Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase.

The ephrins, ligands of Eph receptor tyrosine kinases, have been shown to act as repulsive guidance molecules and to induce collapse of neuronal growth cones. For the first time, we show that the ephrin-A5 collapse is mediated by activation of the small GTPase Rho and its downstream effector Rho kinase. In ephrin-A5-treated retinal ganglion cell cultures, Rho was activated and Rac was downregulated. Pretreatment of ganglion cell axons with C3-transferase, a specific inhibitor of the Rho GTPase, or with Y-27632, a specific inhibitor of the Rho kinase, strongly reduced the collapse rate of retinal growth cones. These results suggest that activation of Rho and its downstream effector Rho kinase are important elements of the ephrin-A5 signal transduction pathway.

Modulation of EphA receptor function by coexpressed ephrinA ligands on retinal ganglion cell axons.

The Eph family is thought to exert its function through the complementary expression of receptors and ligands. Here, we show that EphA receptors colocalize on retinal ganglion cell (RGC) axons with EphA ligands, which are expressed in a high-nasal-to-low-temporal pattern. In the stripe assay, only temporal axons are normally sensitive for repellent axon guidance cues of the caudal tectum. However, overexpression of ephrinA ligands on temporal axons abolishes this sensitivity, whereas treatment with PI-PLC both removes ephrinA ligands from retinal axons and induces a striped outgrowth of formerly insensitive nasal axons. In vivo, retinal overexpression of ephrinA2 leads to topographic targeting errors of temporal axons. These data suggest that differential ligand expression on retinal axons is a major determinant of topographic targeting in the retinotectal projection.

Identification of Elf-1 and B61 as high affinity ligands for the receptor tyrosine kinase MDK1.

Mouse Developmental Kinase 1 (MDK1) is a receptor tyrosine kinase of the eck/eph subfamily expressed in a variety of tissues during early mouse embryogenesis. To obtain further insight into the function of MDK1, we determined identity and localisation of its physiological ligand(s). Staining whole embryos with fusion proteins between the extracellular domain of MDK1 and human secreted alkaline phosphatase revealed areas of high receptor binding in the caudal mesencephalon, the frontal neocortex and the limb buds. This staining was sensitive to treatment with phosphatidylinositol-specific phospholipase C. Using Scatchard analysis, high affinity binding of Elf-1 (1.7 x 10(-10) M) and B61 (2.2 x 10(-10) M) towards MDK1 could be demonstrated. However, the transmembrane ligand Lerk2 displayed no measurable affinity for MDK1. Elf-1 and B61 bind to the three full-length MDK1 isoforms with similar dissociation constants. Slightly lower affinities were observed for the two truncated receptors MDK1-Tl and MDK1-T2. The activation of MDK1 with Elf-1 or B61 leads to the rapid autophosphorylation of MDK1 as well as tyrosine phosphorylation of an unknown 62 kDa phosphoprotein in Rat1 cells. These findings implicate MDK1 in patterning processes during early mouse embryogenesis and suggest MDK1 involvement in early organogenesis and midbrain development.

Identification of alternatively spliced mRNAs encoding variants of MDK1, a novel receptor tyrosine kinase expressed in the murine nervous system.

A novel member of the eck/eph family of receptor tyrosine kinases (RTKs), termed mouse developmental kinase 1 (MDK1), was identified and shown to be closely related to the Eek, Ehk1/Cek7, Ehk2, Cek4/Mek4/hek and Sek/Cek8 subfamily. Northern blot analysis revealed MDK1 mRNA transcripts of 6.8, 5.7, 4.0, 3.2 and 2.6 kb that encode apparent splice variants. Sequence analyses of MDK1 cDNA clones from adult mouse brain predict the existence of at least five isoforms, including two truncated receptor variants lacking the kinase domain. Northern blot and in situ hybridization analysis indicate that in the adult mouse MDK1 RNA expression is restricted to brain, testes and spleen. The distinct patterns of MDK1 gene expression during mouse development suggest an important role in the formation of neuronal structures and possibly other morphogenic processes.

Cloning, characterization, and differential expression of MDK2 and MDK5, two novel receptor tyrosine kinases of the eck/eph family.

