The Journal of Neuroscience, November 15, 1996, 16(22):7182–7192 Detection of Ligands in Regions Anatomically Connected to Neurons Expressing the Eph Receptor Bsk: Potential Roles in Neuron–Target Interaction Jian-Hua Zhang,1 Douglas P. Cerretti,2 Tian Yu,1 John G. Flanagan,3 and Renping Zhou1 1Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855, 2Immunex Research and Development Corporation, Seattle, Washington 98101, and 3Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 Neuron–target interaction is a key feature in the establish- sible for the Bsk-AP binding. In the septum, Elf-1, Lerk3 ment of neuronal networks. However, the underlying mech- (Efl-2), and AL-1/Lerk7 were transcribed. In the hypothala- anism remains unclear. We have shown that at the time of mus, AL-1/Lerk7 was the ligand detected by Bsk-AP. In the target innervation, Bsk, an eph family receptor, is expressed olfactory system, high levels of Lerk3 were detected in the at high levels in several brain regions including the hip- sensory neurons. Both Elf-1 and AL-1/Lerk7 were present in pocampus, olfactory bulb, and retina. To study whether the the tectum. These ligand-positive areas are known to be ligands are expressed in the target tissues, we investigated anatomically connected to Bsk-expressing regions. These the expression of Bsk ligands using a ligand-affinity probe, observations strongly suggest that Bsk and the ligands par- Bsk-AP, which consisted of the extracellular domain of Bsk ticipate in neuron–target interactions in multiple systems and fused in frame with a human placental alkaline phosphatase. provide support for their involvement in topographic These analyses showed that the ligands were expressed at projection. high levels in the developing septum, hypothalamus, olfac- Key words: brain-specific kinase; growth factor receptor; eph tory neural epithelium, and tectum. In situ hybridization stud- family; alkaline phosphatase tagging; neuronal targeting; topo- ies revealed that at least three different factors were respon- graphic projection Neuron–target interaction is a key feature in the establishment of At least seven candidate ligands of the eph family have been neuronal networks and plays a critical role in the development of isolated (Bartley et al., 1994; Beckmann et al., 1994; Cheng et al., topographic connections. Appropriate targeting by neurons re- 1994; Davis et al., 1994; Shao et al., 1994; Bergemann et al., 1995; quires that the guidance cues match terminals with specific cellu- Drescher et al., 1995; Kozlosky et al., 1995; Winslow et al., 1995). lar targets, a requirement accommodated by matching fixed tags The ligands can be divided into two subfamilies, one with mole- on afferents and corresponding targets. Complementarity of mo- cules anchored on the membrane through GPI anchors and the lecular tags on afferents and targets was first postulated by Sperry other with molecules containing a transmembrane domain. Four in his chemoaffinity theory more than half a century ago (Sperry, of the five ligands with GPI anchors—B61, Lerk3 (also named 1943, 1963). EFL-2; Davis et al., 1994), Elf-1, and AL-1/Lerk7—have been Only recently have specific candidate molecules been identified shown to bind to Bsk or its species homologs in vitro (Davis et al., (Cheng et al., 1995; Drescher et al., 1995). Elf-1, a membrane- 1994; Shao et al., 1995; Winslow et al., 1995; Gao et al., 1996). anchored eph family ligand, and one of its putative receptors, However, it is not known whether any of these molecules are Mek4, are expressed in complementary gradients in the chicken accessible to Bsk in vivo, because no detailed in situ expression retina and tectum, respectively, fulfilling a long-standing predic- study has been published. Here we report studies on the expres- tion of Sperry’s chemoaffinity theory (Cheng et al., 1995). Fur- sion of Bsk and its ligands during the development of mouse thermore, an Elf-1-related molecule, the repulsive axonal guid- nervous system. Our studies suggest that Bsk may interact with ance signal (RAGS), is expressed in the chicken tectum in an multiple ligands in vivo and play important roles in the establish- anteroposterior gradient and repels the growth of retinal axons ment and maintenance of projections in multiple systems. (Drescher et al., 1995). The human homolog of RAGS, AL-1/ Lerk7, has been shown previously to be a ligand of the eph family MATERIALS AND METHODS receptor Rek7 (Winslow et al., 1995; Cerretti et al., 1996), a rat Animals. CD-1 mice from embryonic day 8 (E8) to E18 and postnatal day homolog of mouse Bsk (Zhou et al., 1994). These studies strongly 1 (P1) through P180 were used in this study. At least two mice at each age implicate the eph family ligands and receptors in the development were analyzed. The occurrence of a vaginal plug was defined as E1, and the day of birth was defined as P1. Embryos and brains were dissected of topographic mapping. under carbon dioxide anesthesia