SummaryMesenchyme tissue: cells, matrix and soluble factors, influence the morphogenesis, proliferation and differentiation of a variety of embryonic epithelia, e.g. in the tooth, skin, mammary and salivary glands. Mesenchyme derivatives also 'maintain' adult epithelia, e.g. the local proliferation rate and cytokeratin composition of oral mucosa. Abnormalities in such epithelial-mesenchymal interactions lead to a variety of pathologies such as premalignant lesions, e.g. leukoplakia, tumours and psoriasis, whilst therapeutic manipulation of such interactions can prevent the exfoliation of dental implants. In all of these systems it is critical to understand, at the cellular and molecular levels, how the mesenchyme signals to the epithelium and how the latter processes and responds to such signals. We have investigated such questions using the developing embryonic palate both as a model system and as an important organ: failure of mesenchymal signalling leads to the common and distressing birth defect of cleft palate.Bilateral palatal shelves arise from the maxillary processes of embryonic day 11 (E ll) mice, grow initially vertically down the sides of the tongue, elevate on E13-8 to a horizontal position above the dorsum of the tongue and fuse with each other in the midline on E14. The medial edge epithelia of each shelf fuse with each other to form a midline epithelial seam, suprabasal cells die, and the basal (stem) cells synthesize extracellular matrix molecules and turn into mesenchymal cells. Simul taneously the oral epithelia differentiate into stratified squamous cells and the nasal epithelia into pseudostratified ciliated columnar cells. Oral, medial and nasal epithelial differentiation is specified by the underlying mesenchyme in vivo and in vitro. Signalling involves a bifurcating action of a combination of soluble growth factors e.g. T G F-a, TGF-/3, PDGF and FGF on palatal epithelia and mesenchyme. These factors stimulate the synthesis of specific extracellular matrix molecules by palate mesenchyme cells, and the appearance of receptors for such molecules on epithelial cells. In this way, a combination of mesenchymal soluble factors and extracellular matrix molecules direct palatal epithelial differentiation. These signals act on epithelial basal (stem) cells, causing them to synthesize unique proteins, which may direct subsequent differentiation of daughter cells. In the most extreme example, namely the medial edge epithelia, these signals result in the basal epithelial cells transforming into mesenchymal cells, thus demonstrating that they are indeed multipotential stem cells.

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BACKGROUND AND PURPOSEProlongation of the cardiac QRS complex is linked to increased mortality and may result from drug-induced inhibition of cardiac sodium channels (hNaV1.5). There has been no systematic evaluation of preclinical and marketed drugs for their additional potential to cause QRS prolongation via gap junction uncoupling. EXPERIMENTAL APPROACHUsing the human cardiac gap junction connexin 43 (hCx43), a dye transfer 'parachute' assay to determine IC50 values for compound ranking was validated with compounds known to uncouple gap junctions. Uncoupling activity (and hNaV1.5 inhibition by automated patch clamp) was determined in a set of marketed drugs and preclinical candidate drugs, each with information regarding propensity to prolong QRS. KEY RESULTSThe potency of known gap junction uncouplers to uncouple hCx43 was ranked (according to IC50) as phorbol ester>digoxin>meclofenamic acid>carbenoxolone>heptanol. Among the drugs associated with QRS prolongation, 29% were found to uncouple hCx43 (IC50 < 50 μM), whereas no uncoupling activity was observed in drugs not associated with QRS prolongation. In preclinical candidate drugs, hCx43 and hNaV1.5 IC50 values were similar (within threefold). No consistent margin over preclinical Cmax (free) was apparent for QRS prolongation associated with Cx43 inhibition. However, instances were found of QRS prolonging compounds that uncoupled hCx43 with significantly less activity at hNaV1.5. CONCLUSION AND IMPLICATIONSThese results demonstrate that off-target uncoupling activity is apparent in drug and drug-like molecules. Although the full ramifications of Cx inhibition remain to be established, screening for hCx43 off-target activity could reduce the likelihood of developing candidate drugs with a risk of causing QRS prolongation. AbbreviationsCx, connexin; PDBu, phorbol 12,13-dibutyrate

The distribution of acidic and basic fibroblast growth factors (aFGF, bFGF) was mapped during mouse embryonic palate development. Generally, they localised most intensely in the basement membrane and epithelia rather than the mesenchyme. Localisation was predominantly restricted to the palatal nasal, and medial edge epithelia. Staining was particularly intense in the medial edge epithelia at the time of mid-line epithelial seam formation. Intense staining persisted in the epithelia of the degenerating seam and later in the oral and nasal epithelial triangles. Mouse embryonic palate mesenchyme (MEPM) cells cultured in vitro on a variety of substrata (on plastic, on the surface of a collagen gel and within a collagen gel) responded to treatment with aFGF or bFGF. These responses were modulated by the culture substratum. The FGFs stimulated MEPM cell proliferation on plastic and on collagen, but inhibited cell growth in collagen. The FGFs had little effect on protein production when cells were cultured on plastic, but caused a large reduction in on-collagen and incollagen cultures. This reduction was greater in collagenous than non-collagenous proteins. Generally, treatment with FGFs stimulated the production of glycosaminoglycans (GAGs), particularly hyaluronan (HA) and dermatan sulphate (DS). In addition, the size class of HA was shifted to a higher molecular weight form. These data indicate that aFGF and bFGF may play a role in modulating mesenchymal cell matrix biosynthesis, so facilitating palatal epithelial seam degeneration.