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Keywords:

  • chick embryo;
  • Eph;
  • limb muscle;
  • motor neuron;
  • receptor tyrosine kinase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Evidence is accumulating that Eph receptor tyrosine kinases and their ligands regulate cell migration and axonal guidance during development. It was previously found that one of the Eph receptors, EphA4, is transiently expressed in subsets of chick embryonic motor neurons. Here, the expression of EphA and ephrin-A subfamily members was further examined, and the dynamic patterns of expression in chick embryonic motor neurons found. EphA3, EphA4, ephrin-A2, and ephrin-A5 were also expressed in the connective tissues of limb muscles and EphA3 and EphA4 expressing motor neurons innervated EphA3 and EphA4 expressing limb muscles, respectively. These spatiotemporal expression patterns suggest that EphA and ephrin-A proteins play important roles in muscle patterning and motor axonal guidance.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Receptor tyrosine kinases (RTK) play critical roles in a diverse array of cellular responses such as growth, differentiation, and survival in both vertebrate and invertebrate animals ( Schlessinger & Ullrich 1992). The Eph family of RTK, named after the first member ( Hirai et al. 1987 ), now represents the largest group of RTK ( van der Geer et al. 1994 ) with at least 14 members ( Eph Nomenclature Committee 1997). Members of the Eph receptor family were all initially identified as ‘orphan receptors’. However, ligands that bind to and activate Eph family RTK have been identified one by one and found to form a family of eight members now ( Bartley et al. 1994 ; Beckmann et al. 1994 ; Cheng & Flanagan 1994; Davis et al. 1994 ; Shao et al. 1994 ; Bennett et al. 1995 ; Drescher et al. 1995 ; Kozlosky et al. 1995 ; Pandey et al. 1995 ; Shao et al. 1995 ; Winslow et al. 1995 ; Lackmann et al. 1996 ). The ligands are categorized into two groups, ephrin-A and ephrin-B, based on the manner in which they are linked to the plasma membrane. The ephrin-A subclass attaches to the membrane by a glycosylphosphatidyl-inositol (GPI) linkage, and the ephrin-B subclass is linked to the membrane through a transmembrane domain ( Brambilla & Klein 1995; Pandey et al. 1995 ; Gale et al. 1996 ; Eph Nomenclature Committee 1997). The Eph family receptors can also be divided into two groups based on the sequence homologies of their extracellular domains. This grouping also seems to correspond to the preferential binding of the receptors to either the ephrin-A or ephrin-B proteins. The group that comprises receptors interacting preferentially with ephrin-A and ephrin-B is called EphA and EphB, respectively ( Eph Nomenclature Committee 1997).

While ligands for several other families of RTK have either membrane-bound or secreted forms that are both active, soluble forms of the Eph family ligands are not only inactive but in fact may act as antagonists ( Davis et al. 1994 ; Winslow et al. 1995 ). The strict requirement for membrane attachment seems to provide for a specialized mechanism that ensures that the receptor activation is coupled to direct cell–cell contact ( Davis et al. 1994 ), consistent with the finding that Eph family receptors can be highly concentrated at sites of cell–cell contact ( Henkemeyer et al. 1994 ).

Eph family RTK display dynamic and spatially restricted expression patterns during embryogenesis, which suggests that they are involved in a variety of developmental processes. For example, in the central nervous system (CNS), several Eph family RTK are segmentally expressed in the developing hindbrain, indicating that they are involved in the formation of body segments or in regulating the segment-specific properties ( Gilardi-Hebenstreit et al. 1992 ; Nieto et al. 1992 ; Becker et al. 1994 ; Ganju et al. 1994 ; Henkemeyer et al. 1994 ; Ruiz & Robertson 1994; Ellis et al. 1995 ). Many members of this family have been shown to be expressed in developing neuronal tissues ( Pasquale et al. 1992 ; Cheng & Flanagan 1994; Henkemeyer et al. 1994 ; Soans et al. 1994 ; Cheng et al. 1995 ) and some functions in axonal guidance and cell migration have been revealed ( Brambilla & Klein 1995; Friedman & O’Leary 1996; Kilpatrick et al. 1996 ; Ohta et al. 1997 ). Ephrin-A2 and ephrin-A5, two GPI-linked ligands expressed in the developing tectum with an anterior low and posterior high gradient, can function as repulsive guidance molecules for retinal axons both in vitro ( Drescher et al. 1995 ) and in vivo ( Nakamoto et al. 1996 ; Frisen et al. 1998 ). It has also been shown that ephrin-A5 can modulate axon bundling in vitro ( Winslow et al. 1995 ). Mice deficient of EphB2 show commissural axon pathfinding defects in the brain ( Henkemeyer et al. 1996 ) and EphB2/EphB3 double mutants show more severe defects in commissural axon tract formation ( Orioli et al. 1996 ). Mice lacking EphA8 also show aberrant axonal projections ( Park et al. 1997 ). In addition, ephrin-B1, ephrin-B2, EphB1, EphB3, and EphA4 have been reported to mediate repulsive guidance of neural crest migration ( Krull et al. 1997 ; Smith et al. 1997 ; Wang & Anderson 1997). Ephrin-B1, ephrin-B2 ( Wang & Anderson 1997), and ephrin-A5 ( Donoghue et al. 1996 ) inhibit neurite outgrowth from presumable motor neurons in neural tube explants. Thus, Eph family receptor-ligand signaling is likely to play important roles in the patterning of both the central and peripheral nervous systems.

