Reciprocal Expression of the Eph Receptor Cek5 and Its Ligand(s) in the Early Retina

Abstract

Recent evidence suggests that Eph receptor tyrosine kinases and their ligands provide positional information in the developing visual system. We previously found that the Eph receptor Cek5 is more highly expressed in the ventral than dorsal chicken embryonic retina. We now report the identification of a chicken ligand for Cek5 (cCek5-L) that is 75% identical to the ligand LERK2. In situ hybridization experiments do not reveal a dorsoventral gradient of cCek5-L transcripts in the optic tectum at Embryonic Day 8, suggesting that this ligand is not involved in guiding Cek5-expressing axons in the tectum. Surprisingly, it is in the retina that high levels of cCek5-L mRNA are present. In the early retina, cCek5-L is more highly expressed in the dorsal than the ventral aspect. Similarly, a Cek5 Ig chimera labels dorsal but not ventral retina, indicating that even if several Cek5 ligands are present, their overall distribution is complementary to that of Cek5. Hence, Cek5 and cCek5-L may both contribute to define anatomical compartments within the early retina. In contrast, in the 11-day embryonic retina the distributions of Cek5 and its ligand(s) show considerable overlap, suggesting changing functions as development progresses. In dissociated cultures of dorsal or ventral retinal cells seeded on plates coated with either receptor or ligand Ig chimeras, the interaction between Cek5 and its ligand(s) or cCek5-L and its receptor(s) is sufficient to mediate cell adhesion and allows neurite outgrowth.

SHEP1 Function in Cell Migration Is Impaired by a Single Amino Acid Mutation That Disrupts Association with the Scaffolding Protein Cas but Not with Ras GTPases

“…Antibodies-Anti-EphB2 antibodies were obtained using as the antigen a GST fusion protein comprising amino acids 897-995 of chicken EphB2, as described previously (31). Anti-SHEP1 antibodies were made using a GST fusion protein of the SHEP1 SH2 domain, as described previously (14), or a peptide corresponding to the 13 carboxyl-terminal amino acids of SHEP1 cross-linked to bovine serum albumin.…”

In Vivo Tyrosine Phosphorylation Sites of Activated Ephrin-B1 and EphB2 from Neural Tissue

“…Zip tips packed with C 18 were from Millipore Corporation. EphB2 Fc was prepared as previously described (20).…”

Eph/ephrins and N-cadherin coordinate to control the pattern of sympathetic ganglia

“…Embryos for whole-mount staining were fixed as above, washed in PBS, blocked as previously described (Rifkin et al, 2000) and incubated in primary antibodies for 2-3 days at 4°C. Primary antibodies included: Hnk-1 (Developmental Studies Hybridoma Bank, University of Iowa), NCD2 (kind gift of M. Takeichi, Riken Center, Japan), EphB2 [anti-Cek5 (897-995) (Holash et al, 1995)] and ephrinB1 (Koblar et al, 2000). Embryos were then washed and blocked at room temperature for 1 hour before incubation in secondary antibody overnight at 4°C.…”