An Eph-related receptor protein tyrosine kinase gene segmentally expressed in the developing mouse hindbrain.

In search of genes possibly involved in the regulation of hindbrain segmentation, we have isolated mouse cDNA clones corresponding to putative protein kinase genes by polymerase chain reaction amplification of cDNA from 9.5-day-old embryo hindbrains. In situ hybridization analysis revealed that one of these genes, Sek, was expressed in an alternating segment-restricted pattern in the developing hindbrain. Isolation and analysis of Sek cDNAs covering the entire coding sequence indicated that Sek encoded a putative receptor protein tyrosine kinase, belonging to the Eph family. These data are consistent with a role of the Sek gene product in a signal transduction process involved in pattern formation in the hindbrain.

Several receptor tyrosine kinase genes of the Eph family are segmentally expressed in the developing hindbrain.

Pattern formation in the hindbrain involves a segmentation process leading to the formation of metameric units, manifested as successive swellings known as rhombomeres (r). In search for genes involved in cell-cell interactions during hindbrain segmentation, we have screened for protein kinase genes with restricted expression patterns in this region of the CNS. We present the cloning of three novel mouse genes, Sek-2, Sek-3 and Sek-4 (members of the Eph subfamily of putative transmembrane receptor protein tyrosine kinases (RTKs)), the identification of their chromosomal locations, and the analysis of their expression between 7.5 and 10.5 days of development. Before morphological segmentation, Sek-2 is transcribed in a transverse stripe corresponding to prospective r4 and the adjacent mesoderm, suggesting possible roles both in hindbrain segmentation and signalling between neuroepithelium and mesoderm. Sek-3 and Sek-4 have common domains of expression, including r3, r5 and part of the midbrain, as well as specific domains in the diencephalon, telencephalon, spinal cord and in mesodermal and neural crest derivatives. Together with our previous finding that Sek (Sek-1) is expressed in r3 and r5 (Gilardi-Hebenstreit et al., 1992; Nieto et al., 1992), these data indicate that members of the Eph family of RTKs may co-operate in the segmental patterning of the hindbrain.

Heart-specific targeting of beta-galactosidase by the ventricle-specific cardiac myosin light chain 2 promoter using adenovirus vectors.

Adenoviruses are attractive vectors for gene transfer into cardiac muscle. However, their promiscuous tissue tropism, which leads to an ectopic expression of the transgene, is a considerable limitation. To restrict expression to cardiomyocytes, we have constructed two recombinant adenoviruses (Ad-MLC2-250betagal and Ad-MLC2-2100betagal) containing the beta-galactosidase reporter gene under the control of the 250- or 2100-bp rat ventricle-specific cardiac myosin light chain-2v promoter (MLC-2v). Our in vitro and in vivo data have evidenced that the 2100-bp promoter allows stronger beta-galactosidase activity than the 250-bp promoter and that the deleted promoter allows a weak beta-galactosidase expression in skeletal muscle-derived cells in vitro. In contrast to the in vitro results, the highly deleted MLC-2v promoter of 250 pb conserved its heart specificity in in ovo and in vivo when introduced into the adenovirus genome, indicating that the specificity of this promoter is neither altered by the inverted terminal repeat nor by the enhancer of the Ela promoter, both of which located in the 5' flanking region of the promoter. Systemic injections of both recombinant adenoviruses into chicken embryos showed beta-galactosidase expression mainly in the right ventricle of the heart. We have confirmed the cardiac specificity of both promoters in mammalian species after injection of both recombinant adenoviruses into the heart of adult rats in vivo. The comparison of both promoters in vitro and in vivo has shown that the 250-bp MLC-2v promoter is 80% less active than the 2100-bp MLC-2v promoter and has enabled us to conclude that the MLC-2v promoter of 2100 bp is the most appropriate for efficient expression of a reporter gene or a therapeutic cardiac gene (e.g., SERCA2a or minidystrophin gene).

A recombinant E1-deleted canine adenoviral vector capable of transduction and expression of a transgene in human-derived cells and in vivo.

Human adenovirus (HAV) serotypes 2 and 5 are commonly used as vector backbones for adenovirus-mediated gene transfer. However, HAVs were chosen as a backbone for the vectors for historical reasons and have a number of significant disadvantages when used as a shuttle for gene transfer in humans. As an initial trial to circumvent some of the shortcomings of HAV vectors, we have produced an E1-deleted canine adenovirus type 2 (CAV-2) vector for gene transfer. Initially, we demonstrated that CAV-2 undergoes an abortive viral cycle in a wide range of human-derived cell lines. Second, we assayed human sera containing HAV-5 neutralizing antibodies for their ability to inhibit CAV-2-induced plaques on permissive cells. In the cohort tested, our data demonstrate that the humoral response directed against HAV-5 does not inhibit CAV-2 plaque formation in the majority of cases. Canine cell lines expressing the E1 region of CAV-2 were generated and characterized. A recombinant CAV vector (CAVRSVbetagal) deleted in the E1 region and harboring lacZ was constructed. We show that CAVRSVbetagal is able to transduce and direct expression of the transgene in vitro in a variety of mammalian cells, most notably primary human-derived cells. In addition, gene transfer is demonstrated in vivo using chick embryos.

