Disrupted mitochondrial function in the Opa3L122P mouse model for Costeff Syndrome impairs skeletal integrity.

Mitochondrial dysfunction connects metabolic disturbance with numerous pathologies, but the significance of mitochondrial activity in bone remains unclear. We have, therefore, characterized the skeletal phenotype in the Opa3L122P mouse model for Costeff syndrome, in which a missense mutation of the mitochondrial membrane protein, Opa3, impairs mitochondrial activity resulting in visual and metabolic dysfunction. Although widely expressed in the developing normal mouse head, Opa3 expression was restricted after E14.5 to the retina, brain, teeth and mandibular bone. Opa3 was also expressed in adult tibiae, including at the trabecular surfaces and in cortical osteocytes, epiphyseal chondrocytes, marrow adipocytes and mesenchymal stem cell rosettes. Opa3L122P mice displayed craniofacial abnormalities, including undergrowth of the lower mandible, accompanied in some individuals by cranial asymmetry and incisor malocclusion. Opa3L122P mice showed an 8-fold elevation in tibial marrow adiposity, due largely to increased adipogenesis. In addition, femoral length and cortical diameter and wall thickness were reduced, the weakening of the calcified tissue and the geometric component of strength reducing overall cortical strength in Opa3L122P mice by 65%. In lumbar vertebrae reduced vertebral body area and wall thickness were accompanied by a proportionate reduction in marrow adiposity. Although the total biomechanical strength of lumbar vertebrae was reduced by 35%, the strength of the calcified tissue (σmax) was proportionate to a 38% increase in trabecular number. Thus, mitochondrial function is important for the development and maintenance of skeletal integrity, impaired bone growth and strength, particularly in limb bones, representing a significant new feature of the Costeff syndrome phenotype.

Identification and characterization of an inhibitory fibroblast growth factor receptor 2 (FGFR2) molecule, up-regulated in an Apert Syndrome mouse model.

AS (Apert syndrome) is a congenital disease composed of skeletal, visceral and neural abnormalities, caused by dominant-acting mutations in FGFR2 [FGF (fibroblast growth factor) receptor 2]. Multiple FGFR2 splice variants are generated through alternative splicing, including PTC (premature termination codon)-containing transcripts that are normally eliminated via the NMD (nonsense-mediated decay) pathway. We have discovered that a soluble truncated FGFR2 molecule encoded by a PTC-containing transcript is up-regulated and persists in tissues of an AS mouse model. We have termed this IIIa-TM as it arises from aberrant splicing of FGFR2 exon 7 (IIIa) into exon 10 [TM (transmembrane domain)]. IIIa-TM is glycosylated and can modulate the binding of FGF1 to FGFR2 molecules in BIAcore-binding assays. We also show that IIIa-TM can negatively regulate FGF signalling in vitro and in vivo. AS phenotypes are thought to result from gain-of-FGFR2 signalling, but our findings suggest that IIIa-TM can contribute to these through a loss-of-FGFR2 function mechanism. Moreover, our findings raise the interesting possibility that FGFR2 signalling may be a regulator of the NMD pathway.

The expression of Fat-1 cadherin during chick limb development.

Cellular adhesion is fundamental to the behaviour of cell populations during embryonic development and serves to establish correct tissue pattern and architecture. The cadherin superfamily of cell adhesion proteins regulates cellular organization and additionally influences intracellular signalling cascades. Here we present for the first time a detailed account of chick Fat-1 gene expression during embryogenesis visualised by whole-mount in situ hybridisation. In part, we focus on the expression pattern in limb buds that has not been accurately documented. While Fat-1 is generally expressed in epithelial tissues and its Drosophila counterpart Fat-like regulates formation of ectodermally-derived organs, in limb buds we have found that chick Fat-1 is uniquely restricted to mesenchyme. This Fat-1 expression pattern is remarkably dynamic throughout tissue differentiation, limb maturation and pattern formation. Diffuse expression of Fat-1 begins at stage HH17 as the limb bud is forming. It then becomes more proximal as the limb bud grows and is expressed within both tendon and muscle progenitors in the dorsal and ventral subectodermal mesenchyme. Later, Fat-1 transcripts were more abundant in anterior and posterior domains of the limb bud. During hand plate formation, Fat-1 transcripts were expressed in the mesenchyme adjacent to the wrist joint zone and in the interdigit mesenchyme.

The expression of Flrt3 during chick limb development.

The Flrt3 (Fibronectin-Leucine-Rich Transmembrane protein) gene encodes a fibronectin and leucine-rich repeat transmembrane protein whose expression is controlled by fibroblast growth factors (FGFs). FLRT3 has been implicated in neurite outgrowth after nerve damage, as a positive regulator of FGF signalling and in homotypic cell adhesion. Here we describe Flrt3 expression during chick embryonic limb development using whole-mount in situ hybridization. We found very dynamic expression during apical ridge formation and limb bud outgrowth. Initially Flrt3 is expressed in the apical ectodermal ridge and underlying mesenchyme, but then becomes restricted to the dorsal and ventral sides of the apical ridge as a twin stripe. At later stages, abundant expression is seen in the hindlimb and in both the pectoral and pelvic girdle-forming regions. FLRT3 may have a crucial role in regulating cellular adhesion between the epithelial apical ridge and the underlying mesenchyme and in establishing the dorso-ventral position of the ridge.

Expression of csal1 in pre limb-bud chick embryos.

The spalt family of transcriptional repressors has been implicated in limb, heart, ear and kidney development and truncating mutations in a human gene, SALL1, result in the autosomal dominant disorder Townes-Brocks syndrome. Here we show the expression pattern of the chick orthologue of the SALL1 gene, csal1, during early development. We found csal1 expression in the heart and in the pharynx, a source of inductive signals during heart development. Expression was also seen in involuting mesoderm and later in presegmented paraxial mesoderm. We also describe expression in the ectoderm and neural plate of the early embryo and subsequent expression in the neural tube.

FGF-4 signaling is involved in mir-206 expression in developing somites of chicken embryos.

The microRNAs (miRNAs) are recently discovered short, noncoding RNAs, that regulate gene expression in metazoans. We have cloned short RNAs from chicken embryos and identified five new chicken miRNA genes. Genome analysis identified 17 new chicken miRNA genes based on sequence homology to previously characterized mouse miRNAs. Developmental Northern blots of chick embryos showed increased accumulation of most miRNAs analyzed from 1.5 days to 5 days except, the stem cell-specific mir-302, which was expressed at high levels at early stages and then declined. In situ analysis of mature miRNAs revealed the restricted expression of mir-124 in the central nervous system and of mir-206 in developing somites, in particular the developing myotome. In addition, we investigated how miR-206 expression is controlled during somite development using bead implants. These experiments demonstrate that fibroblast growth factor (FGF) -mediated signaling negatively regulates the initiation of mir-206 gene expression. This may be mediated through the effects of FGF on somite differentiation. These data provide the first demonstration that developmental signaling pathways affect miRNA expression. Thus far, miRNAs have not been studied extensively in chicken embryos, and our results show that this system can complement other model organisms to investigate the regulation of many other miRNAs.