We found that EphA4 is expressed on subpopulations of motor neurons innervating limb muscles, but not on those innervating body muscles ( Ohta et al. 1996 ). The neurite growth from purified motor neurons is inhibited by clustered forms of ephrin-A2 or ephrin-A5 but not by their soluble forms ( Ohta et al. 1997 ). By contrast, EphA3 has been reported to be expressed in the cervical and thoracic motor neurons of the medial motor column, innervating receptor-positive axial musculature ( Kilpatrick et al. 1996 ). Interestingly, Gale et al. (1996) have shown that the developing embryo is subdivided into domains defined by reciprocal and apparently mutually exclusive expression of an Eph receptor subclass and its corresponding ligand. Although distributions of the GPI-linked Eph ligands and their receptors in the spinal cord have been reported ( Soans et al. 1994 ; Flenniken et al. 1996 ; Fukushima et al. 1996 ; Kilpatrick et al. 1996 ; Ohta et al. 1996 ; Zhang et al. 1997 ; Araujo et al. 1998 ), they have not been thoroughly documented thus far.

In the present study, we examined the expression of GPI-linked ligands, ephrin-A2 and ephrin-A5, and their receptors, EphA3, EphA4, and EphA5, in the spinal cord of chick embryo systematically by both in situ hybridization and immunohistochemistry, and in the limb by immunohistochemistry. We found that subsets of motor neurons expressed transiently EphA3, EphA4, ephrin-A2, and ephrin-A5, and that the connective tissues of the innervation target muscles of the EphA3- and EphA4-positive motor neurons expressed the same proteins of EphA receptors during the period of intramuscular nerve formation ( Dahm & Landmesser 1991). These results suggest that the interaction between Eph GPI-linked ligands and their receptors affects muscle patterning and motor axonal guidance.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Animals

Chick eggs (White Leghorn) were incubated at 38°C in a forced air incubator, and the embryos were staged according to Hamburger and Hamilton (1951). Embryos at various stages were excised, embedded in Tissue-Tek OCT compound (Miles Elkhart), frozen, and cut at 10–14 μm on a cryostat microtome. For immunohistochemistry the embryos were fixed with 4% paraformaldehyde (PFA) for 1–2 h. Both in situ hybridization (from E3 to E17) and immunohistochemistry (from E5 to E12) were performed on more than three embryos at each stage.

Molecular probes

The XbaI fragment (0.9 kb) of the 3′ untranslated region of the chick ephrin-A2 cDNA ( Ohta et al. 1997 ) was used to prepare an RNA probe. The full-length cDNA of chick ephrin-A5 (868 bp, Drescher et al. 1995 ) was subcloned into pBluescript SK(+), and used to prepare an RNA probe. The chick EphA3 RNA probe was prepared by transcribing nucleotides 187–658 of EphA3 cDNA ( Sajjadi et al. 1991 ). The chick EphA5 RNA probe was prepared by transcribing nucleotides 2371–3132 of EphA5 cDNA ( Siever & Verderame 1994). The chick EphA4 RNA probe was prepared by transcribing an 889 bp BamHI-EcoRI fragment from the 3′ untranslated region of EphA4 cDNA beginning at nucleotide 2711 ( Ohta et al. 1996 ). An Islet-1 RNA probe was generated by transcribing nucleo- tides 317–1308 of the chick Islet-1 cDNA ( Tsuchida et al. 1994 ). These probes were prepared by reverse transcription–polymerase chain reaction. T3, T7 or SP6 RNA polymerase were used in a reaction mix containing 0.83 m M digoxigenin (DIG)-11-UTP (Boehringer Mannheim, Düsseldorf, Germany).

Hybridization

In situ hybridization was performed by a modification of the method of Ohta et al. (1996) . Sections were treated with 1 mg/mL proteinase K for 10 min at 37°C. Hybridization was carried out for 16 h at 55°C in a solution containing 1 μg/mL of each DIG-labeled RNA probe. After washing, the sections were blocked with 10% heat-inactivated sheep serum, then incubated overnight at 4°C with a 1:1000 dilution of alkaline phosphatase-conjugated anti-DIG antibody (Boehringer Mannheim). The antibody was absorbed against embryo powder prepared from stage 27 and 36 embryos. An alkaline phosphatase-mediated color reaction was carried out using 4-nitroblue tetrazolium chloride (Boehringer Mannheim) and 5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim).

Monoclonal antibody production and immunohistochemistry

We generated monoclonal antibodies (mAb) against chick EphA3, ephrin-A2, and ephrin-A5 for immunohistochemistry as described previously for the mAb against EphA4 ( Ohta et al. 1996 ). The chimeric proteins of chick EphA3-Fc, ephrin-A2-Fc, and ephrin-A5-Fc were constructed with the extracellular domain of chick EphA3, ephrin-A2, and ephrin-A5 and the Fc region of a human immunoglobulin, and were transiently expressed in COS cells and purified from the culture supernatants using a protein G-Sepharose column ( Ohta et al. 1997 ). BALB/c female mice were immunized with these chimeric proteins, and mAb were produced as described before ( Ohta et al. 1996 ). The specificity of these mAb was checked by staining transforming cell lines of 293-EphA3, 293-EphA4, 293-ephrin-A2, and 293-ephrin-A5 with the same procedures for sections.