and immediately frozen on powdered dry ice. Coronal and sagittal sections of 14 mm thickness were cut on a Received April 19, 1996; revised Aug. 19, 1996; accepted Aug. 21, 1996. cryostat at 2208C and thaw-mounted onto slides pretreated with This research was supported by National Science Foundation Grant IBN-9409930. triethoxy-3-aminopropyl silane (Sigma, St. Louis, MO; see Ligand- We thank Y. Yue for critical comments on this manuscript. Binding Assay). These slides were then stored at 2808C until use (up to Correspondence should be addressed to Renping Zhou, Laboratory for Cancer 2 months). Research, College of Pharmacy, Rutgers University, Piscataway, NJ 08855. Construction and expression of Bsk-AP. The affinity probe Bsk-AP was Copyright q 1996 Society for Neuroscience 0270-6474/96/167182-11$05.00/0 created as follows. An MroI restriction site was first introduced at the Zhang et al. • Expression of Bsk Ligands in the Developing Brain J. Neurosci., November 15, 1996, 16(22):7182–7192 7183 Figure 1. Differential Bsk expression along the mediolateral hippocampal axis. Bsk mRNA levels detected by in situ hybridization at different posi- tions along the mediolateral axis are shown. A, B, Dark- and bright-field photomicrographs of a medial (septal) hippocampal section. C, D, Dark- and bright-field photomicrographs of a section at an intermediate medio- lateral level. E, F, Dark- and bright-field pictures of a lateral (temporal) hippocampal section. Slides shown here and in the following figures were counterstained with thionin. G, Quantitative analysis of Bsk in situ hybrid- ization signals at different mediolateral hippocampal levels. S, Subiculum; DG, dentate gyrus. Scale bars, 200 mm. junction of the extracellular and transmembrane domain by site-directed Vector (Flanagan and Leder, 1990) using a BglII linker. The vector mutagenesis using an oligonucleotide 59-GATCAAAGCCAGATT- containing the fusion construct was cotransfected into National Institutes CCGGACATCATTGCAGTGTCAG-39 corresponding to nucleotide po- of Health-3T3 cells with pSV2neo plasmid containing the aminoglycoside sition 636–669 of Bsk (Zhou et al., 1994) with a GGA addition to create phosphotransferase gene, which confers G418 resistance. The transfected an MroI site. An EcoRI–MroI DNA fragment containing the entire cells were then selected with 400 mg/ml G418 (Life Technologies, Gaith- coding region of the extracellular domain from the initiation codon to the ersburg, MD), and neo-resistant colonies were first screened for heat- last codon before the transmembrane domain of Bsk was ligated to a resistant alkaline phosphatase activity in the culture supernatant (Flana- secreted form of human placental alkaline phosphatase in the APtag-1 gan and Leder, 1990) and then for metabolically labeled Bsk-AP protein 7184 J. Neurosci., November 15, 1996, 16(22):7182–7192 Zhang et al. • Expression of Bsk Ligands in the Developing Brain Figure 2. Expression of Bsk ligand(s) in the adult septum. A, E, Bsk-AP binding activity in the septum. B, F, Elf-1 expression. C, G, Lerk3 expression. D, H, AL-1/Lerk7 expression. A–D, Coronal sections. E–H, Higher magnifications of the septal regions, showing that the expression of the ligands was restricted to the ventral lateral septum. ac, Anterior commissure; cc, corpus callosum; f, fornix; BST, bed nucleus of the stria terminalis; Cpu, caudate putamen; Ctx, cerebral cortex; LS, lateral septum; MS, medial septum; Se, septum; VDB, vertical diagonal band; Scale bars: A, 1.2 mm; E, 0.6 mm. using immunoprecipitation with a monoclonal antibody against the hu- ACAGGAGGCGTGGCCCTCACC-39, corresponded to nucleotide po- man placental alkaline phosphatase (Medix Biotech). A protein with an sition 283–328 in the Elf-1 coding region (Cheng et al., 1994). Elf-1-06, expected molecular weight of 110 kDa was detected only in the trans- 59-ACCTCATCCCTGTGGCTTGTTCCCTTCCCAGTGTCACCAGC- fected cells in both the media and the cell lysates. The clone with the AATGT-39, corresponded to nucleotide position 926–971 in the 39-end- highest level of expression secreted ;800 OD units z ml21 z hr21 heat- noncoding region of Elf-1 (Cheng et al., 1994). Neither oligonucleotide resistant alkaline phosphatase activity. probe shared any significant homology with other eph family ligands or To examine whether the secreted Bsk-AP was active in ligand binding, any other genes in the gene bank. The two probes gave similar patterns COS-7 cells transiently transfected with Elf-1, a putative ligand of Bsk, of hybridization. The corresponding sense probes were used as controls were stained with Bsk-AP. These experiments showed that Elf-1- and gave no specific signals. expressing cells, but not the cells transfected with the vector alone, Lerk3 probes. Lerk3 is a mouse homolog of the human EFL-2/LERK3, stained positive with Bsk-AP, indicating that the fusion protein retained a ligand isolated through binding to the rat homolog of Bsk, Ehk1 (Davis ligand-binding activity.