Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Accepted manuscripts
    • Issue in progress
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Special issues
    • Subject collections
    • Sign up for alerts
  • About us
    • About Development
    • About the Node
    • Editors and Board
    • Editor biographies
    • Travelling Fellowships
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
    • Biology Open transfer
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contacts
    • Contacts
    • Subscriptions
    • Feedback
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

User menu

  • Log in
  • Log out

Search

  • Advanced search
Development
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

supporting biologistsinspiring biology

Development

  • Log in
Advanced search

RSS  Twitter  Facebook  YouTube 

  • Home
  • Articles
    • Accepted manuscripts
    • Issue in progress
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Special issues
    • Subject collections
    • Sign up for alerts
  • About us
    • About Development
    • About the Node
    • Editors and Board
    • Editor biographies
    • Travelling Fellowships
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
    • Biology Open transfer
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contacts
    • Contacts
    • Subscriptions
    • Feedback
Research Article
Ephrins regulate the formation of terminal axonal arbors during the development of thalamocortical projections
Fanny Mann, Christiane Peuckert, Frank Dehner, Renping Zhou, Jürgen Bolz
Development 2002 129: 3945-3955;
Fanny Mann
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Christiane Peuckert
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Frank Dehner
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Renping Zhou
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jürgen Bolz
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & tables
  • Info & metrics
  • PDF
Loading

Summary

The development of connections between thalamic afferents and their cortical target cells occurs in a highly precise manner. Thalamic axons enter the cortex through deep cortical layers, then stop their growth in layer 4 and elaborate terminal arbors specifically within this layer. The mechanisms that underlie target layer recognition for thalamocortical projections are not known. We compared the growth pattern of thalamic explants cultured on membrane substrates purified from cortical layer 4, the main recipient layer for thalamic axons, and cortical layer 5, a non-target layer. Thalamic axons exhibited a reduced growth rate and an increased branching density on their appropriate target membranes compared with non-target substrate. When confronted with alternating stripes of both membrane substrates, thalamic axons grew preferentially on their target membrane stripes. Enzymatic treatment of cortical membranes revealed that growth, branching and guidance of thalamic axons are independently regulated by attractive and repulsive cues differentially expressed in distinct cortical layers. These results indicate that multiple membrane-associated molecules collectively contribute to the laminar targeting of thalamic afferents. Furthermore, we found that interfering with the function of Eph tyrosine kinase receptors and their ligands, ephrins, abolished the preferential branching of thalamic axons on their target membranes, and that recombinant ephrin-A5 ligand elicited a branch-promoting activity on thalamic axons. We conclude that interactions between Eph receptors and ephrins mediate branch formation of thalamic axons and thereby may play a role in the establishment of layer-specific thalamocortical connections.

  • Wiring molecules
  • Cortical development
  • Thalamocortical projection
  • Ephrin
  • Eph receptor
  • Target recognition
  • Axon guidance
  • Branch formation
  • Mouse

INTRODUCTION

One of the basic features of the mammalian neocortex is its lamina-specific connectivity both with subcortical structures and other cortical areas. For example, in the primary somatosensory area (S1) of the cortex, thalamocortical projections arising from the ventrobasal nucleus (VB) terminate in layer 4 and extend collaterals in layer 6, skipping layer 5. The question of how these connections are established has been examined in the developing cortex of various species, and results indicate that the laminar pattern of thalamocortical projections develops precisely from the outset (Bicknese et al., 1994; Ghosh and Shatz, 1992; Lund and Mustari, 1977; Miller et al., 1993; Agmon et al., 1993; Catalano et al., 1996; Kageyama and Robertson, 1993). In the murine brain, the first thalamic axons enter the cortex at embryonic day (E) 14, and grow along a pathway centered on the subplate layer underlying the undifferentiated cortical plate (Bicknese et al., 1994). Once they reach their target area of the cortex, thalamic axons adopt a radial growth and invade progressively the differentiating cortical layers. At birth, thalamocortical axons extend though developing layers 6 and 5 and start branching within layer 6 (Agmon et al., 1993). They then stop their radial growth at postnatal day (P) 2 after reaching layer 4, form terminal branches and elaborate synaptic contact with their appropriate target cells (Agmon et al., 1993). Over the following week, axons emit more branches within layer 4, but the extent of terminal arbors remains within the width of layer 4 and does not extend across into the adjacent non-target layers (Agmon et al., 1993).

In vitro studies have implicated intracortical cues in directing the lamina-specific growth of thalamic axons. It has been proposed that guidance of thalamic afferents in the subplate results from the expression of growth-promoting factors along their pathway (Götz et al., 1992; Henke-Fahle et al., 1996; Hübener et al., 1995; Kinnunen et al., 1999) and inhibitory signals in the undifferentiated cortical plate (Emerling and Lander, 1996; Tuttle et al., 1995). Later on, upregulation of growth-promoting molecules in the cortical plate allows thalamic axons to enter the cortical layers (Götz et al., 1992; Hübener et al., 1995). The observation of precise layer-specific targeting of thalamocortical projections in organotypic thalamus-cortex co-cultures (Bolz et al., 1992; Molnar and Blakemore, 1991; Yamamoto et al., 1992; Yamamoto et al., 1997) have led to the hypothesis that molecular signals confined to individual cortical layers allow thalamic axons to distinguish between target and non-target cells. In support of this idea, in vitro experiments revealed the existence of layer-specific cues that regulate the assembly of intrinsic cortical circuits (Castellani and Bolz, 1997; Dantzker and Callaway, 1998). However, the molecular identity of the factors that serve this function remains to be elucidated.

Over the past few years, a large number of cell surface and secreted molecules have been identified that orchestrate the development of specific connections in the central nervous system. Among them, the ephrins is a family of putative axon guidance ligands that fall into two subclasses: glycosylphosphatidylinositol (GPI)-linked ephrin-As and transmembrane ephrin-Bs. These interact respectively with A-type and B-type Eph tyrosine kinase receptors. Eph receptors and ephrins have been implicated in a range of developmental processes, including topographic mapping, brain commissure formation and axon guidance at the midline (reviewed by Klein, 2001; Wilkinson, 2001). Recent studies have provided evidence for a key role of ephrins in guiding thalamocortical axons to their appropriate target cells in the developing cortex. A role in regulating topographic mapping of thalamocortical projections within the primary somatosensory area of the neocortex has been proposed based on the analysis of ephrin-A5 knockout mice (Vanderhaeghen et al., 2000). It has also been suggested that ephrin-A5 could function in the patterning of thalamocortical connections between areas of the limbic cortex and neocortex (Gao et al., 1998; Mackarehtschian et al., 1999). In addition, members of the ephrin family may play a role in regulating the laminar pattern of local circuits within the neocortex (Castellani et al., 1998) and the laminar termination of entorhinal afferents in the hippocampus (Stein et al., 1999). Together, these data suggest that ephrins are candidate molecules for providing information for thalamic afferents during targeting to the proper layer in the cortex.

In the present study, we used different in vitro assays to explore the influence of layer-specific cell surface molecules on selective growth and targeting of thalamic axons. We show that multiple membrane-bound signals, acting either as attractive or repulsive cues, cooperate to specify the patterning of thalamocortical projections. Furthermore, our results furnish evidence suggesting that ephrin ligands stimulate branch formation of thalamic axons and thereby may contribute to the formation of terminal arborizations in their target layer.

MATERIALS AND METHODS

Preparation of thalamic explants and dissociated thalamic cells

Embryos at E14 (day of insemination=E1) were removed from anesthetized pregnant OF1 (IFFA-Credo, Lyon, France) and NMRI albino mice (from Harlan Winkelmann, Borchen, Germany), and the brains were transferred in Gey’s Balanced Salt Solution (GBSS) supplemented with 6.5 mg/ml glucose. Neurons in the thalamus are generated following a lateral to medial gradient over a period of time extending from E11 to E16 in the mouse (Angevine, 1970). At E14 most of the neurons in the thalamus are destined to lateral nuclei that provide inputs to the neocortex. In some experiments, medial (limbic) nuclei of the thalamus were isolated from E16 mouse fetuses as previously described (Mann et al., 1998).