Immunohistochemistry for sections was performed according to the protocol from TSA-kit (NEN Life Science Products Inc., Boston, MA, USA). The signal was visualized by means of peroxides-tyramide- fluorescein isothiocyanate (FITC). The antibodies for immunohistochemistry were purified from the ascites, and used at concentrations of 5 μg/mL for mAb ephrin-A2, 20 μg/mL for mAb ephrin-A5, 10 μg/mL for mAb EphA3 and 2.5 μg/mL for mAb EphA4.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

In situ hybridization

Expression of EphA3 mRNA in chick embryonic spinal cord In vertebrates, spinal somatic motor neurons can be divided into two large subpopulations. One innervates axial muscles and forms the medial motor column (MMC) that runs through all levels of the spinal cord. The other innervates limb muscles and forms the lateral motor column (LMC) in the brachial and lumbar segments.

It has been reported that EphA3 is expressed in a subset of axial, but not limb, motor neurons ( Kilpatrick et al. 1996 ), whereas EphA4 is expressed in a subset of limb, but not axial, motor neurons ( Ohta et al. 1996 ). To make a direct comparison of the expression patterns of EphA3 and EphA4, we analyzed transverse sections of chick embryos at various developmental stages by in situ hybridization. Figure 1 shows examples of transverse sections of spinal cords in embryonic day 5 (E5; stage 27), E6 (stage 29), and E7 (stage 32) chick embryos through the brachial ( Fig. 1A–E), thoracic ( Fig. 1F–J), and lumbar ( Fig. 1K–O) regions, which were hybridized with a DIG-labeled probe of either EphA3, EphA4, or Islet-1. The results of the same embryo are presented for each embryonic day.

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Figure 1. Expression of EphA3 messenger ribonucleic acid (mRNA) in chick embryonic spinal cord. Transverse sections of embryonic day 5 (E5, stage 27; A,F,K), E6 (stage 29; B,G,L), and E7 (stage 32; C,H,M) chick spinal cords through the brachial (A–C), thoracic (F–H), and lumbar (K–M) regions were analyzed by in situ hybridization for EphA3. For comparison, the expression patterns of EphA4 (D,I,N) and Islet-1 (E,J,O) mRNA are shown for the same regions of E5 chick embryonic spinal cord. The same embryo was used for each embryonic day. Note that at E5, the EphA3 mRNA is localized to the medial motor column (MMC; arrow in A) and a part of the lateral motor column (LMC) in the brachial region (black arrowhead, A). EphA3 is not expressed in the lumbar region at this stage (K), but at E6 and E7, it is expressed in the MMC (arrows, B,C,M) and a part of the LMC (arrowheads, B,C,M) not only in the brachial (C) and thoracic (G) regions, but also in the lumbar region (L,M). At E5 and E6, EphA3 expression is detected at the ventral ventricular layer in the brachial (A,B), thoracic (F,G), and lumbar (stars K,L) regions. EphA3 is not expressed in dorsal root ganglia (DRG), whereas it is expressed at a high level in the sclerotome around the spinal cord (white arrowheads, A,B,F,G,L). At E5, motor neurons are well developed at all levels as indicated by Islet-1 expression (E,J,O), and EphA4 is specifically expressed in the brachial (D) and lumbar (N) motor neurons, but not in the thoracic ones (I). Islet-1 is expressed in DRG. Bar, 250 μm.

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The EphA3 transcripts were first observed in the ventral ventricular layer of the spinal cord at E4 (data not shown), and the expression lasted until E6 (stars in Fig. 1K,L). In motor neurons, EphA3 was expressed at E5 in the brachial and thoracic ( Fig. 1A,F), but not in the lumbar region ( Fig. 1K). EphA3 seemed to be expressed developmentally later than both EphA4 and the motor neuron marker Islet-1, because EphA3 first appeared at E6 in the lumbar region ( Fig. 1L), but EphA4 ( Fig. 1N) and Islet-1 ( Fig. 1O) were already expressed at E5. As reported by Kilpatrick et al. (1996) , the EphA3 mRNA was expressed in axial motor neurons in the thoracic region ( Fig. 1F–H) and brachial and lumbar regions (arrows in Fig. 1A–C,M), but also in subsets of motor neurons in the LMC, which innervate limb muscles (arrowheads in Fig. 1A–C,M). In the lumbar region, EphA3 was expressed diffusely in the LMC at E6 ( Fig. 1L), then localized to the most lateral and medial parts of the motor column ( Fig. 1M). This expression pattern was observed until E10 (data not shown). As this type of expression was observed in all other embryos examined, it is likely that many motor neurons in the LMC transiently express the EphA3 mRNA. EphA3 was not expressed in dorsal root ganglia (DRG), whereas it was expressed at a high level in the sclerotome around the spinal cord (white arrowheads in Fig. 1A,B,F,G,L).

