Boundaries between cellular compartments often serve as signaling interfaces during embryogenesis. The coronal suture is a major growth center of the skull vault and develops at a boundary between cells derived from neural crest and mesodermal origin, forming the frontal and parietal bones, respectively. Premature fusion of these bones, termed coronal synostosis, is a common human developmental anomaly. Known causes of coronal synostosis include haploinsufficiency of TWIST1 and a gain of function mutation in MSX2. In Twist1(+/-) mice with coronal synostosis, we found that the frontal-parietal boundary is defective. Specifically, neural crest cells invade the undifferentiated mesoderm of the Twist1(+/-) mutant coronal suture. This boundary defect is accompanied by an expansion in Msx2 expression and reduction in ephrin-A4 distribution. Reduced dosage of Msx2 in the Twist1 mutant background restores the expression of ephrin-A4, rescues the suture boundary and inhibits craniosynostosis. Underlining the importance of ephrin-A4, we identified heterozygous mutations in the human orthologue, EFNA4, in three of 81 patients with non-syndromic coronal synostosis. This provides genetic evidence that Twist1, Msx2 and Efna4 function together in boundary formation and the pathogenesis of coronal synostosis.

The flat bones of the vertebrate skull vault develop from two migratory mesenchymal cell populations, the cranial neural crest and paraxial mesoderm. At the onset of skull vault development, these mesenchymal cells emigrate from their sites of origin to positions between the ectoderm and the developing cerebral hemispheres. There they combine, proliferate and differentiate along an osteogenic pathway. Anomalies in skull vault development are relatively common in humans. One such anomaly is familial calvarial foramina, persistent unossified areas within the skull vault. Mutations in MSX2 and TWIST are known to cause calvarial foramina in humans. Little is known of the cellular and developmental processes underlying this defect. Neither is it known whether MSX2 and TWIST function in the same or distinct pathways. We trace the origin of the calvarial foramen defect in Msx2 mutant mice to a group of skeletogenic mesenchyme cells that compose the frontal bone rudiment. We show that this cell population is reduced not because of apoptosis or deficient migration of neural crest-derived precursor cells, but because of defects in its differentiation and proliferation. We demonstrate, in addition, that heterozygous loss of Twist function causes a foramen in the skull vault similar to that caused by loss of Msx2 function. Both the quantity and proliferation of the frontal bone skeletogenic mesenchyme are reduced in Msx2-Twist double mutants compared with individual mutants. Thus Msx2 and Twist cooperate in the control of the differentiation and proliferation of skeletogenic mesenchyme. Molecular epistasis analysis suggests that Msx2 and Twist do not act in tandem to control osteoblast differentiation, but function at the same epistatic level.

Osteogenesis imperfecta is a clinically and genetically heterogeneous brittle bone disorder that results from defects in the synthesis, structure, or posttranslational modification of type I procollagen. Dominant forms of OI result from mutations in COL1A1 or COL1A2, which encode the chains of the type I procollagen heterotrimer. The mildest form of OI typically results from diminished synthesis of structurally normal type I procollagen, whereas moderately severe to lethal forms of OI usually result from structural defects in one of the type I procollagen chains. Recessively inherited OI, usually phenotypically severe, has recently been shown to result from defects in the prolyl-3-hydroxylase complex that lead to the absence of a single 3-hydroxyproline at residue 986 of the alpha1(I) triple helical domain. We studied a cohort of five consanguineous Turkish families, originating from the Black Sea region of Turkey, with moderately severe recessively inherited OI and identified a novel locus for OI on chromosome 17. In these families, and in a Mexican-American family, homozygosity for mutations in FKBP10, which encodes FKBP65, a chaperone that participates in type I procollagen folding, was identified. Further, we determined that FKBP10 mutations affect type I procollagen secretion. These findings identify a previously unrecognized mechanism in the pathogenesis of OI.

To understand the actions of morphogens, it is crucial to determine how they elicit different transcriptional responses in different cell types. Here,we identify a BMP-responsive enhancer of Msx2, an immediate early target of bone morphogenetic protein (BMP) signaling. We show that the BMP-responsive region of Msx2 consists of a core element, required generally for BMP-dependent expression, and ancillary elements that mediate signaling in diverse developmental settings. Analysis of the core element identified two classes of functional sites: GCCG sequences related to the consensus binding site of Mad/Smad-related BMP signal transducers; and a single TTAATT sequence, matching the consensus site for Antennapedia superclass homeodomain proteins. Chromatin immunoprecipitation and mutagenesis experiments indicate that the GCCG sites are direct targets of BMP restricted Smads. Intriguingly, however, these sites are not sufficient for BMP responsiveness in mouse embryos; the TTAATT sequence is also required. DNA sequence comparisons reveal this element is highly conserved in Msx2promoters from mammalian orders but is not detectable in other vertebrates or non-vertebrates. Despite this lack of conservation outside mammals, the Msx2 BMP-responsive element serves as an accurate readout of Dpp signaling in a distantly related bilaterian – Drosophila. Strikingly, in Drosophila embryos, as in mice, both TTAATT and GCCG sequences are required for Dpp responsiveness, showing that a common cis-regulatory apparatus can mediate the transcriptional activation of BMP-regulated genes in widely divergent bilaterians.

On page 555 under the section titled Mutations in FKBP10 cause Recessive OI, there are two errors in the nomenclature for the identified mutations. The FKBP10 (NM_021939.3) mutation isolated in the Turkish cases (proband R06-113A) is c.321_353 del and is predicted to result in the deletion of eleven amino acids in the protein, p.Met107_Leu117 del. In the second paragraph of the subheading, the mutation in the Mexican-American family (proband R93-188) should be