Twist1 is expressed in the tissues of the maxillary eminence and the mandibular arch, this finding suggests that the requirement for Twist1 is not the same in all neural crest derivatives. The effect of the loss of Twist1 function is not restricted to neural crest-derived bones, since the predominantly mesoderm-derived parietal and interparietal bones are also affected, presumably as a consequence of lost interactions with neural crest-derived tissues. In contrast, the formation of other mesodermal skeletal derivatives such as the occipital bones and most of the chondrocranium are not affected by the loss of Twist1 in the neural crest cells.

EphA4 as an effector of Twist1 in the guidance of osteogenic precursor cells during calvarial bone growth and in craniosynostosis.

Heterozygous loss of Twist1 function causes coronal synostosis in both mice and humans. We showed previously that in mice this phenotype is associated with a defect in the neural crest-mesoderm boundary within the coronal suture, as well as with a reduction in the expression of ephrin A2 (Efna2), ephrin A4 (Efna4) and EphA4 in the coronal suture. We also demonstrated that mutations in human EFNA4 are a cause of non-syndromic coronal synostosis. Here we investigate the cellular mechanisms by which Twist1, acting through Eph-ephrin signaling, regulates coronal suture development. We show that EphA4 mutant mice exhibit defects in the coronal suture and neural crest-mesoderm boundary that phenocopy those of Twist1(+/-) mice. Further, we demonstrate that Twist1 and EphA4 interact genetically: EphA4 expression in the coronal suture is reduced in Twist1 mutants, and compound Twist1-EphA4 heterozygotes have suture defects of greater severity than those of individual heterozygotes. Thus, EphA4 is a Twist1 effector in coronal suture development. Finally, by DiI labeling of migratory osteogenic precursor cells that contribute to the frontal and parietal bones, we show that Twist1 and EphA4 are required for the exclusion of such cells from the coronal suture. We suggest that the failure of this process in Twist1 and EphA4 mutants is the cause of craniosynostosis.

Landmark-based analysis of the morphological relationship between endocranial shape and traces of the middle meningeal vessels.

The morphogenesis and evolution of the cranium are the result of structural interactions among its components, leading to covariance between traits. Soft and hard tissues exert a reciprocal physical and physiological influence, leading to the final phenotype in terms of both ontogeny and evolution. The middle meningeal vessels, interfacing the brain and the braincase, provide an opportunity to study this network, even in extinct human species. Between and within-species variations of the vascular patterns may be mechanically influenced by the cranial morphology (structural hypothesis) or else by actual physiological responses and adaptations, mostly related to oxygen supply and/or thermoregulation (functional hypothesis). In this analysis, we tested the relationship between neurocranial shape and the general morphology of the traces of the middle meningeal vessels in a modern human population, by using landmark-based geometrical models. Although there are some neurocranial differences between groups with different vascular patterns, they are very small or not statistically significant. Only the depth of the imprints may be more influenced by the endocranial morphology. Even if the neurocranial differences among extinct hominids are definitely larger than those within the modern species, the present analysis suggests that it is unlikely that the differences in vascular patterns among the human species are related only to the effects of different neurocranial geometry. This is rather relevant when the marked development of the meningeal network in Homo sapiens is taken into account, compared with the patterns described for nonmodern human species.

Differential FGF ligands and FGF receptors expression pattern in frontal and parietal calvarial bones.

The mammalian skull vault consists mainly of 5 flat bones, the paired frontals and parietals, and the unpaired interparietal. All of these bones are formed by intramembranous ossification within a layer of mesenchyme, the skeletogenic membrane, located between the dermal mesenchyme and the meninges surrounding the brain. While the frontal bones are of neural crest in origin, the parietal bones arise from mesoderm. The present study is a characterization of frontal and parietal bones at their molecular level, aiming to highlight distinct differences between the neural crest-derived frontal and mesodermal-derived parietal bone. We performed a detailed comparative gene expression profile of FGF ligands and their receptors known to play crucial role in skeletogenesis. This analysis revealed that a differential expression pattern of the major FGF osteogenic molecules and their receptors exists between the neural crest-derived frontal bone and the paraxial mesoderm-derived parietal bone. Particularly, the expression of ligands such as Fgf-2, Fgf-9 and Fgf-18 was upregulated in frontal bone on embryonic day 17.5, postnatal day 1 and postnatal day 60 mice. Frontal bone also elaborated higher levels of Fgf receptor 1, 2 and 3 transcripts versus parietal bone. Taken together, these data suggest that the frontal bone is a domain with higher FGF-signaling competence than parietal bone.