To prepare thalamic explants, blocks of diencephalic tissue were isolated and cut in 200 μm3 explants with a McIlwain tissue chopper. To prepare dissociated thalamic cells, thalami were excised and collected in ice cold Hank’s Balanced Salt Solution (HBSS) containing 6.5 mg/ml glucose and 0.025% trypsin, then incubated at 37°C for 25 minutes. Thalamic tissue was dissociated into single cells and filtered through a nylon gauze to remove cell aggregates. Cells were centrifuged at 8 g for 8 minutes and cell density was adjusted to 2×106 cells/ml.

Isolation of cortical layers

Mice at P6-8 (day of birth=P0) were decapitated and the brains were transferred in GBSS with 6.5 mg/ml glucose. The dorsal neocortex was dissected and cut in 200 μm slices with a McIlwain tissue chopper. Cortex slices from the primary somatosensory area in which a barrel pattern in layer 4 was clearly visible were selected. Cortical layer 4 and layer 5 were isolated as described previously (Castellani and Bolz, 1997; Götz et al., 1992).

NIH3T3 cells

NIH3T3 cell line transfected with a retroviral vector, pLIG*, containing the human ephrin-A5 gene (EPHA5) or, as a control, transfected with the vector alone (Gao et al., 1996) were grown in the following medium: DMEM/F12 (1:1), 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin.

Membrane preparation and functional assays

Membranes from isolated cortical layers and confluent NIH3T3 cells were prepared as described previously (Mann et al., 1998). To prepare homogenous substrates of membrane, pairs of glass coverslips were coated with 1.9 μg of laminin and 0.1 μg of poly-L-lysine in 100 μl GBSS as a ‘sandwich’ for 1 hour at 37°C, then incubated for 2 hours at 37°C with 100 μl of membrane suspension used at a concentration of 60 μg/ml. In some experiments, a 1:1 mixture of layer 4 and layer 5 membranes was used as a substrate for neurite growth. In the latter case, the total membrane concentration was adjusted to 120 μg/ml. Thus, the amount of target membranes was unchanged when layer 4 membranes were used individually or mixed with layer 5 membranes. For stripe assay, polycarbonate filters containing alternating lanes of two membrane species were prepared according to Walter et al. (Walter et al., 1987). In some cases, membrane stripes were transferred on glass coverslip as described by Wizenmann et al. (Wizenmann et al., 1993). For the enzyme treatment, membranes were incubated with phosphatidylinositol-specific phospholipase C (PI-PLC; 3 U/5 mg proteins) for 1 hour at 37°C and washed in phosphate-buffered saline (PBS) before use in substrate preparation. Control membranes were processed in the same way but without PI-PLC. Coated coverslips were placed in Petriperm dishes in 750 μl of medium (60% Eagle’s basal medium, 30% HBSS and 10% fetal bovine serum, supplemented with 0.1 mM glutamine, 1 mg/ml glucose, 4 mg/ml methylcellulose, 100 U/ML penicillin and 100 μg/ml streptomycin). Thalamic explants or dissociated thalamic cells were pipetted onto the substrate and incubated for 10-30 minutes at 37°C before adding 1.25 ml of medium. In some experiments, purified ephrin-A5-Fc fusion protein was added to the culture medium at a final concentration of 2 μg/ml. Cultures were grown at 37°C in an incubator with a humid atmosphere containing 5% CO2 and fixed after 2 days in 2 ml of 4% paraformaldehyde with 30% sucrose.

Immunostaining

To visualize processes extending from the dissociated thalamic cells and to confirm their neuronal origin, thalamic cultures were immunostained with the neuron-specific marker SMI 312 (Sternberger Monoclonals, Lutherville) using the following protocol. After washing three times for 20 minutes in 0.5% Triton X-100/PBS, thalamic cell cultures were incubated with the first antibody SMI 312 at 1:1000 in blocking solution (0.5% Triton X-100, 1% bovine serum albumin in PBS) for 48 hours at 4°C. After washing, cells were incubated with the secondary antibody (Cy3-conjugated goat anti mouse IgG (H+L), Jackson Immuno Research) diluted at 1:1000 together with 50 ng/ml DAPI in blocking solution for 24 hours at 4°C. Cultures were washed and mounted in moviol containing n-propylgallate.

Quantification of axonal outgrowth, branching and guidance

The number of axons extending from thalamic explants was counted under an inverted microscope with 20× phase-contrast objective [Zeiss Plan-Neofluar, numerical aperture (NA) 0.50]. Because fiber outgrowth is very dense close to the explant, only main axons extending on the substrate were taken into account. To estimate axonal elongation, the five longest axons extending from each explant were measured from the external border of the explant to the tip of their growth cone. Statistical comparison between average number of axons and average axonal length under different culture conditions were determined using a two-tailed Student’s t-test.

Axonal arborization was examined with a 40× phase-contrast optic (Zeiss Plan-Neofluar, NA 0.75) in combination with additional magnification lenses (1.6× Optovar). For each explant, five isolated and unfasciculated axons were randomly chosen. Axonal length was measured and the number of side branches was counted. In the stripe assays with dissociated neurons, SMI 312 labeled axons were traced using a high-power continuous-focus camera lucida, and we reported on the same drawings, the position of the stripe boundaries. Side branches were counted for single axons which extended about orthogonal to the membrane stripes and crossed at least two membrane lanes. The branching density was calculated as the ratio between total number of branches and total axonal length. Statistical differences were determined with a Fisher’s permutation tests.

Finally, axon guidance in stripe assays was assessed by counting for a pair of stripes the number of axons extending on each type of membrane species. Axonal numbers were compared with a two tailed Student’s t-test and the data are presented in percentage. Values reported in the present study represent results collected in two to four independent experiments.

In situ hybridization with human ephrin-A5 probes

For in situ hybridization, fresh-frozen brains of P6 animals were coronally sectioned into 20 μm thick slices. Alternating cryostat sections containing the barrel field of the somatosensory cortex were stained with Cresyl Violet, stained for cytochrome oxidase and used for in situ hybridization respectively. For in situ analysis, sections were thaw-mounted onto Superfrost Plus slides (Menzel Gläser, Germany), dried at 55°C for 3 hours and fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature. Permeabilisation was done in 0.2 M HCl for 10 minutes. Sections were digested with 1 μg/ml proteinase K (Roche) in 0.1 M Tris HCl (pH 8.0) for 5 minutes at 37°C and treated with 5 mM acetanhydride in 0.1 M triethanolamine (pH 8.0) for 10 minutes at room temperature. Between each of these steps, slices were rinsed with PBS. Digoxigenin-labeled riboprobes were made by in vitro transcription from pBlueskript SK(–) hephnA5 kindly provided by J. G. Flanagan and used for hybridization at a final concentration of 3 ng/μl (Vanderhaegen et al., 2000). To allow optimal tissue penetration, sense and antisense riboprobes were hydrolyzed at 60°C for 32.5 minutes. The approximate length of the hydrolyzed probes was 0.2 kb. The in situ hybridization was performed as described by Weth et al. (Weth et al., 1996). Only background staining was obtained when the sense probe was used as a negative control.

Receptor and ligand affinity probe staining

To assay the expression of EphA receptors and ephrin-A ligands on thalamic axons and in cortical layers, supernatant from COS cells expressing the zebrafish ephrin-A5 ectodomain fused to alkaline phosphatase (ephrin-A5-AP) (kindly provided by Caroline Brennan) and the EphA3-Fc fusion protein (R&D Systems) were used as described (Flanagan and Leder, 1990; Marcus et al., 2000).