Using a polymerase chain reaction-based strategy for the cloning of developmentally regulated receptor tyrosine kinases, we identified two novel members of the eck/eph-related subfamily which, in analogy with the recently identified mouse developmental kinase 1 (MDK1), were designated MDK2 and MDK5. MDK2 is highly homologous to the mouse kinase Myk-1 and the human kinase Htk, whereas MDK5 represents the mouse homologue of human Hek2. Northern blot analyses of adult mouse tissues revealed a 4.7 kb transcript of MDK2 and a 4.8 kb transcript of MDK5 in various organ systems, including lung, liver, kidney, intestine, muscle, heart, and, in the case of MDK5, also the brain. In addition to the full-length transcripts, smaller fragments were identified that probably represent truncated receptors. Northern blot analysis and in situ hybridization of mouse embryos indicated abundant expression during embryonic development, with preferential involvement of tissues of epithelial and endothelial origin for both kinases and of the spinal cord gray matter for MDK5. Unlike most other members of the eck/eph-related subfamily, the expression of MDK2 and MDK5 is not primarily restricted to neuronal structures, and their abundant presence in various organ systems during embryonic development suggests an important role in gestational growth and differentiation.

Isolation and developmental expression analysis of Enx-1, a novel mouse Polycomb group gene.

Members of the Polycomb group (Pc-G) of genes encode transcriptional regulators that control the expression of key developmental effector genes in Drosophila melanogaster. Although multiple Pc-G genes have been identified and characterized in Drosophila, information about these important regulatory proteins in vertebrates, including their precise expression patterns, has remained scarce. We report here the cloning of Enx-1, a novel vertebrate Pc-G gene, which encodes the murine homolog of the Drosophila Enhancer of zeste (E(z)) gene. Drosophila E(z) controls the expression of several homeobox genes as well as some segmentation genes and its disruption causes multiple phenotypes in Drosophila development. Analysis of the primary structure of murine Enx-1 reveals the conservation of several regions, including the previously described SET domain and a newly defined CXC domain. In addition, we find the SET domain to be conserved in evolutionarily distant species ranging from vertebrates to plants and fungi. The expression pattern analysis of Enx-1 reveals ubiquitous expression throughout early embryogenesis, while in later embryonic development Enx-1 expression becomes restricted to specific sites within the central and peripheral nervous system and to the major sites of fetal hematopoiesis. In adult stages we also find Enx-1 expression to be restricted to specific tissues, including spleen, testis and placenta.

Differential expression of MAM-subfamily protein tyrosine phosphatases during mouse development.

The MAM-subfamily of type II transmembrane protein tyrosine phosphatases (PTPases) currently comprises the enzymes PTPkappa, PTPmu and PCP2. In an effort to elucidate the individual physiological roles of these closely related proteins we performed a detailed analysis of their mRNA transcript distributions at different stages of mouse embryogenesis and postnatal brain development. Our in situ hybridization studies revealed distinct and complementary expression patterns of PTPkappa, PTPmu and PCP2 transcripts. Based on our results and previous reports we discuss MAM-PTPases as a new class of morphoregulatory molecules.

Eph receptors and ephrins demarcate cerebellar lobules before and during their formation.

The formation of the ten cerebellar lobules is an unsolved problem in brain development. We report a screen for the four subfamilies of Eph receptors and their ligands (ephrins) in developing mouse cerebellum, using soluble receptor-immunoglobulin and ligand-immunoglobulin fusion proteins, and antibodies against EphA and ephrin-B proteins. Our results identify Eph receptors and ephrins as the first molecules known to demarcate individual lobules during development. Staining for ephrin-A ligands is in lobule VIII as it forms, across the whole width of the cerebellum. Staining for three EphA receptors approximately coincides with presumptive lobules VI and/or VII before and just after birth, whereas a fourth EphA receptor (EphA4, which binds ligands of both subfamilies) has more widespread expression. Staining for EphB receptors is in lobules VII, VIII, and IX. Staining for ephrin-B ligands is much weaker, becomes detectable only after birth, and does not appear to be lobule-specific. Staining for all subfamilies spreads to at least some adjacent lobules as maturation proceeds. The lobule-specific patterns appear before the lobules form, and initially extend across the width of the cerebellum, in spite of the lesser conservation of the lateral extensions of the lobules. These expression patterns define previously unknown developmental units and suggest that Eph family proteins may contribute to cerebellar morphogenesis.