Expression of EphA5 mRNA in chick embryonic spinal cord Although the EphA subfamily in chick currently comprises EphA3, A4, A5, and A7, EphA7 ( Araujo & Nieto 1997; Sefton et al. 1997 ) was not known when we started the current study, and was not examined. Expression of EphA5 in the brachial and thoracic regions of the spinal cord is shown in Fig. 2. The expression was seen on the ventral side of the ventricular layer from E4 on (data not shown), and spread dorsally with development. The distribution of EphA5-positive cells was diffuse, especially in the ventral spinal cord, and was not restricted to motor neurons ( Fig. 2). EphA5 was also expressed in DRG (arrows in Fig. 2B,C,E,F).

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Figure 2. Expression of EphA5 messenger ribonucleic acid (mRNA) in chick embryonic spinal cord. Transverse sections of embryonic day 5 (E5, stage 27; A,D), E6 (stage 29; B,E), and E7 (stage 32; C,F) chick spinal cords through the brachial (A–C) and thoracic (D–F) regions were analyzed by in situ hybridization for EphA5. EphA5 expression is observed on the ventral side of the ventricular layer from E4 on, and spread dorsally with development (A–F). EphA5 does not seem to be expressed in the motor column in the thoracic and brachial regions (B,C,E,F), whereas it is expressed in the dorsal root ganglion (DRG) from E6 on (arrows). Bar, 250 μm.

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Oligodendrocyte precursors originate in the ventral ventricular zone of the developing vertebrate spinal cord and subsequently migrate dorsally and radially to populate presumptive white matter ( Ono et al. 1995 ). EphA5 was expressed in the ventral ventricular layer at first (E4; data not shown) and then its expression spread dorsally and radially ( Fig. 2A,D). These expression patterns suggest that the oligodendrocyte precursors express EphA5, but further examinations in combination with oligodendrocyte markers are necessary.

Expression of ephrin-A2 and ephrin-A5 mRNA in chick embryonic spinal cord The ligands for the EphA receptors comprise the GPI-linked ephrin-A subfamily among which only ephrin-A2 and ephrin-A5 are known in chick ( Eph Nomenclature Committee 1997). We examined the distribution of these ligands in the spinal cord of chick embryos from E3 to E17. The ephrin-A2 mRNA was localized in motor neurons of the brachial and lumbar LMC from E5 to E10 and only faintly at E14 (data not shown), but not in the cervical and thoracic regions ( Fig. 3). Ephrin-A2 was expressed in the DRG and surrounding sclerotome as well as in the ventricular cell layer throughout the spinal cord from E4 to E17 (data not shown).

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Figure 3. Expression of ephrin-A2 messenger ribonucleic acid (mRNA) in embryonic day 5 (E5) chick embryonic spinal cord. Transverse sections of E5 (stage 27) chick spinal cord through the cervical (A), brachial (B), thoracic (C) and lumbar (D) regions were analyzed by in situ hybridization for ephrin-A2. Note that the ephrin-A2 mRNA is localized at the motor neurons of the brachial and lumbar, but not the cervical and thoracic regions. Ephrin-A2 is also expressed in the ventricular cell layer throughout the spinal cord, and in the dorsal root ganglion (DRG) in all regions examined. Bar, 250 μm.

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The expression pattern of ephrin-A5 mRNA is dynamic compared to that of ephrin-A2. Its expression started from E4 in motor neurons from brachial to lumbar levels ( Fig. 4E,I,M), but no expression was observed in the cervical region from E4 to E6 ( Fig. 4A–C). At E7 ephrin-A5 expression was detected in the dorsal part of the spinal cord in the cervical region ( Fig. 4D), and in other regions as well ( Fig. 4H,L,P). This expression pattern was observed throughout the spinal cord until E10, but disappeared at E14 (data not shown). Ephrin-A5 expression in thoracic motor neurons was transient and disappeared by E6, whereas its expression in the preganglionic column of Terni started at E6 and lasted until E10 (data not shown; arrowheads in Fig. 4K,L). The signals were observed in motor neurons in the brachial and lumbar LMC from E4 to E10, but the expression was not uniform in the LMC. The ephrin-A5 mRNA was localized in the medial half of the LMC at E6 ( Fig. 4O). This expression in the motor column could be observed at E10, but not at E14 (data not shown). Ephrin-A5 was also expressed in the DRG at all levels until E14, and the expression completely disappeared by E17 (data not shown).

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Figure 4. Expression of ephrin-A5 messenger ribonucleic acid (mRNA) in chick embryonic spinal cord. Transverse sections of embryonic day 4 (E4, stage 23; A,E,I,M), E5 (stage 27; B,F,J,N), E6 (stage 29; C,G,K,O) and E7 (stage 32; D,H,L,P) chick spinal cords through the cervical (A–D), brachial (E–H), thoracic (I–L) and lumbar (M–P) regions were analyzed by in situ hybridization for ephrin-A5. The same embryo was used for each embryonic day. Note that the ephrin-A5 mRNA is expressed at the medial motor column (MMC) and lateral motor column (LMC) in the brachial (E,F), thoracic (I,J) and lumbar (M,N) regions at E4 and E5. Ephrin-A5 expression in the MMC disappears at E6 (G,K,O), while its expression starts at the same stage in the preganglionic column of Terni in the thoracic region (arrowhead, K,L). In the cervical region (A–D), ephrin-A5 is not detected in the motor column. At E7 (D,H,L,P) ephrin-A5 is expressed in cells of the dorsal side throughout the spinal cord. Ephrin-A5 is also expressed in the dorsal root ganglion (DRG). Bar, 250 μm.

