In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases

Abstract

The results of previous in vitro experiments indicate that a glycosylphosphatidylinositol (GPI)-anchored protein may play an important role in the guidance of temporal retinal axons during the formation of the topographically ordered retinotectal projection. We have purified and cloned a GPI-anchored, 25 kDa glycoprotein that is a good candidate for a molecule involved in this process. During the time of innervation by retinal ganglion cells, this protein is gradedly expressed in the posterior part of the developing tectum. In two different in vitro assay systems, the recombinant protein induces growth cone collapse and repulsion of retinal ganglion cell axons. These phenomena are observed for axons of temporal as well as nasal origin, indicating that an additional activity may be necessary to confer the nasotemporal specificity observed in previous assays. We named the protein RAGS (for repulsive axon guidance signal). The sequence of RAGS shows significant homology to recently identified ligands for receptor tyrosine kinases of the Eph subfamily.

EphrinA5 protein distribution in the developing mouse brain

“…Moreover, the presence of potential ephrinA5 receptors such as EphA3 [40], EphB2 [41], EphA5 [35,42,43], EphA7 and EphA4 [35] has been also described in the mitral cells during embryogenesis. This suggests that ephrinA5 and its receptors are not expressed in complementary gradient as it has been described for other systems such as the retinotectal one [8,44]. Instead, we showed that ephrinA5 is uniformly expressed in this structure as for its receptors [40-42,35,20].…”

“…EphrinA5 RNA is known to be expressed in a caudal (high) to rostral (low) gradient in the mouse superior colliculus [44,50,55]. This gradient is thought to be at the basis of the chemorepellent action of ephrinA5 on retinal ganglion cell's axons arising from the nasal part of the retina [50].…”

The Eek receptor, a member of the Eph family of tyrosine protein kinases, can be activated by three different Eph family ligands

“…Elf-1/Cek7-L is highly expressed at the midbrain tectum in a decreasing caudal-rostral gradient (Cheng and Flanagan, 1994;Cheng et al, 1995). AL-1/RAGS is also highly expressed in the caudal tectum (Drescher et al, 1995). Ehk1-L/E¯-2/Lerk3 is highly expressed in the brain although its speci®c localization in the brain has not yet been reported (Davis et al, 1994).…”

“…Therefore, we used degenerate oligonucleotides designed from conserved regions of the Eph family ligands to amplify related sequences from mouse brain cDNA since the expression of the Eek receptor is brain-speci®c. Three known ligands, Elf-1/Cek7-L (Cheng and Flanagan, 1994;Shao et al, 1995), Ehk1-L/E¯-2/Lerk3 (Davis et al, 1994) and AL-1/RAGS (Winslow et al, 1995;Drescher et al, 1995), were ampli®ed and subsequently their full-length cDNAs were cloned from a mouse brain cDNA library. To investigate whether these ligands can bind to the Eek receptor, we constructed expression vectors containing the entire coding sequence of the ligands, except for the hydrophobic sequence of each ligands fused to the Fc portion of human immunoglobulin G1 (Figure 5a).…”

“…A study of AL-1/RAGS function using in vitro cultures of cortical neurons expressing the Rek7 receptor tyrosine kinase, a rat homologue of mouse Bsk, has suggested that AL-1/RAGS functions actively to stimulate axon fasciculation (Winslow et al, 1995). In addition, RAGS, a chick homologue of AL-1, was demonstrated to induce growth cone collapse and repulsion of retinal ganglion cell axons (Drescher et al, 1995). Recently it was reported that, in mice lacking Nuk, axons of the anterior commissure fail to reach their targets and project aberrantly to the¯oor of the brain .…”