Cell lineage in mammalian craniofacial mesenchyme.

We have analysed the contributions of neural crest and mesoderm to mammalian craniofacial mesenchyme and its derivatives by cell lineage tracing experiments in mouse embryos, using the permanent genetic markers Wnt1-cre for neural crest and Mesp1-cre for mesoderm, combined with the Rosa26 reporter. At the end of neural crest cell migration (E9.5) the two patterns are reciprocal, with a mutual boundary just posterior to the eye. Mesodermal cells expressing endothelial markers (angioblasts) are found not to respect this boundary; they are associated with the migrating neural crest from the 5-somite stage, and by E9.5 they form a pre-endothelial meshwork throughout the cranial mesenchyme. Mesodermal cells of the myogenic lineage also migrate with neural crest cells, as the branchial arches form. By E17.5 the neural crest-mesoderm boundary in the subectodermal mesenchyme becomes out of register with that of the underlying skeletogenic layer, which is between the frontal and parietal bones. At E13.5 the primordia of these bones lie basolateral to the brain, extending towards the vertex of the skull during the following 4-5 days. We used DiI labelling of the bone primordia in ex-utero E13.5 embryos to distinguish between two possibilities for the origin of the frontal and parietal bones: (1) recruitment from adjacent connective tissue or (2) proliferation of the original primordia. The results clearly demonstrated that the bone primordia extend vertically by intrinsic growth, without detectable recruitment of adjacent mesenchymal cells.

Molecular and cellular characterization of mouse calvarial osteoblasts derived from neural crest and paraxial mesoderm.

BACKGROUND: Cranial skeletogenic mesenchyme is derived from two distinct embryonic sources: mesoderm and cranial neural crest. Previous studies have focused on molecular and cellular differences of juvenile and adult osteoblasts. METHODS: To further understand the features of mouse-derived juvenile osteoblasts, the authors separated calvarial osteoblasts by their developmental origins: frontal bone-derived osteoblasts from cranial neural crest, and parietal bone-derived osteoblasts from paraxial mesoderm. Cells were harvested from a total of 120 mice. RESULTS: Interestingly, the authors observed distinct morphologies and proliferation potential of the two populations of osteoblasts. Osteogenic genes such as alkaline phosphatase, osteopontin, collagen I, and Wnt5a, which was recently identified as playing a role in skeletogenesis, were abundantly expressed in parietal bone-derived osteoblasts versus frontal bone-derived osteoblasts. In addition, fibroblast growth factor (FGF) receptor 2, and FGF-18 were more highly expressed in the parietal bone-derived osteoblasts, suggesting a more differentiated phenotype. In contrast, FGF-2, and adhesion molecules osteoblast cadherins and bone morphogenetic protein receptor IB, the bone tissue-specific type receptor were overexpressed in frontal bone-derived osteoblasts compared with parietal bone-derived osteoblasts. CONCLUSIONS: The authors observed that although neural crest-derived osteoblasts represented a population of less differentiated, faster growing cells, they formed bone nodules more rapidly than parietal bone-derived osteoblasts. This in vitro study suggests that embryonic tissue derivations influence postnatal in vitro calvarial osteoblast cell biology.

Concerted action of Msx1 and Msx2 in regulating cranial neural crest cell differentiation during frontal bone development.