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Distribution and temporal changes of the EphA3, EphA4, ephrin-A2, and ephrin-A5 mRNA expression are summarized in Fig. 5. These results demonstrate that motor neurons express both Eph receptors and ligands during the period of their axonal growth.

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Figure 5. Summary of spatial (A) and temporal (B) expression patterns of the EphA3, EphA4, ephrin-A2, and ephrin-A5 messenger ribonucleic acid in the motor columns and dorsal root ganglion.

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Immunohistochemistry

To further analyze the expression of EphA and ephrin-A subfamilies we developed mAb against the extracellular domains of EphA3, EphA4, ephrin-A2, and ephrin-A5. Although these molecules are similar to each other in terms of cDNA sequence, anti-EphA3 mAb stained 293-EphA3 transformant cells, but not 293-EphA4 cells, while anti-EphA4 mAb did the opposite ( Fig. 6). The same is true with mAb against the ephrin-A2 and ephrin-A5 proteins. We therefore concluded that these antibodies specifically recognize and distinguish each protein. However, it is still possible that these antibodies bind to the other subfamily members.

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Figure 6. Staining specificity of monoclonal antibodies (mAb) against EphA3, EphA4, ephrin-A2 and ephrin-A5. The mAb anti-EphA3 stains 293-EphA3 transformant cells (A), but not 293-EphA4 cells (C). The mAb anti-EphA4 stains 293-EphA4 cells (D), but not 293-EphA3 cells (B). The mAb anti-ephrin-A2 stains 293-ephrin-A2 cells (E), but not 293-ephrin-A5 cells (G). The mAb anti-ephrin-A5 stains 293-ephrin-A5 cells (H), but not 293-ephrin-A2 cells (F).

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Expression in chick embryonic spinal cord By using these specific mAb, we examined the expression patterns of EphA3, EphA4, ephrin-A2, and ephrin-A5 at the protein level. We found that these proteins were expressed in essentially the same patterns as their transcripts with some exceptions. The outside of the dorsal spinal cord and parts of the limb buds were intensely stained with anti-EphA3 mAb( Fig. 7A). At a higher magnification, the stainings in the ventral ventricular layer of the cord was observed throughout the spinal cord at E5 (open arrowheads in Fig. 7E,I,M), and those in the ventral horn were observed in the brachial and thoracic regions, but not in the lumbar region ( Fig. 7E,I,M). However, EphA3 expression in the lumbar ventral horn was observed at E6 (data not shown). At E8 staining in the motor column and ventral root was clear ( Fig. 7Q). These expression patterns are consistent with the results of in situ hybridization ( Fig. 1). One exception is that DRG cells, where the transcripts were not detected, expressed EphA3 protein in the dorsal funiculi and axons (arrowhead in Fig. 7I).

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Figure 7. EphA3, EphA4, ephrin-A2 and ephrin-A5 protein expression in embryonic day 5 (E5, stage 27) and E8 (stage 34) chick embryos. (A–D) Immunostaining of transverse sections at the lumbar level of a stage 27 chick embryo with monoclonal antibodies (mAb) against EphA3, EphA4, ephrin-A2, and ephrin-A5. (A) The EphA3 protein is strongly expressed on the dorsal side of the spinal cord, which are parts of the neural arch of the vertebra and muscles, and parts of the limb buds. The staining is not observed in the spinal cord and motor neurons. (B) EphA4 is expressed in the lateral motor column (LMC) and ventricular layer of the spinal cord, ventral root, and the dorsal side of the cord like anti-EphA3. It is also widely expressed in the limb buds. (C) Ephrin-A2 is expressed in the whole spinal cord, dorsal and ventral roots, dorsal root ganglion (DRG), and limb buds. (D) Ephrin-A5 is expressed in parts of the limb buds, whereas its expression in the spinal cord is very weak. (E–S) Examples of adjacent sections of the spinal cord and DRG at a higher magnification are shown for E5 brachial (E–H), E5 thoracic (I–L), E5 lumbar (M–P), and E8 brachial (Q–S) regions. EphA3 is expressed at the ventral part of the spinal cord in the brachial (E) and thoracic (I) regions, but not in the lumbar (M) at E5. Motor neurons in the LMC and ventral root (arrow in Q) are immunopositive at E8. Moreover, dorsal funiculi (arrowhead in I) and DRG axons are immuno-positive. EphA3 expression is observed in the ventral ventricular layer throughout the spinal cord (open arrowheads in E,I,M), but not detected at E8 (Q). The EphA4 protein is expressed on both cell bodies and axons of motor neurons in the brachial and lumbar regions at E5 (NF, star; arrow, N) and E8 (arrow, R), but not in the thoracic region (J). It is not observed in the DRG (F,J,N,R) and its axons in the dorsal funiculi (arrowhead in J). The ephrin-A2 protein is expressed broadly in the spinal cord, ventral nerve, DRG, and its axons in the dorsal funiculi (arrowhead in K,S). There is no obvious difference in the ephrin-A2 protein expression between the thoracic and lumbar regions. Immunoreactivity in the dorsal spinal cord is stronger than that in the ventral half (G,K,O,S). The ephrin-A5 protein is weakly expressed in the whole spinal cord and DRG (H,L,P), but expression in the LMC is relatively clear (H,P). s.c., spinal cord; d, dorsal root ganglion. Bars, 500 μm (A–D), 100 μm (E–S).