RESULTS

Thalamic fibers outgrowth and branching on membrane substrates from isolated cortical layers

We used an in vitro strategy to examine the molecular mechanisms underlying lamina-specific termination of thalamocortical projections in the primary somatosensory cortex. As a first step, we asked whether membrane-bound molecules expressed in the cortical target layer of thalamocortical axons provide signals that cause thalamic afferents to stop their growth and elaborate terminal arbors. To this end, thalamic explants harvested from E14 mouse embryos were cultured on membrane substrates from isolated cortical layers. For technical reasons, cortical layers were dissected at P6-8, shortly after the initial growth of thalamic axons in layer 4, when cortical layers are well differentiated. Previous co-culture experiments have shown that the factors allowing target recognition are present in the cortex during the first postnatal week (Götz et al., 1992). Fig. 1 shows photomicrographs of typical growth patterns observed on membranes from layer 4, the target layer for thalamic inputs, and on membranes from layer 5, a non-target layer that thalamic axons normally cross without branching. Thalamic explants exhibited differential growth patterns on both membrane substrates, with a reduced outgrowth on membranes from layer 4 compared with layer 5. Moreover, examination of individual fibers at high magnification indicated a profuse branching of those axons that grew on layer 4 membranes. To quantify these observations, mean number of fibers per explant and average axonal length were measured. On target membranes, we found 44% less thalamic fibers (Fig. 2A) and a 19% reduction in axonal length (Fig. 2B) compared with non-target membranes. Consistent with our qualitative observations, measurements of branching frequency indicated that thalamic axons emitted 57% more side branches on membranes isolated from layer 4 than on membranes from layer 5 (Fig. 2C).

    Fig. 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 1.

Comparison of thalamic axons growth on surfaces coated with membrane extracts isolated from different cortical layers. (A,B) Photomicrographs of E14 thalamic explants cultured for 2 days on membranes from cortical layer 4 and cortical layer 5. The general outgrowth of thalamic explants was reduced on membranes from layer 4 (A) (which is their target substrate) compared with membranes from layer 5 (B). (C-J) Photomicrographs and schematic drawings illustrating the branching pattern of individual growing axons on layer 4 and layer 5 membrane substrates. Thalamic axons exhibit numerous side branches on membranes from their target layer (C-F) and much less on their non-target substrate (G-J). Scale bar: 100 μm in A,B; 10 μm in C-J.

    Fig. 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 2.

Quantitative analysis of thalamic axon growth and branching on membranes from cortical layer 4 (black bars) and cortical layer 5 (white bars). Histograms depict the number of fibers per explant (A), axon length (B) and branching density (C) on each type of membrane substrate. (D) Stripe assay with thalamic axons growing perpendicular to membrane stripes (see Fig. 3). The histogram depicts the branching density observed in each set of stripes. n, number of explants analyzed (A); n, number of fibers examined (B-D). Error bars represent the s.e.m.

In a second set of experiments, dissociated thalamic cells were placed on a substrate consisting of alternating stripes of membranes from cortical layers 4 and 5. This assay allowed us to analyze branch formation of single thalamic axons that grew perpendicular to the membrane stripes and thus encountered successively non-target and target substrates, as they normally do in vivo. As shown in Fig. 3, thalamic axons extended on both type of membranes and freely crossed the stripe borders, but branch formation was higher on stripes from target layer than from non-target layer (Fig. 3A,B). Quantitative analysis indicated a 35% increased in branching density on layer 4 membranes compared with layer 5 membranes (Fig. 2D). Taken together, our findings indicate the presence of cell surface cues restricted to individual cortical layers that control the elongation and branch formation of thalamocortical axons.

    Fig. 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 3.

Camera lucida drawings of representative axons crossing stripes with membranes from cortical layer 4 (dark gray) and membranes from cortical layer 5 (light gray). In A, thalamic axons extend first on a membrane stripe from their target layer, whereas in B the axons encounter first a membrane stripe from a non-target layer. In each case, thalamic axons branch preferentially on their appropriate target membranes. Scale bar: 50 μm.

Influence of ephrins in layer-specific sprouting of thalamic axons

Recent studies have provided evidences for a role of GPI-anchored ephrin ligands as topographic guidance cues in the developing thalamocortical system (Gao et al., 1998; Mackarehtschian et al., 1999; Vanderhaeghen et al., 2000). We were interested in further exploring the possibility that Eph ligands might also function in setting up the layer specificity of thalamocortical connections. Vanderhaeghen et al. (Vanderhaeghen et al., 2000) analyzed ephrin-A5 mRNA expression from E18 to P3 in mouse brains, and they report that at postnatal stages labeling in the somatosensory cortex was strongest in layer 6 and layer 4, with only moderate expression in layer 5 especially in the motor areas. We examined whether ephrin-A5 is also present at later postnatal stages, when thalamocortical arbors are still being elaborated. For this in situ hybridization on coronal sections from P6 brains was performed to detect ephrin-A5 mRNA. Adjacent sections were stained with Cresyl Violet to identify the cortical layers and stained for cytochrome oxidase to reveal the barrel pattern in S1, the primary somatosensory cortex. As illustrated in Fig. 4A, at P6 ephrin-A5 mRNA is clearly detectable in layer 4 and there is also a somewhat weaker staining in layer 6 of S1. In the adjacent motor cortex, ephrin-A5 mRNA is most prominently expressed in some large cells in layer 5. Ephrin-A ligand protein distribution in cortical layers was determined by affinity probe in situ with an EphA3-Fc fusion protein. In P6 somatosensory cortex, EphA3-Fc binding activity was restricted to layer 4 and layer 6, similar to ephrin-A5 transcripts (Fig. 4B).

    Fig. 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 4.

Ephrin-A5 expression within primary somatosensory and motor cortex at P6 and EphA receptor labeling on E14 thalamic axons in vitro. (A) Serial coronal sections of the right hemisphere (medial is left) stained with Cresyl Violet, ephrin-A5 in situ hybridization and cytochrome oxidase histochemistry. The boundaries between the barrel field of the somatosensory cortex (S) and the motor cortex (M) are indicated by the arrowheads. Bars show the boundaries between cortical layers 4, 5 and 6. (B) Coronal section through the barrel field of S1 stained with Hoechst and EphA3-Fc fusion protein. (C) Phase-contrast and bright-field images of ephrin-A5-AP stained thalamic axons.

Finally, to detect EphA receptors on thalamic axons, we used a probe consisting of the ephrin-A5 ectodomain fused to alkaline phosphatase to label thalamic explants from E14 animals in vitro. Labeling was seen on virtually all thalamic neurites (Fig. 4C), confirming the presence of EphA receptors on growing thalamic axons and growth cones.

The detection of ephrin-A ligands in the cortical layers where thalamocortical axons elaborate branches suggest a role of ephrins as branching factors for thalamic afferents. To address directly a possible function of ephrin-A5 in stimulating branch formation for thalamic axons, we compared the branching pattern of thalamic fibers on membrane substrates from NIH3T3 cells expressing the transfected ephrin-A5 ligand or from control cells. As depicted in Fig. 5, there was a strong effect of the recombinant ephrin-A5 on the branching behavior of thalamic fibers. On a total number of 181 axons examined, there was an overall increase by 101% of the branching density on ephrin-A5 containing membranes compared to control membranes (Fig. 6C). By contrast, we found no significant effect of ephrin-A5 on the average growth of thalamic axons (Fig. 6A,B). However, previous experiments showed that a subset of fibers from the ventrobasal nucleus of the thalamus was repelled by ephrin-A5 in vitro (Vanderhaeghen et al., 2000). We found that short thalamic axons (<100 μm), which may represent a subset of fibers that is sensitive to the inhibitory activity of ephrin-A5, exhibited a similar branching density (39.1±4.9 branches/mm, n=29) as did axons that grew longer on NIH3T3-ephrin-A5 membranes (>300 μm, 34.0±4.3 branches/mm, n=33). This suggests that all or most of the thalamic axons showed a strong branching response to ephrin-A5 that was independent of their growth rate on the ephrin-A5 substrate. This branching response was abolished after removal of ephrin-A5 from NIH3T3 cell membranes using the enzyme PI-PLC (Fig. 6D). These data demonstrate that ephrin-A5 acts as a branch promoter for thalamic axons in vitro.

    Fig. 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 5.

Recombinant ephrin-A5 promotes collateral formation for thalamic axons. Phase-contrast photomicrographs and schematic drawings of individual axons from thalamic neurons cultured on membrane substrates isolated from NIH3T3-vector cells (A-D) and NIH3T3-ephrin-A5 cells (E-H). In the presence of ephrin-A5 ligand, thalamic axons present a profuse branching, whereas only few branches form under control conditions. Scale bar: 10 μm.

