Mistargeting hippocampal axons by expression of a truncated Eph receptor

Abstract

Topographic mapping of axon terminals is a general principle of neural architecture that underlies the interconnections among many neural structures. The Eph family tyrosine kinase receptors and their ligands, the ephrins, have been implicated in the formation of topographic projection maps. We show that multiple Eph receptors and ligands are expressed in the hippocampus and its major subcortical projection target, the lateral septum, and that expression of a truncated Eph receptor in the mouse brain results in a pronounced alteration of the hippocamposeptal topographic map. Our observations provide strong support for a critical role of Eph family guidance factors in regulating ontogeny of hippocampal projections.

Fig 1.

Differential expression of the Eph receptors in the medial and lateral hippocampus. Sagital mouse brain sections of different developmental stages were hybridized with radio-labeled anti-sense probes of 6 EphA receptors (EphA3-A8). The expression of EphA1 and -A2 was not examined because they lack significant expression in developing brain (8). Top two panels on the left show bright field photomicrographs of E18 medial and lateral hippocampus, respectively. The hybridized parasagittal sections were stained with thionin to show hippocampal cytoarchitecture. Other panels show darkfield images of the E18 brain sections hybridized with anti-sense receptor probes indicated. Sense controls showed no specific signals. Medial, medial hippocampus; Lateral, lateral hippocampus. (Scale bar: 250 μm.)

Fig 2.

EphA receptor protein expression in the hippocampus. (A and B) Ephrin-A2 binding revealed high EphA receptor protein levels in the medial hippocampus (MH), and low levels in the lateral hippocampus (LH). A neighboring section was stained with thionin to reveal major cytoarchitecture features (A). (C–H) EphA5-LacZ expression in the medial (C and F), intermediate (D and G), and lateral (E and H) CA1 (C–E) and CA3 (F–H) regions. (I) EphA3 protein levels in the medial (M) and lateral (L) hippocampal lysates. EphA3 protein was first precipitated from 200 μg of E18 medial or lateral hippocampal lysates, and then analyzed with Western blot using EphA3 antibody. (Scale bars: A, 500 μm; H, 62.5 μm.)

Fig 3.

Spatial and temporal patterns of expression of three A-ephrins in the developing septum. (A–C) Expression of ephrin-A2, -A3, and -A5 in the P7 mouse septum. Note that at this stage, no ephrin-A2 and -A3 were detected. (D–F) Expression of ephrin-A2, -A3, and -A5 in the P14 septum. Ctx, cerebral cortex; Cpu, caudate putamen; Se, septum. (Scale bar: 1.5 mm.)

Fig 4.

Expression of a truncated EphA5 receptor in the mouse brain. (A) Schematic illustration of the transgene construct. The extracellular (EC) and transmembrane domains (TM) of EphA5 was fused in frame to an enhanced GFP. The EphA5–GFP fusion gene is placed under the control of the neuron-specific α-tubulin promoter (Tα1). (B) Detection of transgene DNA. Two independent transgenic mouse lines express high levels of transgene, as shown by PCR. PCR analysis produced an expected 298-bp fragment that is highest in homozygous mice (lanes 1 and 4), intermediate in heterozygous mice (lanes 2 and 5). No PCR fragments were detected in the wild-type littermates (lanes 3 and 6). DNA probes from the EphA5 and GFP regions as indicated were also used in Southern blot analysis to confirm PCR results. (C) mRNA and protein expression of EphA5–GFP transgene in the hippocampus. The mRNA was detected with antisense probe specific to the GFP portion of the transgene. The fusion protein was visualized by using fluorescence microscopy. There are no differences in expression between the medial and lateral hippocampal pyramidal neurons. Control nontransgenic hippocampus showed only faint background fluorescence. (Scale bar: 50 μm.)

Fig 5.

Inhibition of wild-type EphA3 and A5 activation by a truncated EphA5 receptor. (A) Cotransfection of EphA5(K−) DNA inhibits activation of EphA3 receptor tyrosine kinase. (B) Cotransfection of EphA5(K−) DNA inhibits activation of EphA5 tyrosine kinase. (Top) Tyrosine kinase activation in the absence of EphA5(K−) and the lack of activation in the presence of Eph(K−), as indicated in Bottom (DNA transfected in μg). (Middle) Similar wild-type receptor protein synthesis in the presence and absence of EphA5(K−). The proteins were labeled with [³⁵S]methionine and cysteine for analysis of expression levels (Middle). (C and D) Tyrosine phosphorylation of EphA3 (C) and EphA5 (D) is reduced in the trangenic hippocampus. Hippocampi from each wild-type control (WT) or transgenic (T1-T7) mice were analyzed separately. (Upper) Eph receptor immunoprecipitates analyzed with anti-phosphotyrosine antibody. (Lower) The blots were reprobed with anti-EphA3 (C) or EphA5 (D) antibody to show the protein levels of the receptors.

Fig 6.

Mistargeting of hippocampal axons in the lateral septum. (A) Position of DiI injection in the medial hippocampus. (B and D) Bright-field and fluorescence pictures of septal sections from control mice with DiI injected in the medial hippocampus. (C and E–G) Bright-field and fluorescence photomicrographs of septal sections from three different transgenic mice with medial hippocampal DiI tracing. Note the shift of the terminal zone ventrally and laterally. Anatomic landmarks are marked with white lines in D–G for reference. Ctx, cerebral cortex; LHip, lateral hippocampus; MHip, medial hippocampus; LV, lateral ventricle; LS, lateral septum; MS, medial septum; cc, corpus callosum. Arrows indicate terminal area of hippocampal axons. Arrowheads denote retrograde labeling of septo-hippcampal axons and neurons. (Scale bar: 500 μm.)