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The EphA4 protein was expressed in the spinal cord and limb buds in essentially the same pattern as reported before ( Ohta et al. 1996 ; Fig. 7B). Motor neurons in the brachial and lumbar regions expressed EphA4 on both cell bodies and axons ( Fig. 7F, star and arrow in N), but thoracic motor neurons ( Fig. 7J) and DRG cells did not seem to express it ( Fig. 7F,J,N,R; arrowhead in J). At E8 brachial and lumbar motor neurons and axons intensely expressed EphA4 ( Fig. 7R).

Anti-ephrin-A2 mAb stained the whole spinal cord and limb buds ( Fig. 7C), but the staining intensity in LMC was not high, and no differences in the staining of motor neurons between thoracic and lumbar regions were observed ( Fig. 7K,O), which was against the results of in situ hybridization ( Fig. 3). Compared with EphA4 ( Fig. 7B,F,N,R), the staining intensity of ephrin-A2 in the motor column was not high, whereas ventral roots were immunopositive ( Fig. 7C,G,K,O,S). As the dorsal funiculus, which consists of nerve fibers from the DRG, was clearly immunopositive with ephrin-A2 in contrast to EphA4 (arrowheads in Fig. 7J,K), we conclude that the ephrin-A2 protein was expressed on axons of both motor neurons and DRG cells. The ephrin-A2 expression in the dorsal half of the spinal cord was higher than that of ventral side ( Fig. 7G,K,O,S).

The expression of ephrin-A5 was strong in the limb buds, but very weak in the spinal cord ( Fig. 7D). At a higher magnification, the immunoreactivities were observed in the LMC and DRG, but the whole spinal cord was also weakly immunopositive ( Fig. 7H,L,P). The clear difference in the expression detected by in situ hybridization ( Fig. 4) was not obvious with immunohistochemistry. We produced several different clones of mAb against EphA3, EphA4, ephrin-A2, and ephrin-A5 for immunohistochemistry. These different clones presumably recognized different epitopes of these molecules, but did not show any significant differences. We therefore consider it unlikely that the epitopes of mAb were masked by the interaction of these antigens with other proteins.

The present results demonstrate that the localization of the mRNA of Eph family in motor neurons is not necessarily identical to those of the proteins, especially for Eph ligands. These results agree with the expression analysis of mouse ephrin-A5 in the spinal cord. Although the mouse ephrin-A5 transcripts were expressed in a subset of motor neurons in the embryonic spinal cord ( Flenniken et al. 1996 ), it was unlikely that the ephrin-A5 protein was expressed on the motor neurons, because the Eph receptor-Fc chimeric protein failed to bind to the ventral spinal cord ( Gale et al. 1996 ). Present antibody studies more properly represent protein expression than the studies of Fc-chimeric protein, which requires intact forms of the molecules for binding.

One possible explanation for these discrepancies between in situ hybridization and immunohistochemistry is that the amount of the ephrin-A2 or ephrin-A5 protein produced by motor neurons is too low to be detected by immunohistochemistry. Another possibility is that the ephrin-A2 or ephrin-A5 protein is produced by motor neurons in high quantities, but is exclusively transported to axons and growth cones. To distinguish these two possibilities, we examined their expression in the limb by immunohistochemistry, and found that both ephrin-A2 and ephrin-A5 immunostaining were not associated with nerve fibers (cf. Figs 9,10). From these results, it seems unlikely that the protein is localized only in the axons, but not cell bodies.

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Figure 9. EphA3, EphA4, ephrin-A2 and ephrin-A5 protein expression in the hindlimb at the embryonic day stage 8 (E8, stage 34) and E12 (stage 38). Thigh muscles at the adjacent cross-sections were stained with monoclonal antibodies (mAb) against EphA3 (B,O), EphA4 (C,I), ephrin-A2 (E,J,L), ephrin-A5 (F,M), muscle marker 13F4 (A,G,K). myosin MF30 (N) and neurofilament 82E10 (D,H). (A–M), E8; (N,O), E12. (G–M) Enlargements of the corresponding staining in (A,C–F). The schema indicates muscle connective tissues. (B) The EphA3 protein is specifically expressed in FTI and a part of FTE, PIL, IF, at a more proximal region than in other sections. Furthermore, immunostaining can be seen at a part of the sciatic nerve (arrowhead) and intramuscular nerve (arrows). The EphA3 protein is clearly expressed in the perimysium, surrounding myotube clusters (N,O, and schema). (C) The EphA4 protein is expressed in FTI, FTE, PIL, IF, PIFL, FCLP, nerve trunk (arrowhead) and axons within the muscles. Arrows in (C) and (D) indicate corresponding axons. The nerve trunks are stained in patches (H,I). EphA4 is also localized in the perimysium and epimysium (G,I, and schema). Arrowheads in (G–J) indicate nerve trunk. (E) The ephrin-A2 protein is expressed diffusely all over the section, but its expression in muscles of anti-EphA3 and anti-EphA4 staining positive is relatively weak. The nerves do not express ephrin-A2 (J). At higher magnification ephrin-A2 is expressed in AIL and its surrounding connective tissue (L). (F,M) The ephrin-A5 protein is expressed outside the IC and AIL. The white dots in (L,M) indicate muscles as shown in (K). The individual muscles were identified by the muscle marker 13F4 (A). Dorsal muscles: FTI, femorotibialis internus; FTM, femorotibialis medialis; FTE, femorotibialis externus; AMB, ambiens; IC, iliotibialis cranialis; AIL, iliotibialis lateralis preacetabularis; PIL, iliotibialis lateralis postacetabularis; and IF, iliofibularis. Ventral muscles are PIFL, pubo-ischio-femoralis pars lateralis; PIFM, pubo-ischio-femoralis pars medialis; FCLP, flexor cruris lateralis par pelvica; and FCM, flexor cruris medialis ( Schroeter & Tosney 1991; Kardon 1998). Bars, 500 μm (A–F); 100 μm (G–O).