    Fig. 6.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 6.

Quantitative analysis of thalamic axon growth on membranes from NIH3T3-ephrin-A5 cells (black bars) or from control cells (white bars). (A) Number of axons extending from thalamic explants, (B) analysis of axonal length, (C) branch formation under control condition and (D) branch formation after treatment of membranes with PI-PLC enzyme. n, number of explants scored (A); n, number of fibers examined (B-D). ‘n.s.’ indicates not significant and error bars represent the s.e.m.

To test for a possible functional role of ephrins in the patterning of thalamocortical projections, we used two different in vitro approaches. First, we examined the growth and branching of thalamic axons on membranes from layer 4 and layer 5 after removal of GPI-anchored molecules with the enzyme PI-PLC. Enzyme treatment increased the growth of thalamic axons both on layer 5 and layer 4 membranes, compared to control substrates (Table 1). However, the differential growth of thalamic explants on target and non-target membranes was unaffected, as axons grew shorter on treated layer 4 than on treated layer 5 membranes (Table 1). On PI-PLC treated layer 4 membranes, branching of thalamic fibers, decreased by 43% compared to control layer 4 membranes (Fig. 7A). By contrast, elimination of GPI-linked molecules did not affected axonal sprouting on layer 5 membranes (Fig. 7A). This indicates that GPI-anchored branching factors are expressed in the target layer for thalamocortical fibers.

    Fig. 7.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 7.

Histograms of thalamic axons sprouting on membranes from distinct cortical layers with PI-PLC enzyme treatment (A) and in the presence of soluble ephrin-A5-Fc (B). Bars represent the percentage of branches formed on membranes from cortical layer 4 (black bars) and cortical layer 5 (white bars). Data are presented in percentage and normalized to 100% for values obtained on membranes from layer 5. The ability of layer 4 membranes to support thalamic axons sprouting decreases after removal of GPI-anchored molecules and after exposure to ephrin-A5-Fc fusion protein. By contrast, none of these treatments influences the branching of thalamic axons on layer 5 membranes. (C) Histogram of branch formation of thalamic axons cultured on cortical membranes from layer 4 (black bar), layer 5 (white bar), and on a mixture (1:1) of both types of membranes (gray bar). On the latter substrate, thalamic axons exhibit a branching density similar to the density observed on layer 5 membranes alone, suggesting that the branch-promoting activity in membranes from layer 4 is inhibited by the membrane extracts from layer 5. n, number of fibers examined.

View this table:
  • View inline
  • View popup
  • Download powerpoint
Table 1.

Second, to test whether these GPI-anchored factors belong to the subfamily of GPI-linked ephrin-A ligands, thalamic explants were grown on cortical membranes in the presence of soluble ephrin-A5-Fc fusion protein, added to the culture medium at 2 μg/ml. Exogenous ephrin-A5-Fc did not affect the general growth rate of thalamic explants (Table 1) and, unlike membrane-bound ephrin-A5 (which elicited a strong branch promoting activity on thalamic axons), soluble ephrin-A5-Fc did not increase thalamic axon branching. This suggests that membrane attachment (or higher cluster forms) might be required for ephrin-A5 to act as a branching factor. Instead, we found that in the presence of ephrin-A5-Fc the branching rate of thalamic fibers on layer 4 membranes decreased by 48%, and was similar to the branching level observed on layer 5 membranes with or without ephrin-A5-Fc (Fig. 7B). This finding suggests that ephrin-A5-Fc interferes with normal Eph/ephrin interactions in cultures of thalamic axons on their target membranes. The inability of thalamic axons to preferentially branch on layer 4 membranes could be explained by the internalization of EphA receptors after activation by ephrin-A5-Fc and the subsequent desensitization of thalamic axons to ephrin-A cues expressed in their target membranes (Ciossek et al., 1998).

Influence of branch inhibitors in layer-specific sprouting of thalamic axons

The experiments described above indicate that membrane-associated factors promote the branching of thalamocortical axons in their target layer. It is also possible that additional signals in layer 5 may prevent thalamic afferents to branch in this inappropriate layer. Such an inhibition of branch formation in inappropriate territories has already been demonstrated in the formation of topographic retinotectal projection (Roskies and O’Leary, 1994). To address this issue, thalamic explants were confronted with a substrate consisting of a 1:1 mixture of layer 4 and layer 5 cortical membranes (see Materials and Methods). Under this condition, the branching density of thalamic axons was not significantly different from the branching observed on layer 5 membranes alone (Fig. 7C). This result provides evidence for the presence of inhibitory signals in non-target layer 5 membranes that mask or reduce the branching properties of layer 4 membranes.

Guidance of thalamic fibers on membranes from isolated cortical layers

Terminal branches of thalamic axons are confined to layer 4 and do not extend across into the adjacent non-target layers, suggesting that mechanisms operate that guide axon collateral extension within the boundaries of their appropriate territory. To test this hypothesis, the in vitro stripe assay was used to offer growing thalamic axons a choice between alternating lanes of membranes from their target layer 4 and non-target layer 5. We observed that thalamic axons that grew parallel to the membrane stripes tend to restrict their growth on their target substrate. On a total number of 126 pair of stripes and 586 axons examined, we found that 339 fibers (58%) grew on membranes from cortical layer 4 and only 247 (42%) on layer 5 membranes (Fig. 8A,B). PI-PLC treatment of membranes from layer 5, but not from layer 4, leads to an equal distribution of growing axons on target and non-target membranes (Fig. 8B). As enzyme treatment of non-target membranes abolished the preferential growth of thalamic axons on their target membranes, we postulated that GPI-anchored molecule(s) in layer 5 act as repulsive guidance cue(s) for thalamocortical axons.

    Fig. 8.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 8.

(A) Outgrowth of thalamic explants confronted with parallel membrane stripes from cortical layer 4 (L4) and cortical layer 5 (L5). (B) Distribution of thalamic axons on membrane stripes from cortical layer 4 (black bars) and cortical layer 5 (white bars) under control conditions and after PI-PLC treatment of one type of membrane. Bars indicate the percentage of axons growing on one type of stripe. Results demonstrate that thalamic axons preferentially extend on their target membrane stripes, and this choice can be blocked by treatment of layer 5 membranes with PI-PLC enzyme. n, number of pairs of stripes analyzed. Scale bar: 100 μm.

Effects of ephrin-A5 on limbic thalamic neurons

All experiments described so far were performed with explants from somatomotor thalamic nuclei that project to the neocortex (see Materials and Methods). In a previous study, it was demonstrated that the Eph ligand ephrin-A5 selectively inhibited outgrowth of a sub-population of thalamic neurons that project to the limbic cortex (Gao et al., 1998). We were interested in further examining the response of limbic thalamic axons to recombinant ephrin-A5 and to compare it with the behavior of non-limbic fibers described above. In accordance with the results of Gao et al. (Gao et al., 1998), we found a 13% reduction in the length of limbic thalamic axons on NIH3T3-ephrin-A5 cell membranes compared with control membranes (Table 2). In addition, in the stripe assay, limbic thalamic fibers extended preferentially on control lanes and avoided the membrane stripes containing recombinant ephrin-A5 (Fig. 9B), indicating that ephrin-A5 functions as a repellent guidance cue for limbic thalamic axons. We found no guidance effect of ephrin-A5 on fibers extending from explants prepared from the whole non-limbic thalamus (Fig. 9A). Finally, whereas in the stripe assay ephrin-A5 exhibits a repellent guidance activity on limbic fibers, on homogenous membrane preparations we found that ephrin-A5 activates branch formation of limbic thalamic axons, as measured by a 63% increased branching on NIH3T3-ephrin-A5 membranes versus control membranes (Table 2).

    Fig. 9.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 9.