Segregation of the receptor EphA7 from its tyrosine kinase-negative isoform on neurons in adult mouse brain.

The EphA7 gene encodes not only a typical receptor tyrosine kinase (TK+) but also an isoform lacking the tyrosine kinase domain (TK-). We have made antibodies to localise EphA7 TK+ and TK- isoforms in mouse brain. The TK- isoform was not detectable prenatally, despite reported expression of the TK- mRNA in the embryo. However, both TK+ and TK- isoforms showed striking distributions in adult brain. TK+ receptor immunoreactivity was strong in neuropil throughout most of the telencephalon, probably on fine arborisations from neurons which expressed EphA7 during development (in cerebral cortex, hippocampus, and striatum). In contrast, TK- receptor immunoreactivity was conspicuous on cell bodies and proximal dendrites of a limited number of neuronal types, some of which carried EphA7 TK+ receptor on their axons. This suggests that the TK- receptor, acting as a dominant negative antagonist, may ensure that the TK+ receptor only responds to signals encountered by the growing extremities of axons or dendrites.

Distribution of the receptor EphA7 and its ligands in development of the mouse nervous system.

EphA7 is a receptor tyrosine kinase of the Eph family. We have mapped EphA7 immunoreactivity and ligand binding in mouse embryo heads and developing brain. Immunoreactivity for the full-length receptor is found in all the cell populations that express EphA7 mRNA. In particular, it is located on growing axons from EphA7-expressing neurons, both in the trigeminal nerve and in developing brain. In many cases it persists in terminal fields in adult brain. Ligand is detected in a largely complementary distribution in embryos, but is surprisingly weak or undetectable in the target regions of many EphA7-positive axons postnatally.

Characterization of KIF1C, a new kinesin-like protein involved in vesicle transport from the Golgi apparatus to the endoplasmic reticulum.

Kinesins comprise a large family of microtubule-based motor proteins, of which individual members mediate specific types of motile processes. Using the ezrin domain of the protein-tyrosine phosphatase PTPD1 as a bait in a yeast two-hybrid screen, we identified a new kinesin-like protein, KIF1C. KIF1C represents a member of the Unc104 subfamily of kinesin-like proteins that are involved in the transport of mitochondria or synaptic vesicles in axons. Like its homologues, the 1103-amino acid protein KIF1C consists of an amino-terminal motor domain followed by a U104 domain and probably binds to target membranes through carboxyl-terminal sequences. Interestingly, KIF1C was tyrosine-phosphorylated after peroxovanadate stimulation when overexpressed in 293 or NIH3T3 fibroblasts or in native C2C12 cells. Using immunofluorescence, we found that KIF1C is localized primarily at the Golgi apparatus. In brefeldin A-treated cells, the Golgi membranes and KIF1C redistributed to the endoplasmic reticulum (ER). This brefeldin A-induced flow of Golgi membranes into the ER was inhibited in cells transiently overexpressing catalytically inactive KIF1C. In conclusion, our data suggest an involvement of tyrosine phosphorylation in the regulation of the Golgi to ER membrane flow and describe a new kinesin-like motor protein responsible for this transport.

Eph receptor-ligand interactions are necessary for guidance of retinal ganglion cell axons in vitro.

Previous results of an in vitro guidance test, the stripe assay, have demonstrated the presence of a repulsive axon guidance activity for temporal retinal axons in the posterior part of the vertebrate optic tectum. Ephrin-A5 and Ephrin-A2 are ligands for the EphA subfamily of Eph receptor tyrosine kinases, which are expressed in overlapping gradients in the posterior part of the tectum. When recombinantly expressed, both proteins have been shown to guide retinal ganglion cell axons in the stripe assay. While these results suggest that Ephrin-A5 and Ephrin-A2 form part of the posterior repulsive guidance activity, they do not elucidate whether they are necessary components. Here we report that soluble forms of the ligands at nanomolar concentrations completely abolish this repulsive activity. Similar results were obtained with the soluble extracellular domain of EphA3, which is a receptor for Ephrin-A2 and Ephrin-A5, but not with the corresponding domain of EphB3, a receptor for the transmembrane class of Eph ligands. These experiments show that the repulsive axon guidance activity seen in the stripe assay is mediated by Ephrin-A ligands.