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image

Figure 10. EphA3, EphA4, ephrin-A2, and ephrin-A5 protein expression in the shank at embryonic day 8 (E8, stage 34). Adjacent cross-sections through the mid-shank region were stained with monoclonal antibody (mAb) against EphA3 (B), EphA4 (C), ephrin-A2 (E), ephrin-A5 (F), muscle marker 13F4 (A), and neurofilament 82E10 (D). Orientations: top, dorsal; bottom, ventral; right, external; left, internal. The EphA3 protein is specifically expressed in FB, GI, and FP3 (B). The EphA4 protein is expressed in almost all shank muscles except GM, EDL, and FDL (C). The arrows indicate corresponding axons (C,D). EphA4 is localized in the perimysium and epimysium as shown in the thigh. Expression of ephrin-A2 is diffuse in the entire shank (E) and ephrin-A5 localized around the tibia and fibula (F). Individual muscles were identified by the muscle marker 13F4 (A). Dorsal muscles: FL, fibularis longus; FB, fibularis brevis; TC, tibialis cranialis; EDL, extensor digitorum longus. Ventral muscles: GM, gastrocnemius intermedius; GI, gastrocnemius internus; GE, gastrocnemius externus; FDL, flexor digitorum longus; FP3, flexor perforatus 3 ( Kardon 1998). Bar, 500 μm.

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Expression in chick hindlimb muscles Expression patterns of the Eph receptors and their ligands in the muscles were described with the focus on the thigh and shank regions. Limb mesenchyme cells expressed EphA3, EphA4, ephrin-A2, and ephrin-A5 as observed in Fig. 7(A–D). At the transverse sections of the proximal–distal axis, dorsal and ventral muscle masses were observed with muscle marker mAb 13F4 staining ( Fig. 8A). Expression of EphA3 was restricted in narrow regions of the limb bud as shown in Fig. 7(A) and was not seen in this section ( Fig. 8B). EphA4 expression was closely associated with muscle mass, especially with the dorsal one, but only a part of the ventral one in this section ( Fig. 8C). These expression patterns suggest a possibility that EphA receptors have a role to stop myoblast migration and accumulate there, although ephrin-A2 and ephrin-A5 seemed not to be expressed on myogenic cells ( Fig. 8E,F) and it is unknown whether myogenic cells express other Eph ligands. As the distribution of ephrin-A2 seemed to be complementary to the expression of EphA4 ( Fig. 8C,E), it raised the possibility that ephrin-A2 forms a boundary for myoblast migration. The expression of ephrin-A5 was localized in small areas ( Fig. 8F). Both ephrin-A2 and ephrin-A5 were not expressed intensely in nerve trunks at E5 (arrows in Fig. 8D,E,G,I), but EphA4 was clearly expressed ( Fig. 8G,H).

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Figure 8. The EphA3, EphA4, ephrin-A2, and ephrin-A5 protein expression in embryonic day 5 (E5, stage 27) hindlimb bud. The cross-sections were analyzed by immunohistochemistry for EphA3 (B), EphA4 (C,H), ephrin-A2 (E,I), ephrin-A5 (F), muscle marker (mAb 13F4; A), and neurofilament (mAb 82E10; Go et al. 1989 ; D,G). Dorsal side is top and ventral is lower. (A) Muscle marker shows dorsal and ventral muscle mass separated by femur (f). (B) EphA3 is not expressed at this level along the proximal–distal axis. (C) EphA4 closely co-localize with parts of myogenic cells. EphA4 expression is not restricted to the dorsal side, and could be seen on the ventral side at other levels. (E) The ephrin-A2 expression seems to be complementary with that of EphA4. (F) The ephrin-A5 is expressed in very restricted regions at this level. Parts of (D), (C) and (E) are enlarged as (G–I), respectively. Arrows indicate the corresponding axon. EphA4 staining is co-localized with axons, but that of ephrin-A2 is not. Bar, 500 μm (A–F); 100 μm (G–I).

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After muscle differentiation the expression patterns of EphA3, EphA4, ephrin-A2, and ephrin-A5 reflected their localization at E5. EphA3 was expressed in only a few muscles in the thigh ( Fig. 9B) and shank ( Fig. 10B) regions. Its expression in femorotibialis externus (FTE) and femorotibialis internus (FTI) was observed, but that in iliotibialis lateralis postacetabularis (PIL) and iliofibularis (IF) was not observed at a level the same as in Fig. 9(A). The section in Fig. 9(B) was from a more proximal region than in Fig. 9(A), and EphA3 expression was detected in PIL and IF. The staining was observed in only a part of IF. Thus, EphA3 was not expressed evenly in each muscle. Compared with neurofilament staining (data not shown), EphA3 was expressed by nerve fibers (arrows and arrowhead in Fig. 9B). At a higher magnification EphA3 was clearly expressed by the muscle connective tissue, perimysium, but not by the myotubes ( Fig. 9N,O and schema in Tono-oka et al. 1996 ), consistent with the origin of the connective tissues.