Recombinant ephrin-A5 ligand elicits distinct guidance effects on non-limbic and limbic thalamic axons in stripe assay. Outgrowth of thalamic fibers from non-limbic (A,B) and limbic (C,D) thalamic explants (see Materials and Methods) confronted with parallel membrane stripes from NIH3T3-ephrin-A5 cells and from untransfected control cells. Upper panels: photomicrographs of representative examples of axonal outgrowth. Lower panels: quantitative analysis of axonal preference. The histograms depict the percentage of axons growing on ephrin-A5 containing stripes (black bars) and on control stripes (white bars). Non-limbic thalamic axons grow equally on both types of stripes, whereas limbic thalamic axons avoid ephrin-A5 ligand and prefer control stripes. Scale bar: 100 μm; n, number of pair of stripes examined.

View this table:
  • View inline
  • View popup
  • Download powerpoint
Table 2.

DISCUSSION

The layer-specific patterning of thalamocortical projections develops with a high degree of specificity. The present in vitro experiments provide evidence for molecular cues selectively expressed in either target or non-target layers that regulate elongation, branching and targeting of thalamic axons. Blocking endogenous ephrin-A ligands abolishes the preferential branching of thalamic axons on substrates from their target layer, while ephrin-A5, which is expressed in layer 4 of the somatosensory cortex, induces collateral formation of thalamic axons. This suggests that ephrin-As are instrumental for the formation of terminal arborizations of thalamic axons in their cortical target layer.

Previous experiments revealed that thalamic axons terminate in layer 4 of cortical slice cultures (Bolz et al., 1992; Götz et al., 1992; Molnar and Blakemore, 1991; Yamamoto et al., 1992). Together with the fact that thalamic growth cones often pause and transiently collapse at the time they reach layer 4 (Yamamoto et al., 1997), these observations support the argument that thalamic fibers recognize a ‘stop signal’ expressed at the surface of their target cells in layer 4 (Molnar and Blakemore, 1991). Götz et al. (Götz et al., 1992) have shown that axonal outgrowth from thalamic explants on membrane substrates from layer 4 is reduced compared with the outgrowth on membranes from the deep cortical layers. The present study confirms this result.

Shortly after thalamic axons have ceased their extension in their target layer, lateral branches emerge at a short distance behind the leading growth cone (Yamamoto et al., 1997). This process, called backbranching, was initially described in the frog visual system (Harris et al., 1987), and appears to be a common mechanism used to elaborate terminal arbors. We show that cultured thalamic axons form more lateral branches on membranes from their target layer than on membranes from layer 5, a non-target layer. Our experiments implicated the activity of both branch-promoting cues in layer 4 and branch-inhibiting cues in layer 5. It is thought that the formation of the filopodia-like extensions we observed along the shaft of thalamic axons represents the initial stage of new branch formation (Davenport et al., 1999). In our culture system, however, these lateral extensions remained short (2-10 μm), with only a few of them developing a new growth cone, suggesting that additional cues might be necessary in vivo for the stabilization and the expansion of these branches into terminal arborization.

Removal of GPI-linked molecules and addition of exogenous ephrin-A5-Fc strongly inhibited thalamic axon branching on membranes from their target layer, but did not interfere with the differential outgrowth of thalamic explants on membranes from layer 4 and layer 5. Thus, growth arrest and arborization in layer 4 are separate processes controlled by independent molecular mechanisms. This might be a common principle in the development of layer-specific neuronal projections. In the tectum, for example, cues for the laminar-specific termination of retinal axons comprise N-cadherin and Vicia villosa agglutinin-B4 binding glycoconjugates, whereas neurotrophins regulate the size of retinal arbors without affecting their laminar distribution (Inoue and Sanes, 1997). Similarly, in the hippocampus, the extracellular molecule Reelin acts as a branching factor for entorhinal afferents. However, in the absence of Reelin, entorhinal axons still terminate in the appropriate layer (Borrell et al., 1999; Del Rio et al., 1997).

When confronted with a choice between target and non-target substrates, thalamic fibers are able to distinguish between layers 4 and 5 membranes and progressively restrict their growth on their appropriate substrate. Thus, although on uniform substrates the outgrowth of thalamic axons is reduced on layer 4 membranes compared with layer 5 membranes, when given a choice the axons prefer to grow on their target layer substrate. A similar behavior has been observed in co-cultures of thalamus and cortex: thalamic axons entering the lateral side of a cortical slice follow a pathway centered on layer 4 and avoid the adjacent non-target layers (Yamamoto et al., 1992). Our results with stripe assays indicate that this preferential growth on target membranes is at least in part due to repulsive guidance signals expressed in non-target layers.

Taken together, the present results provide evidence for the existence of multiple membrane-associated molecules expressed in individual layers that influence the growth and branching of thalamic axons. The cooperation of positive and negative cues allows the layer-specific termination of thalamocortical afferents and restricts the extension of terminal arbors within the appropriate target layer.

The molecular nature of factors involved in setting up layer specificity of thalamocortical connections is not known. In experiments with soluble ephrin-A5-Fc, we showed that branching of thalamic axon is strongly reduced on membranes from cortical layer 4, consistent with the idea that A-type ephrin ligands mediate branch formation of thalamic afferents in their appropriate target layer. This result is reminiscent of previous studies showing that several ephrin-A ligands act as branch factors for cortical, hippocampal and retinal neurons in vitro (Castellani et al., 1998; Gao et al., 1999; Davenport et al., 1999). Interestingly, ephrin-A5 was found to specifically promote branching of cortical axons that target the cortical layer 4, whereas cortical axons that normally avoid layer 4 show no branching responses to ephrin-A5 (Castellani et al., 1998). Growth cone collapse induced by ephrin-As was shown to be the leading event that initiates the formation of backbranches along the axonal shaft (Davenport et al., 1999). However, a separate study has demonstrated that ephrin-A ligands can also inhibit branch formation in the retinotectal system (Yates et al., 2001).

Our experiments do not indicate which individual ephrin-A ligand or which specific combination of ligands is likely to operate during thalamic axon branching in layer 4 of the cortex. As recombinant ephrin-A5 stimulates thalamic axons sprouting in our culture system, it is tempting to speculate that ephrin-A5 influences the formation of terminal arborization in vivo. There is some controversy, however, about the laminar expression of ephrin-A5 in the developing cortex. Studies by Castellani et al. (Castellani et al., 1998) and Vanderhaeghen et al. (Vanderhaeghen et al., 2000) in the mouse primary somatosensory area reported ephrin-A5 expression in cortical layers 6 and 4, whereas Yabuta et al. (Yabuta et al., 2000) described that ephrin-A5 is most intensively distributed in the deep cortical layers but not in layer 4. In the present study, we re-examined ephrin-A5 expression in the mouse barrel cortex with a more sensitive in situ hybridization technique using hydrolyzed riboprobes. Our results confirmed expression in cortical layers innervated by thalamic axons (layers 6 and 4), suggesting a possible involvement of ephrin-A5 in thalamic afferent branching. ephrin-A5–/– (Efna5–/–) knockout mice show no gross defects in layer-specific thalamocortical targeting in S1 (Vanderhaeghen et al., 2000), and anterograde tracing of VB afferents revealed terminal arbors forming correctly in layer 4 (Muehlfriedel et al., 2000). However, VB terminal arbors are less complex and contain fewer branches in the ephrin-A5–/– mice than in control mice (Muehlfriedel et al., 2000) (D. Uziel and J. Bolz, unpublished), indicating that ephrin-A5 regulates branch formation of thalamocortical axons in cortical layer 4.

Supporting the idea that ephrin ligands might play a role on thalamocortical branch formation in other areas of the neocortex, studies investigating the developmental expression of Eph ligands in rodents have reported significant expression of ephrin-As across sensory and motor areas (Mackarehtschian et al., 1999; Vanderhaeghen et al., 2000). In some neocortical regions, however, expression was also found outside layer 4 (Mackarehtschian et al., 1999; Vanderhaeghen et al., 2000) (present study). How can these observations be integrated into the idea that ephrin-A ligands are involved in layer-specific branching of thalamic afferents? One possibility is that branch-inhibiting factors co-expressed in non-target layers might mask the branch-promoting activity of ephrin-A ligands. We show that signals present in membranes from layer 5 are sufficient to interfere in vitro with the increased branching of thalamic axons induced on membranes from layer 4, a result that is consistent with this interpretation. Potential candidates for such inhibitory function are the semaphorins, a family of cell-surface and secreted molecules that can function as inhibitors or chemorepellents. In the developing cortex, several members of the semaphorin family exhibit a layer-specific distribution restricted to deep layers 6 and 5 and superficial layers 2/3 (Skaliora et al., 1998). As some semaphorins also inhibit axonal arborization (Bagnard et al., 1998; Kolodkin et al., 1992; Matthes et al., 1995), it has been suggested that they might prevent ingrowing thalamic fibers to form and extend collaterals in inappropriate cortical layers (Skaliora et al., 1998).