The homeobox genes Msx1 and Msx2 function as transcriptional regulators that control cellular proliferation and differentiation during embryonic development. Mutations in the Msx1 and Msx2 genes in mice disrupt tissue-tissue interactions and cause multiple craniofacial malformations. Although Msx1 and Msx2 are both expressed throughout the entire development of the frontal bone, the frontal bone defect in Msx1 or Msx2 null mutants is rather mild, suggesting the possibility of functional compensation between Msx1 and Msx2 during early frontal bone development. To investigate this hypothesis, we generated Msx1(-/-);Msx2(-/-) mice. These double mutant embryos died at E17 to E18 with no formation of the frontal bone. There was no apparent defect in CNC migration into the presumptive frontal bone primordium, but differentiation of the frontal mesenchyme and establishment of the frontal primordium was defective, indicating that Msx1 and Msx2 genes are specifically required for osteogenesis in the cranial neural crest lineage within the frontal bone primordium. Mechanistically, our data suggest that Msx genes are critical for the expression of Runx2 in the frontonasal subpopulation of cranial neural crest cells and for differentiation of the osteogenic lineage. This early function of the Msx genes is likely independent of the Bmp signaling pathway.

Frontomaxillary facial angle at 11 + 0 to 13 + 6 weeks: effect of plane of acquisition.

OBJECTIVE: To determine the range of positions of the fetal head in which a three-dimensional (3D) volume is acquired for subsequent successful assessment of the frontomaxillary facial (FMF) angle. METHOD: We obtained 3D volumes of the fetal head at 11 + 0 to 13 + 6 weeks. The volumes were acquired with the head in different positions and reconstructed to obtain a mid-sagittal section and demonstrate the maxilla, palate and frontal bone, which constitute the landmarks for the assessment of the FMF angle. RESULTS: In the reconstructed mid-sagittal sections, it was possible to demonstrate the landmarks that define the FMF angle in most of the cases when the 3D volume acquisition plane was: (a) mid-sagittal, with the angle between the face of the transducer and the direction of the fetal nose being about 0-99 degrees, 150-199 degrees and 330-359 degrees; (b) transverse at the level of the biparietal diameter when the angle between the transducer and the midline echo of the brain was 0-29 degrees; and (c) oblique around the crown-rump axis when the angle from the mid-sagittal plane was 0-49 degrees. However, the measurement of the FMF angle was artificially increased when in the mid-sagittal plane the angle was 40-99 degrees and 150-199 degrees. CONCLUSION: Successful assessment of the FMF angle by 3D ultrasound is dependent on the plane and angle of the volume acquisition.

Frontomaxillary facial angle at 11+0 to 13+6 weeks' gestation-reproducibility of measurements.

OBJECTIVE: To assess the intra- and interobserver reproducibility in the measurement of the frontomaxillary facial (FMF) angle at 11+0 to 13+6 weeks' gestation and to investigate the effect of deviations from the exact mid-sagittal view on these measurements. METHODS: Three-dimensional (3D) volumes of the fetal face were used by two operators to measure the FMF angle in 50 chromosomally normal and 50 trisomy 21 fetuses. The measurements were taken in the exact mid-sagittal view and repeated after lateral rotation of the head by 5 degrees, 10 degrees and 15 degrees away from the vertical position of the occipitofrontal diameter axis. Mean difference and 95% limits of agreement between paired measurements of FMF angle by the same and by two different sonographers were determined. RESULTS: In the mid-sagittal plane the maxillary bone was rectangular shaped. Rotation away from this plane became easily recognizable because at a mean of 7 degrees (range, 4-10 degrees) the shape of the maxilla changed with the appearance of the zygomatic process of the maxilla and at a mean of 8 degrees (range, 4-12 degrees) the tip of the nose became invisible. In both the normal and trisomy 21 fetuses the FMF angle measured at 5-15 degrees was not significantly different from the one measured in the mid-sagittal plane. In 95% of the cases, the difference between paired measurements of the FMF angle by the same sonographer at the mid-sagittal plane was between -2.3 degrees and 3.0 degrees and at 15 degrees it was -1.0 degrees to 6.8 degrees. At the mid-sagittal plane, the difference in measurements between two sonographers was -3.1 to 3.0 degrees. CONCLUSION: The landmarks that define the mid-sagittal plane of the fetal face are the tip of the nose and the rectangular shaped maxilla. Measurement of the FMF angle is highly reproducible.