At E8 the EphA4 protein was expressed mainly on the posterior side of the thigh region ( Fig. 9C) and almost all shank muscles except a few muscles ( Fig. 10C). EphA4 was also expressed by nerve trunks and intramuscular nerves (arrowhead in Fig. 9C,D,H,I and arrows in Figs 9C,D,10C,D) and by muscle connective tissues, perimysium and epimysium, but not by myotubes ( Fig. 9G,I). From the staining in Fig. 10(C) it was evident that EphA4 expressing motor axons innervated EphA4 expressing muscles. In the shank, gastrocnemius intermedius (GM), extensor digitorum longus (EDL) and flexor digitorum longus (FDL) did not express EphA4 and were innervated by EphA4-negative axons ( Fig. 10C,D). The same is true for EphA3. Thus, subsets of limb innervating motor neurons expressed EphA3 and EphA4 during the period of intramuscular nerve formation ( Dahm & Landmesser 1991). It is possible that the motor neurons and their target muscles, which did not express EphA3 or EphA4, express other subfamily members.

Ephrin-A2 was expressed throughout the limb in various quantities, and its expression in the anti-EphA3 and anti-EphA4 mAb positive muscles was relatively low ( Fig. 9E) as shown in Fig. 8(C,E), indicating reciprocal expression of a receptor subclass and its corresponding ligand ( Gale et al. 1996 ). In the thigh ephrin-A2 protein was expressed by muscle connective tissue ( Fig. 9L), but not by nerve fibers at E8 ( Fig. 9J). The expression of ephrin-A5 was observed in a relatively restricted muscle connective tissue ( Fig. 9F,M). The expression of ephrin-A2 was diffuse at the shank ( Fig. 10E) and that of ephrin-A5 was observed around the tibia and fibula ( Fig. 10F).

The present results showed that the Eph receptors and their ligands are co-expressed on motor neurons in chick embryos, although the ephrin-A2 and ephrin-A5 proteins did not seem to be expressed in high quantities. The co-localization of a receptor and its ligand on motor neurons during the same period has also been described in the case of collapsin/semaIII and neuropilin ( Luo et al. 1993 ; Messersmith et al. 1995 ; Takagi et al. 1995 ). The interaction between the receptor, neuropilin, and its ligand, collapsin/semaIII, is likely to play similar roles to the Eph/ephrin interaction during neural development ( Messersmith et al. 1995 ). In the case of collapsin/semaIII and neuropilin, however, the interaction between the receptor and its ligand on the same cells is not considered to be functionally important thus far, mainly because the ligand is a secreted protein. By contrast, in the case of EphA and ephrin-A, because the ligands are membrane anchored proteins, co-localization on the same cells could be functionally important. For example, in the retinotectal projection in chick embryos, ephrin-A2 and ephrin-A5 are not only expressed in the tectum ( Monschau et al. 1997 ), but also co-localized with EphA receptors on a subset of retinal ganglion cell axons, in a manner similar to the case of motor neurons as described before ( Marcus et al. 1996 ; Connor et al. 1998 ). Although the functional significance of this co-localization in vivo is not well understood at this point, it is possible that ephrin-A2 and ephrin-A5 on the retinal ganglion cell axons are to modulate sensitivity of the EphA receptors on the same axons ( Hornberger et al. 1999 ). Furthermore, ephrin-A2 and ephrin-A5 might have functions as receptors for the EphA receptors, as shown in the EphB and ephrin-B subfamily ( Henkemeyer et al. 1996 ).

Another striking result in the present study is that EphA3 and EphA4 were expressed in the muscle connective tissues and their expression patterns were not necessarily even in a single muscle. Furthermore, EphA3 or EphA4 expressing motor axons innervated the same receptor expressing hindlimb muscles as has been suggested in the case of EphA3 and EphA3- positive axial muscles ( Kilpatrick et al. 1996 ). Muscle connective tissues are considered to function in muscle patterning ( Tono-oka et al. 1996 ) and also in intramuscular nerve formation ( Phelan & Hollyday 1990). As Eph receptor–receptor interactions are unlikely, myoblasts and myotubes presumably express some Eph ligands and interact with surrounding connective tissues and motor axons. We are now undertaking an analysis of the application of functional blocking monoclonal antibodies against ephrin-A2, EphA3, and EphA4. From these analyses it might be possible to reveal the roles of ephrin-A2, EphA3, or EphA4 on the motor neuron axons and the muscle patterning.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

We thank Dr M. Go for his critical reading of this manuscript, Drs S. Hirano and S. Chiba (Niigata University) for advice on muscle identification, and K. Hirokawa and M. Kumamaru for their technical assistance. mAb 13F4 and MF30 were from the Developmental Studies Hybridoma Bank and mAb 82E10 was from Dr S. Fujita (Mitsubishi Chemicals). This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, and Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan to H. T.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
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