As suggested previously, we find that the Eph ligand ephrin-A5 selectively inhibits outgrowth of neurons from limbic thalamus (Gao et al., 1998). Moreover, results from stripe assay experiments in this study indicate that ephrin-A5 also serves as a repulsive guidance cue for limbic thalamic axons, but not for non-limbic thalamic axons. How might the selective effect of ephrin-A5 on axon guidance be related to the development of thalamocortical projections in vivo? During embryonic development, ephrin-A5 expression occurs predominately as a gradient across the subplate zone and the cortical plate of sensory and motor cortex, but there is very little expression in the limbic cortex (Gao et al., 1998; Mackarehtschian et al., 1999), suggesting that ephrin-A5 could function in the patterning of thalamocortical connections to specific cortical areas. In an earlier study we reported the existence of membrane-bound signals differentially expressed in limbic cortex and neocortex that allow thalamic afferents to distinguish between target and non-target cortical regions (Mann et al., 1998). The limbic-associated membrane protein (LAMP), a glycoprotein that is selectively expressed in the limbic cortex and other limbic regions, has been suggested to function as a recognition signal for limbic thalamic afferents and as a repulsive signal for thalamic afferents which project to neocortical areas (Barbe and Levitt, 1992; Mann et al., 1998). The present study also implicates ephrin-A5 as a positional cue in neocortical areas, which through repulsive activity prevents limbic thalamic afferents from invading inappropriate neocortical region.

The observation of multiple cell type specific activities of ephrin-A5 raises the question of how a single ligand can mediate these different responses. Because of the redundancy in Eph receptor and ephrin interactions (Gale et al., 1996), the mechanisms by which ephrin-A5 stimulates branching or repels growth cones may involve distinct Eph receptors. Three putative receptors for ephrin-A5 are expressed in the developing thalamus: EphA3 and EphA4 are found in almost all thalamic nuclei, whereas little expression of EphA5 is found outside the limbic thalamus (Gao et al., 1998; Mackarehtschian et al., 1999; Vanderhaeghen et al., 2000; Zhou et al., 1994). Thus EphA5 is a potential candidate to mediate the repulsive activity of ephrin-A5 for limbic fibers. By contrast, the branching response to ephrin-A5 may occur independently of EphA5 expression, as both populations of limbic and non-limbic neurons recognize ephrin-A5 as a branch promoter. Another possibility is that multiple cellular responses require the recruitment of distinct co-factors functionally associated to Eph receptors. Such a link between Eph receptor activation and function of member of the L1 family of cell adhesion molecules have been suggested (Zisch et al., 1997), and co-expression of ephrins and Eph receptors on axons has been found to regulate responsiveness to ephrin ligands in vitro (Hornberger et al., 1999). Finally, differences between intrinsic properties of axonal populations could govern different responses to ephrin-A5 ligand through a single receptor mechanism. For example, it has been shown that by increasing cytosolic concentration of either cAMP or cGMP, repulsive actions of some axonal guidance signals are converted to attractive effects (Ming et al., 1997; Song et al., 1998).

The present results indicate that the effects of ephrin-A5 on limbic thalamic fibers depend critically on the spatial context in which it is presented. When presented uniformly, ephrin-A5 increased axon branching. By contrast, when distributed as a sharp boundary, it repelled limbic axons. Similarly, in a recent study it was found that cortical axons respond differently to semaphorins when they grow towards increasing or decreasing semaphorin gradients (Bagnard et al., 2000). It is now an intriguing question to examine whether the spatial distribution of wiring molecules in the environment of growing axons influences expression levels or turnover rates of receptors, co-factors and/or second messengers, and thereby causes differential responses to the same extrinsic signal.

Acknowledgments

We thank Caroline Brennan for providing the ephrin-A5-AP probe, John Flanagan for providing the ephrin-A5 plasmid, and Naura Chounlamountri for excellent technical assistance. Special thanks to Fanco Weth for his enduring support in the in situ experiments, to Bill Harris and Christine Holt for their help and advice during the publication process. This work was supported by HFSP and IZKF Jena (J. B.), and a Fellowship from the French Ministry of Research (F. M.).

Footnotes

    • Accepted May 21, 2002.
  • © 2002.

