Teneurin-2 is expressed in tissues that regulate limb and somite pattern formation and is induced in vitro and in situ by FGF8.

Teneurin-2 is a member of a novel family of transmembrane proteins characterized to date in fish, birds, mammals, and Drosophila (e.g., the pair-rule gene product Ten-m). We have shown that teneurin-2 is expressed by neurons in the developing avian visual system in a pattern complementary to the expression of teneurin-1 and that recombinant teneurin-2 induces morphologic changes in neuronal cells in culture (Rubin et al., 1999). Here we have used cRNA probes to two newly identified splice variants and a teneurin-2-specific antibody to determine whether teneurin-2 is also expressed outside the nervous system. Both reverse transcriptase-polymerase chain reaction and in situ hybridization indicate that the three splice variants known so far are coexpressed at sites of pattern formation during development. Teneurin-2 mRNAs and protein are found in the developing limbs, somites, and craniofacial mesenchyme. In addition to expression of teneurin-2 by the apical ectodermal ridge, teneurin-2 transcripts also appear transiently at sites of tendon development. Teneurin-2 expression patterns were strikingly similar to those of fibroblast growth factor 8 (FGF8). In agreement with the overlapping expression pattern, FGF8-coated beads implanted into chicken limb buds induced the ectopic expression of teneurin-2 and soluble FGF8 induced teneurin-2 in limb explant cultures. Thus, teneurin-2 could act downstream of FGF8 during morphogenesis.

Nuclear localization of the testis determining gene product SRY.

We have studied the expression of the human SRY protein (termed p27SRY) in two different cell lines by using specific antibodies. Confocal microscopy enabled us to localize p27SRY precisely in the nucleus in a discrete punctuate pattern. Furthermore, through microinjection experiments, we have demonstrated that the localization of the p27SRY protein into the nucleus was an event involving the NH2-terminal part of the high mobility group (HMG) domain. With the help of several synthetic peptides and various p27SRY mutants, we have characterized a bipartite basic motif in this part of the protein corresponding to a nuclear localization signal. This nuclear localization signal appears to be highly conserved in SRY box- and HMB box-containing proteins, suggesting common properties of nuclear targeting within the HMG box protein family.

Expression and subcellular localization of dystrophin in skeletal, cardiac and smooth muscles during the human development.

Dystrophin, the product of the DMD gene, is present in all muscle types in normal individuals. Its function has yet to be elucidated, but its absence or the presence of a truncated version of the protein is responsible for the appearance of Duchenne and Becker muscular dystrophies. Using monoclonal antibodies raised against distinct regions of the dystrophin protein, we have examined its expression and subcellular distribution during the human development in skeletal and smooth muscles. We show that both dystrophin expression and its association to the plasma membrane take place earlier in cardiac and smooth muscles (8 weeks of gestation) than in skeletal muscle. In skeletal muscle, dystrophin is first detected in the cytoplasm, and progressively localizes to the plasma membrane from 10 weeks onwards. Since we have obtained marked differences in staining when using antibodies against either a central region of the protein or the C-terminal part, we suggest that different fetal and adult dystrophin isoforms are expressed, probably differing in their C-terminal domain. These findings are discussed in the context of the pathology of Duchenne muscular dystrophy.

Electrotransfer of microsomal cytochrome P450 after isoelectric focusing (IEF blots): resolution of human 2A and 3A isozymes.

In this article a procedure is described which allows us to achieve electroblots of membrane-associated proteins after isoelectric focusing. Proteins are resolved in vertical acrylamide slab gels containing ampholines, urea, and nonionic detergents (dodecyl maltoside and Tergitol NP10). After migration the focusing gel is reinforced by a carrier gel (10% acrylamide) polymerized on only one face of the slab. Electroelution of the proteins toward a hydrophobic membrane (Immobilon P) is obtained by adding denaturing agents to the carrier gel (sodium dodecyl sulfate and mercaptoethanol) and by increasing the methanol concentration (60%) in the transfer buffer. This method is applied to analyzing P450 2A and 3A cytochromes from human liver microsomes.

[Expression and localization of dystrophin during human skeletal, cardiac and smooth muscle development]. / Expression et localisation de la dystrophine au cours du dévelopement des muscles squelettique, cardiaque et lisse humains.

Dystrophin, the Duchenne muscular Dystrophy gene product, is a large cytoskeletal protein associated with a complex of membrane proteins, the Dystrophin Glycoprotein Complex (DGC). Dystrophin is localized to the sarcolemmal membrane of all normal muscle types, but is absent from muscles of DMD patients. Using monoclonal antibodies raised against distinct regions of the dystrophin, we studied its expression and subcellular localization during human skeletal, cardiac and smooth muscle development. We have shown that the expression and the association of dystrophin with the plasma membrane take place earlier in cardiac and smooth muscles (8 weeks of gestation) than in skeletal muscles. In skeletal muscles, dystrophin is first observed in the cytoplasm, and is progressively localized to the plasma membrane from 10 weeks onwards. We obtained differences in staining when using antibodies against either the central part of the protein or the carboxy-terminal domain, and we suggested that isoforms of dystrophin, probably differing in their carboxy-terminal end and their capacity to associate with the plasma membrane were differentially expressed during development and in different tissue-types (7). These findings are discussed in the context of the pathology of Duchenne Muscular Dystrophy.

Becker muscular dystrophy: demonstration of the carrier status of a female by immunoblotting and immunostaining.

Becker muscular dystrophy (BMD) often results from in-frame mutations of the dystrophin gene, leading to the production of an altered-sized protein. We examined the expression of dystrophin in a BMD patient and in his asymptomatic mother by Western blot and immunofluorescence. The combination of these techniques allowed us to demonstrate the presence of two different dystrophins, normal-sized or reduced-sized in the muscular fibers of the asymptomatic carrier. This result emphasizes the value of dystrophin analysis for carrier detection and genetic counselling of families with Becker muscular dystrophy.

Molecular deletion patterns in families from southern France with Duchenne/Becker muscular dystrophies.

We studied 38 unrelated patients from southern France with Duchenne (DMD) or Becker (BMD) muscular dystrophy for intragenic deletions of the DMD/BMD gene. We used both multiplex amplification of selected exons and cDNA probes. Of the 26 (68%) unrelated individuals found to have deletions, 24 (92%) were detected by multiplex polymerase chain reaction. All these deletions have been delineated with regard to the exon-containing HindIII fragments revealed by cDNA probes, and in two cases, junction fragments of altered size were seen. The correlation between phenotype and type of deletion agreed with the reading frame theory, except for two BMD and two DMD cases.