No abstract

The interpretation of positional information can provide the basis for pattern formation in limb morphogenesis. The gradient in positional information along the antero-posterior axis, which is specified with respect to a localised boundary region, can be modified by grafting this region to successive positions along the axis. The pattern of digits obtained is consistent with a model based on diffusion of a labile morphogen and is thus similar to models proposed for the development of pattern in invertebrates.

Myf5 is the first myogenic regulatory factor to be expressed in the mouse embryo and it determines the entry of cells into the skeletal muscle programme. A region situated between -58 kb and -48 kb from the gene directs Myf5 transcription at sites where muscles will form. We now show that this region consists of a number of distinct regulatory elements that specifically target sites of myogenesis in the somite, limbs and hypoglossal cord, and also sites of Myf5 transcription in the central nervous system. Deletion of these sequences in the context of the locus shows that elements within the region are essential, and also reveals the combinatorial complexity of the transcriptional regulation of Myf5. Both within the-58 kb to -48 kb region and elsewhere in the locus, multiple sequences are present that direct transcription in subdomains of a single site during development, thus revealing distinct phases of myogenesis when subpopulations of progenitor cells enter the programme of skeletal muscle differentiation.

Within the mammalian genome, there are many multimember gene families that encode membrane proteins with extracellular leucine rich repeats which are thought to act as cell adhesion or signalling molecules. We previously showed that the members of the NLRR gene family are expressed in a developmentally restricted manner in the mouse with NLRR-1 being expressed in the developing myotome. The FLRT gene family shows a similar genomic layout and predicted protein secondary structure to the NLRRs so we analysed expression of the three FLRT genes during mouse development. FLRTs are glycosylated membrane proteins expressed at the cell surface which localise in a homophilic manner to cell-cell contacts expressing the focal adhesion marker vinculin. Each member of the FLRT family has a distinct, highly regulated expression pattern, as was seen for the NLRR family. FLRT3 has a provocative expression pattern during somite development being expressed in regions of the somite where muscle precursor cells migrate from the dermomyotome and move into the myotome, and later in myotomal precursors destined to migrate towards their final destination, for example, those that form the ventral body wall. FLRT3 is also expressed at the midbrain/hindbrain boundary and in the apical ectodermal ridge, regions where FGF signalling is known to be important, suggesting that the role for FLRT3 in FGF signalling identified in Xenopus is conserved in mammals. FLRT1 is expressed at brain compartmental boundaries and FLRT2 is expressed in a subset of the sclerotome, adjacent to the region that forms the syndetome, suggesting that interaction with FGF signalling may be a general property of FLRT proteins. We confirmed this by showing that all FLRTs can interact with FGFR1 and FLRTs can be induced by the activation of FGF signalling by FGF-2. We conclude that FLRT proteins act as regulators of FGF signalling, being induced by the signal and then able to interact with the signalling receptor, in many tissues during mouse embryogenesis. This process may, in part, be dependent on homophilic intercellular interactions between FLRT molecules.

Retinoic acid may be the natural morphogen used to generate digit pattern in the chick limb bud. It has been proposed that retinoic acid acts by binding to a cellular retinoic acid-binding protein (CRABP) and then entering the nucleus to alter the pattern of gene activity. High-affinity receptors that bind both retinoic acid and DNA and are analogous to the steroid receptors have been identified. But the concentration of endogenous retinoic acid in the limb and the binding coefficient of the nuclear receptors indicate that they are saturated throughout the limb. Here we investigate the CRABP distribution in the developing chick limb bud. We find CRABP in the area of intense morphogenetic activity at the tip, with a differential distribution across the anteroposterior axis, the high point being at the anterior margin. Retinoic acid also forms a concentration gradient across the limb bud, but is highest on the posterior side. We propose that CRABP could be reducing the effective concentration of retinoic acid reaching the nucleus to a level appropriate for the differential regulation of gene transcription, providing a spatially modulated morphogenetic gradient of information for digit formation.