References

  1. ↵
    Agmon, A., Yang, L. T., O’Dowd, D. K. and Jones, E. G. (1993). Organized growth of thalamocortical axons from the deep tier of terminations into layer IV of developing mouse barrel cortex. J. Neurosci. 13, 5365-5382.
    OpenUrlAbstract
  2. ↵
    Angevine, J. B. (1970). Time of neuron origin in the diencephalon of the mouse. An autoradiographic study. J. Comp. Neurol. 139, 129-187.
    OpenUrlCrossRefPubMedWeb of Science
  3. ↵
    Bagnard, D., Lohrum, M., Uziel, D., Puschel, A. W. and Bolz, J. (1998). Semaphorins act as attractive and repulsive guidance signals during the development of cortical projections. Development 125, 5043-5053.
    OpenUrlAbstract
  4. ↵
    Bagnard, D., Thomasset, N., Lohrum, M., Puschel, A. W. and Bolz, J. (2000). Spatial distributions of guidance molecules regulate chemorepulsion and chemoattraction of growth cones. J. Neurosci. 20, 1030-1035.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Barbe, M. F. and Levitt, P. (1992). Attraction of specific thalamic input by cerebral grafts depends on the molecular identity of the implant. Proc. Natl. Acad. Sci. USA 89, 3706-3710.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Bicknese, A. R., Sheppard, A. M., O’Leary, D. D. and Pearlman, A. L. (1994). Thalamocortical axons extend along a chondroitin sulfate proteoglycan- enriched pathway coincident with the neocortical subplate and distinct from the efferent path. J. Neurosci. 14, 3500-3510.
    OpenUrlAbstract
  7. ↵
    Bolz, J., Novak, N. and Staiger, V. (1992). Formation of specific afferent connections in organotypic slice cultures from rat visual cortex cocultured with lateral geniculate nucleus. J. Neurosci. 12, 3054-3070.
    OpenUrlAbstract
  8. ↵
    Borrell, V., del Rio, J. A., Alcantara, S., Derer, M., Martinez, A., D’Arcangelo, G., Nakajima, K., Mikoshiba, K., Derer, P., Curran, T. et al. (1999). Reelin regulates the development and synaptogenesis of the layer-specific entorhino-hippocampal connections. J. Neurosci.19, 1345-1358.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Castellani, V. and Bolz, J. (1997). Membrane-associated molecules regulate the formation of layer-specific cortical circuits. Proc. Natl. Acad. Sci. USA 94, 7030-7035.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Castellani, V., Yue, Y., Gao, P. P., Zhou, R. and Bolz, J. (1998). Dual action of a ligand for Eph receptor tyrosine kinases on specific populations of axons during the development of cortical circuits. J. Neurosci. 18, 4663-4672.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Catalano, S. M., Robertson, R. T. and Killackey, H. P. (1996). Individual axon morphology and thalamocortical topography in developing rat somatosensory cortex. J. Comp. Neurol. 367, 36-53.
    OpenUrlCrossRefPubMedWeb of Science
  12. ↵
    Ciossek, T., Monschau, B., Kremoser, C., Loschinger, J., Lang, S., Muller, B. K., Bonhoeffer, F., Drescher, U. (1998). Eph receptor-ligand interactions are necessary for guidance of retinal ganglion cell axons in vitro. Eur. J. Neurosci. 10, 1574-1580.
    OpenUrlCrossRefPubMedWeb of Science
  13. ↵
    Dantzker, J. L. and Callaway, E. M. (1998). The development of local, layer-specific visual cortical axons in the absence of extrinsic influences and intrinsic activity. J. Neurosci. 18, 4145-4154.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Davenport, R. W., Thies, E., Cohen, M. L. (1998) Neuronal growth cone collapse triggers lateral extensions along trailing axons. Nat. Neurosci. 3, 254-259.
    OpenUrlCrossRef
  15. ↵
    Del Rio, J. A., Heimrich, B., Borrell, V., Forster, E., Drakew, A., Alcantara, S., Nakajima, K., Miyata, T., Ogawa, M., Mikoshiba, K. et al. (1997). A role for Cajal-Retzius cells and reelin in the development of hippocampal connections. Nature385, 70-74.
    OpenUrlCrossRefPubMed
  16. ↵
    Emerling, D. E. and Lander, A. D. (1996). Inhibitors and promoters of thalamic neuron adhesion and outgrowth in embryonic neocortex: functional association with chondroitin sulfate. Neuron 17, 1089-1100.
    OpenUrlCrossRefPubMedWeb of Science
  17. ↵
    Flanagan, J. G. and Leder, P. (1990). The kit ligand: a cell surface molecule altered in steel mutant fibroblasts. Cell 63, 185-194
    OpenUrlCrossRefPubMedWeb of Science
  18. ↵
    Gale, N. W., Holland, S. J., Valenzuela, D. M., Flenniken, A., Pan, L., Ryan, T. E., Henkemeyer, M., Strebhardt, K., Hirai, H., Wilkinson, D. G. et al. (1996). Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis. Neuron17, 9-19.
    OpenUrlCrossRefPubMedWeb of Science
  19. ↵
    Gao, P. P., Zhang, J. H., Yokoyama, M., Racey, B., Dreyfus, C. F., Black, I. B. and Zhou, R. (1996). Regulation of topographic projection in the brain: Elf-1 in the hippocamposeptal system. Proc. Natl. Acad. Sci. USA 93, 11161-11166.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Gao, P. P., Yue, Y., Zhang, J. H., Cerretti, D. P., Levitt, P. and Zhou, R. (1998). Regulation of thalamic neurite outgrowth by the Eph ligand ephrin-A5: implications in the development of thalamocortical projections. Proc. Natl. Acad. Sci. USA 95, 5329-5334.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Gao, P. P., Yue, Y., Cerretti, D. P., Dreyfus, C. and Zhou, R. (1999). Ephrin-dependent growth and pruning of hippocampal axons. Proc. Natl. Acad. Sci. USA 96, 4073-4077.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Ghosh, A. and Shatz, C. J. (1992). Pathfinding and target selection by developing geniculocortical axons. J. Neurosci. 12, 39-55.
    OpenUrlAbstract
  23. ↵
    Götz, M., Novak, N., Bastmeyer, M. and Bolz, J. (1992). Membrane bound molecules in rat cerebral cortex regulate thalamic innervation. Development 116, 507-519.
    OpenUrlAbstract
  24. ↵
    Harris, W. A., Holt, C. E. and Bonhoeffer, F. (1987) Retinal axons with and without their somata, growing to and arborizing in the tectum of Xenopus embryos: a time-lapse video study of single fibres in vivo. Development 101, 123-133.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Henke-Fahle, S., Mann, F., Götz, M., Wild, K. and Bolz, J. (1996). Dual action of a carbohydrate epitope on afferent and efferent axons in cortical development. J. Neurosci. 16, 4195-4206.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Hornberger, M. R., Dutting, D., Ciossek, T., Yamada, T., Handwerker, C., Lang, S., Weth, F., Huf, J., Wessel, R., Logan, C. et al. (1999). Modulation of EphA receptor function by coexpressed ephrinA ligands on retinal ganglion cell axons. Neuron22, 731-742.
    OpenUrlCrossRefPubMedWeb of Science
  27. ↵
    Hübener, M., Götz, M., Klostermann, S. and Bolz, J. (1995). Guidance of thalamocortical axons by growth-promoting molecules in developing rat cerebral cortex. Eur. J. Neurosci. 7, 1963-1972.
    OpenUrlCrossRefPubMedWeb of Science
  28. ↵
    Inoue, A. and Sanes, J. R. (1997). Lamina-specific connectivity in the brain: regulation by N-cadherin, neurotrophins, and glycoconjugates. Science 276, 1428-1431.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Kageyama, G. H. and Robertson, R. T. (1993). Development of geniculocortical projections to visual cortex in rat: evidence early ingrowth and synaptogenesis. J. Comp. Neurol. 335, 123-148.
    OpenUrlCrossRefPubMedWeb of Science
  30. ↵
    Klein, R. (2001). Excitatory Eph receptors and adhesive ephrin ligands. Curr. Opin. Cell Biol. 13, 196-203.
    OpenUrlCrossRefPubMedWeb of Science
  31. ↵
    Kinnunen, A., Niemi, M., Kinnunen, T., Kaksonen, M., Nolo, R. and Rauvala, H. (1999). Heparan sulphate and HB-GAM (heparin-binding growth-associated molecule) in the development of the thalamocortical pathway of rat brain. Eur. J. Neurosci. 11, 491-502.
    OpenUrlCrossRefPubMedWeb of Science
  32. ↵
    Kolodkin, A. L., Matthes, D. J., O’Connor, T. P., Patel, N. H., Admon, A., Bentley, D. and Goodman, C. S. (1992). Fasciclin IV: sequence, expression, and function during growth cone guidance in the grasshopper embryo. Neuron 9, 831-845.
    OpenUrlCrossRefPubMedWeb of Science
  33. ↵
    Lund, R. D. and Mustari, M. J. (1977). Development of the geniculocortical pathway in rats. J. Comp. Neurol. 173, 289-306.
    OpenUrlCrossRefPubMedWeb of Science
  34. ↵
    Mackarehtschian, K., Lau, C. K., Caras, I. and McConnell, S. K. (1999). Regional differences in the developing cerebral cortex revealed by ephrin-A5 expression. Cereb. Cortex 9, 601-610.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Mann, F., Zhukareva, V., Pimenta, A., Levitt, P. and Bolz, J. (1998). Membrane-associated molecules guide limbic and nonlimbic thalamocortical projections. J. Neurosci. 18, 9409-9419.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Marcus, R. C., Matthews, G. A., Gale, N. W., Yancopoulos, G. D., Mason, C. A. (2000). Axon guidance in the mouse optic chiasm: retinal neurite inhibition by ephrin ‘A’-expressing hypothalamic cells in vitro. Dev. Biol. 221, 132-147.
    OpenUrlCrossRefPubMedWeb of Science
  37. ↵
    Matthes, D. J., Sink, H., Kolodkin, A. L. and Goodman, C. S. (1995). Semaphorin II can function as a selective inhibitor of specific synaptic arborizations. Cell 81, 631-639.
    OpenUrlCrossRefPubMedWeb of Science
  38. ↵
    Miller, B., Chou, L. and Finlay, B. L. (1993). The early development of thalamocortical and corticothalamic projections. J. Comp. Neurol. 335, 16-41.
    OpenUrlCrossRefPubMedWeb of Science
  39. ↵
    Ming, G. L., Song, H. J., Berninger, B., Holt, C. E., Tessier-Lavigne, M. and Poo, M. M. (1997). cAMP-dependent growth cone guidance by netrin-1. Neuron 19, 1225-1235.
    OpenUrlCrossRefPubMedWeb of Science
  40. ↵
    Molnar, Z. and Blakemore, C. (1991). Lack of regional specificity for connections formed between thalamus and cortex in coculture. Nature 351, 475-477.
    OpenUrlCrossRefPubMed
  41. ↵
    Muehlfriedel, S., Uziel, D., Zarbalis, K., Aschoff, A. P., Wurst, W. and Bolz, J. (2000). Development of thalamocortical arbors in the barrel cortex of ephrin-A5 knockout mice. Soc. Neurosci. Abstr. 26, 575.
    OpenUrl
  42. ↵
    Roskies, A. L. and O’Leary, D. D. (1994). Control of topographic retinal axon branching by inhibitory membrane-bound molecules. Science 265, 799-803.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Skaliora, I., Singer, W., Betz, H. and Puschel, A. W. (1998). Differential patterns of semaphorin expression in the developing rat brain. Eur. J. Neurosci. 10, 1215-1229.
    OpenUrlCrossRefPubMedWeb of Science
  44. ↵
    Song, H., Ming, G., He, Z., Lehmann, M., McKerracher, L., Tessier-Lavigne, M. and Poo, M. (1998). Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides. Science 281, 1515-1518.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Stein, E., Savaskan, N. E., Ninnemann, O., Nitsch, R., Zhou, R. and Skutella, T. (1999). A role for the Eph ligand ephrin-A3 in entorhino-hippocampal axon targeting. J. Neurosci. 19, 8885-8893.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    Tuttle, R., Schlaggar, B. L., Braisted, J. E. and O’Leary, D. D. (1995). Maturation-dependent upregulation of growth-promoting molecules in developing cortical plate controls thalamic and cortical neurite growth. J. Neurosci. 15, 3039-3052.
    OpenUrlAbstract
  47. ↵
    Vanderhaeghen, P., Lu, Q., Prakash, N., Frisen, J., Walsh, C. A., Frostig, R. D. and Flanagan, J. G. (2000). A mapping label required for normal scale of body representation in the cortex. Nat. Neurosci. 3, 358-365.
    OpenUrlCrossRefPubMedWeb of Science
  48. ↵
    Walter, J., Kern-Veits, B., Huf, J., Stolze, B. and Bonhoeffer, F. (1987). Recognition of position-specific properties of tectal cell membranes by retinal axons in vitro. Development 101, 685-696.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    Weth, F., Nadler, W. and Korsching, S. (1996). Nested expression domains for odorant receptors in zebrafish olfactory epithelium. Proc. Natl. Acad. Sci. USA 93, 13321-13326.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    Wilkinson, D. (2001). Multiple roles of EPH receptors and ephrins in neural development. Nat. Rev. Neurosci. 2, 155-164.
    OpenUrlPubMedWeb of Science
  51. ↵
    Wizenmann, A., Thies, E., Klostermann, S., Bonhoeffer, F. and Bahr, M. (1993). Appearance of target-specific guidance information for regenerating axons after CNS lesions. Neuron 11, 975-983.
    OpenUrlCrossRefPubMedWeb of Science
  52. ↵
    Yabuta, N. H., Butler, A. K. and Callaway, E. M. (2000). Laminar specificity of local circuits in barrel cortex of ephrin-A5 knockout mice. J. Neurosci. (Online) 20, RC88.
  53. ↵
    Yamamoto, N., Yamada, K., Kurotani, T. and Toyama, K. (1992). Laminar specificity of extrinsic cortical connections studied in coculture preparations. Neuron 9, 217-228.
    OpenUrlCrossRefPubMedWeb of Science
  54. ↵
    Yamamoto, N., Higashi, S. and Toyama, K. (1997). Stop and branch behaviors of geniculocortical axons: a time-lapse study in organotypic cocultures. J. Neurosci. 17, 3653-3663.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    Yates, P. A., Roskies, A. L., McLaughlin, T., O’Leary, D. D. (2001). Topographic-specific axon branching controlled by ephrin-As is the critical event in retinotectal map development. J. Neurosci. 21, 8548-8563.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Zhou, R., Copeland, T. D., Kromer, L. F. and Schulz, N. T. (1994). Isolation and characterization of Bsk, a growth factor receptor-like tyrosine kinase associated with the limbic system. J. Neurosci. Res. 37, 129-143.
    OpenUrlCrossRefPubMedWeb of Science
  57. ↵
    Zisch, A. H., Stallcup, W. B., Chong, L. D., Dahlin-Huppe, K., Voshol, J., Schachner, M. and Pasquale, E. B. (1997). Tyrosine phosphorylation of L1 family adhesion molecules: implication of the Eph kinase Cek5. J. Neurosci. Res. 47, 655-665.
    OpenUrlCrossRefPubMedWeb of Science
View Abstract
Previous ArticleNext Article
Back to top
Previous ArticleNext Article