Characterisation of the Sek-1 receptor tyrosine kinase.

We present an initial characterisation of the mouse Sek-1 protein, a member of the Eph subfamily of putative receptor tyrosine kinases, which has been proposed to play a role in the segmentation of both the hindbrain and the mesoderm. Antibodies raised against the protein have been used to confirm the early embryonic expression pattern previously established by mRNA in situ hybridisation. These antibodies, together with the expression of the Sek-1 gene in a baculovirus system, were instrumental in demonstrating that the protein carries a tyrosine kinase activity and that it is presented at the cell surface with its N-terminal (putative ligand-binding) domain outside of the cell. Therefore, as expected from its amino acid sequence, Sek-1 conforms to the general model of receptor-type tyrosine kinases.

Construction of a defective adenovirus vector expressing the pseudorabies virus glycoprotein gp50 and its use as a live vaccine.

The gene encoding the pseudorabies virus glycoprotein gp50 was cloned at the very left end of the genome of adenovirus type 5 to give a recombinant adenovirus (Ad-gp50) defective for the E1A gene. Ad-gp50 expressed high levels of gp50 in cells which either complemented (293 cells) or did not complement (Vero and HeLa cells) the E1A gene. Surprisingly, over an extended period, higher levels of gp50 were produced in HeLa cells which lack the E1A gene. Rabbits and mice inoculated with Ad-gp50 showed a strong antibody response against gp50. Some of them were protected from a virulent challenge with pseudorabies virus.

How to build a vertebrate hindbrain. Lessons from genetics.

During vertebrate embryogenesis, the hindbrain is the site of a segmentation process which leads to the formation, along the anterior-posterior axis, of 7-8 metameres called rhombomeres. This phenomenon plays an essential role in early hindbrain regionalisation and in the specification of the pattern of developing structures in this region of the brain. Data accumulated during the last 10 years have also shown that rhombomeres are units of gene expression and of cell lineage. Hence, a number of regulatory genes are expressed according to segment-specific patterns in the hindbrain and have been implicated in the pattern formation process. In this review, we focus on the analysis of the function and regulation of these genes along the different steps of hindbrain segmentation, from segment delimitation to acquisition of positional identity. On this basis, we propose a model for the control of early hindbrain development.

Krox-20 patterns the hindbrain through both cell-autonomous and non cell-autonomous mechanisms.

The Krox-20 gene encodes a zinc finger transcription factor, which has been shown previously, by targeted inactivation in the mouse, to be required for the development of rhombomeres (r) 3 and 5 in the segmented embryonic hindbrain. In the present work, Krox-20 was expressed ectopically in the developing chick hindbrain by use of electroporation. We demonstrate that Krox-20 expression is sufficient to confer odd-numbered rhombomere characteristics to r2, r4, and r6 cells, presumably in a cell-autonomous manner. Therefore, Krox-20, appears as the major determinant of odd-numbered identity within the hindbrain. In addition, we provide evidence for the existence of a non cell-autonomous autoactivation mechanism allowing recruitment of Krox-20-positive cells from even-numbered territories by neighboring Krox-20-expressing cells. On the basis of these observations, we propose that Krox-20 regulates multiple, intertwined steps in segmental patterning: Initial activation of Krox-20 in a few cells leads to the segregation, homogenization, and possibly expansion of territories to which Krox-20 in addition confers an odd-numbered identity.

Repression of the viral latent promoter BC-R2 in Epstein-Barr virus negative cell lines.

The regulation of the latent promoter BC-R2 of Epstein-Barr virus (EBV) was examined using the chloramphenicol acetyl transferase (CAT) gene reporter system. A 5' deletion analysis of BC-R2 promoter sequences has been used to characterize a region, described previously as a transcriptional enhancer in EBV positive cell lines, which can repress the BC-R2 transcriptional activity in EBV negative cell lines.

A receptor protein tyrosine kinase implicated in the segmental patterning of the hindbrain and mesoderm.

