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A TWIST in the fate of human osteoblasts identifies signaling molecules involved in skull development
Ethylin Wang Jabs
Center for Craniofacial Development and Disorders, Institute of Genetic Medicine, Departments of Pediatrics, Medicine, and Plastic Surgery, The Johns Hopkins University School of Medicine, Children’s Medical and Surgical Center 1004, 600 North Wolfe Street, Baltimore, Maryland 21287-3914, USA.
Phone: (410) 955-4160; Fax: (410) 955-0484; E-mail: ewjabs@jhmi.edu.
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Published May 1, 2001 - More info
J Clin Invest. 2001;107(9):1075–1077. https://doi.org/10.1172/JCI12853.
© 2001 The American Society for Clinical Investigation
The twist gene was first recognized by the Nobel Prize–winning laboratory of Nüsslein-Volhard (1) and described in Drosophila as a zygotic gene required for dorsoventral patterning (2). Along the dorsoventral axis during the blastoderm stages of Drosophila, the most ventral nuclei constitute the mesodermal anlage which gives rise to cells that invaginate at gastrulation. With the loss of twist expression, homozygous mutant embryos fail to develop a mesoderm layer, resulting in few if any mesodermally derived internal organs. The head segments appear everted due to abnormal head involution, and the anterior end of the embryo is “twisted” in the egg.
In mice, Twist expression also is initially observed along the dorsoventral gradient and then in the mesoderm and neural crest cell derivatives (3). Later, Twist is found in the somites, head mesenchyme, aortic arches, branchial arches, limb buds, and mesenchyme underneath the epidermis (4). Twist is expressed in the undifferentiated cells committed to muscle and cartilage development and inhibits their differentiation. Its transcripts are present in primary osteoblastic cells of the newborn mouse calvaria (5). Mouse embryos homozygous for the disruption of the Twist gene exhibit defects in the head mesenchyme, failure of neural tube closure in the cranial region, and pyknotic nuclei found in cranial nerves (6). Heterozygous mice display skull defects of poorly developed squamosal bones and of overdeveloped interparietal bones and limb abnormalities (7).
The conservation of the Twist mutant phenotype also extends to humans. The heterozygous loss of the TWIST gene or its function results in a common autosomal dominant syndrome, Saethre-Chotzen, which is characterized by craniosynostosis, facial dysmorphisms, and hand and foot abnormalities (8, 9). Of these developmental abnormalities, craniosynostosis — the premature fusion of cranial sutures — is the most notable. Among the cranial sutures, the coronal suture across the width of the skull is most often prematurely synostosed, leading to a brachycephalic skull — one that is box-shaped and of shortened length. Mineralization of the cranium normally occurs directly from membrane-derived paraxial mesoderm. Ossification centers coalesce to form bones, and their osteogenic fronts meet to induce sutures along their lines of approximation. The skull continues to grow by appositional growth at the suture, with deposition of premineralized bone matrix along the suture margins. Several hypotheses have been proposed that premature synostosis is due to the abnormal development of the mesodermal blastema, accelerated bone growth, and/or increased differentiation. The discovery of the new disease gene TWIST generates an important tool that potentially could help in elucidating the signaling pathway and pathogenesis involved in craniosynostosis.
A study reported in this issue of the JCI is evidence that this potential is being realized. Yousfi and colleagues from the Marie laboratory (10) identified TWIST target genes using human mutant calvaria osteoblastic cells from a child with Saethre-Chotzen syndrome. This individual carries a previously reported and apparently recurrent point mutation in TWIST that introduces a premature stop codon (8, 9, 11). The product of this Y103X mutant allele would be predicted to be truncated upstream of the basic helix-loop-helix (bHLH) domain, which is normally required for dimerization and for DNA binding. However, since the patient’s osteoblasts showed decreased TWIST mRNA and protein levels, it seems likely that mRNAs carrying this mutation do not survive nonsense-mediated decay and that the Y103X allele is a true null. In vitro and in vivo experiments with TWIST mutant cells showed increased cell growth and ability to form bonelike nodular structures. These cells and aggregates exhibited increased expression of osteoblastic markers, alkaline phosphatase, and type I collagen, independent of cell growth. However, TWIST mutant cells showed reduction of osteocalcin mRNA expression during osteogenesis. The altered expression of these osteoblastic genes with craniosynostosis is consistent with previously published observations (5, 12, 13). Overexpression of TWIST by transfection studies in osteosarcoma cells may inhibit osteoblast differentiation (12), and reduction of murine Twist expression occurs with osteoblastic maturation (5). Osteocalcin-deficient mice have increased bone formation (13). Therefore, the significance of the findings from the Marie laboratory (10) is that the premature ossification of cranial sutures in Saethre-Chotzen syndrome patients with TWIST mutations is caused by increased osteoblastic cell proliferation and differentiation (Figure 1).
Figure 1Elements involved in the molecular pathway of sutural osteoblast differentiation (modified from ref. 15). Top: normal cellular differentiation and relationship of TWIST and FGFR signaling. Bottom: altered gene expression in TWIST Y103X mutant osteoblasts from a Saethre-Chotzen syndrome patient and in FGFR2 S252W mutant osteoblasts from an Apert syndrome patient. Bone sialoprotein is a mature osteoblast marker.
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