This Issue

 Download PDF

Email

Thank you for your interest in spreading the word on Development.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Ephrins regulate the formation of terminal axonal arbors during the development of thalamocortical projections
(Your Name) has sent you a message from Development
(Your Name) thought you would like to see the Development web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Research Article
Ephrins regulate the formation of terminal axonal arbors during the development of thalamocortical projections
Fanny Mann, Christiane Peuckert, Frank Dehner, Renping Zhou, Jürgen Bolz
Development 2002 129: 3945-3955;
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
Research Article
Ephrins regulate the formation of terminal axonal arbors during the development of thalamocortical projections
Fanny Mann, Christiane Peuckert, Frank Dehner, Renping Zhou, Jürgen Bolz
Development 2002 129: 3945-3955;

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Alerts

Please log in to add an alert for this article.

Sign in to email alerts with your email address

Article navigation

  • Top
  • Article
    • Summary
    • INTRODUCTION
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • Acknowledgments
    • Footnotes
    • References
  • Figures & tables
  • Info & metrics
  • PDF

Related articles

Cited by...

More in this TOC section

  • Ceramides mediate positional signals in Arabidopsis thaliana protoderm differentiation
  • Zebrafish model for spondylo-megaepiphyseal-metaphyseal dysplasia reveals post-embryonic roles of Nkx3.2 in the skeleton
  • Ror2-mediated non-canonical Wnt signaling regulates Cdc42 and cell proliferation during tooth root development
Show more RESEARCH ARTICLES

Similar articles

Other journals from The Company of Biologists

Journal of Cell Science

Journal of Experimental Biology

Disease Models & Mechanisms

Biology Open

Advertisement

Kathryn Virginia Anderson (1952-2020)

Developmental geneticist Kathryn Anderson passed away at home on 30 November 2020. Tamara Caspary, a former postdoc and friend, remembers Kathryn and her remarkable contribution to developmental biology.


Zooming into 2021

In a new Editorial, Editor-in-Chief James Briscoe and Executive Editor Katherine Brown reflect on the triumphs and tribulations of the last 12 months, and look towards a hopefully calmer and more predictable year.


Read & Publish participation extends worldwide

Over 60 institutions in 12 countries are now participating in our Read & Publish initiative. Here, James Briscoe explains what this means for his institution, The Francis Crick Institute. Find out more and view our full list of participating institutions.


Upcoming special issues

Imaging Development, Stem Cells and Regeneration
Submission deadline: 30 March 2021
Publication: mid-2021

The Immune System in Development and Regeneration
Guest editors: Florent Ginhoux and Paul Martin
Submission deadline: 1 September 2021
Publication: Spring 2022

Both special issues welcome Review articles as well as Research articles, and will be widely promoted online and at key global conferences.


Development presents...

Our successful webinar series continues into 2021, with early-career researchers presenting their papers and a chance to virtually network with the developmental biology community afterwards. Sign up to join our next session:

10 February
Time: 13:00 (GMT)
Chaired by: preLights

Articles

  • Accepted manuscripts
  • Issue in progress
  • Latest complete issue
  • Issue archive
  • Archive by article type
  • Special issues
  • Subject collections
  • Sign up for alerts

About us

  • About Development
  • About the Node
  • Editors and board
  • Editor biographies
  • Travelling Fellowships
  • Grants and funding
  • Journal Meetings
  • Workshops
  • The Company of Biologists

For authors

  • Submit a manuscript
  • Aims and scope
  • Presubmission enquiries
  • Article types
  • Manuscript preparation
  • Cover suggestions
  • Editorial process
  • Promoting your paper
  • Open Access
  • Biology Open transfer

Journal info

  • Journal policies
  • Rights and permissions
  • Media policies
  • Reviewer guide
  • Sign up for alerts

Contact

  • Contact Development
  • Subscriptions
  • Advertising
  • Feedback

 Twitter   YouTube   LinkedIn

© 2021   The Company of Biologists Ltd   Registered Charity 277992