Pattern formation in the hindbrain and paraxial mesoderm of vertebrates occurs by the formation of a series of repeated segments. These processes of segmentation appear different at the morphological level, since hindbrain segments, the rhombomeres, form by the subdivision of the neural epithelium into compartments, whereas the mesodermal somites form by the sequential aggregation of mesenchymal cells into epithelial balls. Previous studies have implicated genes encoding transcription factors in the development of hindbrain segments, but nothing is known of genes involved in the formation of somites. Cellular interactions and signal transduction must be an important aspect of hindbrain segmentation, so we have screened for tyrosine kinases expressed in rhombomere-restricted patterns in the developing mouse embryo. We have identified a receptor protein tyrosine kinase, Sek, that has high relative levels of expression in rhombomeres 3 and 5. This alternating pattern is established coincidentally, both spatially and temporally, with the expression of Krox-20, a zinc-finger gene expressed prior to the morphological formation of rhombomeres. In addition, Sek expression occurs in several other developing tissues, including a dynamic regulation in the developing forebrain, spinal cord, early mesoderm and anterior presomitic mesoderm (segmental plate). The latter expression occurs in two stripes that correlate with, and presage, the formation of somites. Sek expression initially occurs throughout the presumptive somite, then becomes restricted anteriorly, and finally is down-regulated as the definitive somite is formed. These data suggest that despite the morphological differences in the segmentation of the hindbrain and mesoderm, Sek is involved in the segmental patterning of both of these tissues.

Segmental expression of the EphA4 (Sek-1) receptor tyrosine kinase in the hindbrain is under direct transcriptional control of Krox-20.

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres (r) with distinct identities. Recent studies have uncovered regulatory links between transcription factors governing this process, but little is known of how these relate to molecules mediating cell-cell signalling. The Eph receptor tyrosine kinase gene EphA4 (Sek-1) is expressed in r3 and r5, and function-blocking experiments suggest that it is involved in restricting intermingling of cells between odd- and even-numbered rhombomeres. We have analysed the cis-acting regulatory sequences of the EphA4 gene in transgenic mice and identified a 470 bp enhancer element that drives specific expression in r3 and r5. Within this element, we have identified eight binding sites for the Krox-20 transcription factor that is also expressed in r3 and r5. Mutation of these binding sites abolishes r3/r5 enhancer activity and ectopic expression of Krox-20 leads to ectopic activation of the enhancer. These data indicate that Krox-20 is a direct transcriptional activator of EphA4. Together with evidence that Krox-20 regulates Hox gene expression, our findings reveal a mechanism by which the identity and movement of cells are coupled such that sharply restricted segmental domains are generated.

[Role of the Krox-20 gene in the development of rhombencephalon]. / Rôle du gène Krox-20 dans le développement du rhombencéphale.

In the hindbrain region of the developing CNS, anteroposterior patterning involves a transient segmentation process which leads to the formation of morphological bulges called rhombomeres. The rhombomeres constitute cell lineage restriction units and participate in the establishment of a metameric pattern which is responsible for the segmental organisation of motor and reticular neurons. Like Drosophila compartments, rhombomeres also constitute domains of specific gene expression. Genes expressed in a rhombomere-specific manner so far identified encode various types of putative regulatory molecules, including transcription factors, like Hox proteins, the zinc finger protein Krox-20 and the basic domain leucine-zipper protein kreisler, and receptor type molecules, like Sek-1, a member of the EPH family of tyrosine kinase receptors. Such genes are thought to play a role either in the definition of segmental territories or in the specification of the identity of the rhombomeres. Initial analysis of the function of some of these genes have indeed supported this hypothesis. This is the case for the Krox-20 gene. It is expressed within the developing hindbrain in two transverse domains which prefigure and then coincide with r3 and r5. We have inactivated Krox-20 by homologous recombination in ES cells and demonstrated that the mutation leads to the deletion of r3 and r5. The mutation introduced into the Krox-20 gene involved the in-frame insertion of the lacZ coding sequence. This allowed us to follow the late expression pattern of the gene and to identify two additional phenotypes, affecting myelination of the peripheral nervous system and endochondral ossification. The lacZ reporter also permitted a detailed analysis of the expression of Krox-20 in peripheral glial cells, revealing important steps in the control of their development. Recently we have performed a detailed analysis of specific neuronal populations affected by the mutation which shed new light on the role of Krox-20 in the segmentation and on the physiological consequences of its inactivation. We have also identified several new members of the Sek-1 family of receptor tyrosine kinases, which are also expressed in a rhombomere-specific manner. Finally, we have provided evidence that Krox-20 is as a key regulator of r3/r5-specific transcription, controlling the expression of at least five other regulator genes in these rhombomeres. In three cases, Hoxb-2, Hoxa-2 and Sek-1, we could demonstrate that Krox-20 was directly involved in the transcriptional activation of these genes.