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Expression of Receptor Protein Tyrosine Phosphatases in Embryonic Chick Spinal Cord
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Molecular and Cellular Neuroscience 16, 470 – 480 (2000) doi:10.1006/mcne.2000.0887, available online at http://www.idealibrary.com on
Expression of Receptor Protein Tyrosine Phosphatases in Embryonic Chick Spinal Cord John K. Chilton* ,† and Andrew W. Stoker* ,1 *Neural Development Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, United Kingdom; and †Department of Human Anatomy and Genetics, Oxford University, South Parks Road, Oxford OX1 3QX, United Kingdom
Receptor-like protein tyrosine phosphatases potentially play a crucial role in axon growth and targeting. We focus here on their role within the embryonic avian spinal cord, in particular the development and outgrowth of motorneurons. We have used in situ mRNA hybridization to examine the spatiotemporal expression of eight receptorlike protein tyrosine phosphatases and find that it is both dynamic and highly varied, including novel, isoform-specific expression patterns. CRYP␣1 is expressed in all of the ventral motorneuron pools, whereas CRYP2, RPTP␥, and RPTP␣ are only expressed in specific subsets of these neurons. CRYP␣2, RPTP⌿, and RPTP␦ are neuronally expressed elsewhere in the cord, but not in ventral motorneurons, whereas RPTP is unique in being restricted to capillaries. The developmentally regulated expression of these genes strongly suggests that the encoded phosphatases play numerous roles during neurogenesis and axonogenesis in the vertebrate spinal cord.
INTRODUCTION During spinal cord development many cell types are born in a defined spatial and temporal order, followed by migration of these cells to appropriate positions and the extension of axons and dendrites. Fine control is therefore needed over the birth of adequate numbers of given cell types, their subsequent migration, and, finally, the extension of neurites to appropriate synaptic partners. Recent research into spinal cord patterning during development has identified many transcription factors characteristic of its cells in terms of both general spa-
1 To whom correspondence and reprint requests should be addressed. Fax: 0207 831 4366. E-mail: [email protected].
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tiotemporal qualities and specific subtypes (reviewed in Lumsden and Krumlauf, 1996; Tanabe and Jessell, 1996). The initial dorsoventral distinctions within the neural tube are regulated to a large degree by the action of Sonic hedgehog (Shh) (reviewed in Briscoe and Ericson, 1999). Regional expression of other transcription factors subsequently builds upon this specification, producing progressively more complex patterns of cell types. Members of the LIM homeodomain (LIM-HD) family of transcription factors are expressed in motorneurons where their combinatorial expression correlates with motorneuron subtype (Tsuchida et al., 1994; Fig. 1A). One member, Islet-1 (Isl1), is required for motorneuron generation and, consequently, associated interneurons (Pfaff et al., 1996). Other members serve to refine the subtype identity of motorneurons and their projection patterns (Sharma et al., 1998). Control of interneuron identity is less well established. The combinatorial expression of PAX2 by interneurons in various combinations with the LIM1/2 proteins, Engrailed-1 (EN-1) and EVX-1 defines distinct populations (Burrill et al., 1997). Additionally, GDF-7, a member of the BMP family of proteins is necessary for the generation of specific dorsal interneurons (Lee et al., 1998). The control of cell movement is also vitally important in development of the spinal cord. Cells initially migrate laterally from the ventricular zone along the midline and later turn along the dorsoventral axis as the neural tube matures (Leber et al., 1990; Leber and Sanes, 1995). Therefore, transcription factor combinations must maintain positional and functional identity during the cell mixing that occurs during cell migration. For example, EN-1 appears not to be required for initial specification of identity (Matise and Joyner, 1997) but instead regulates later interactions with the secreted factor netrin-1 and the formation of appropriate axonal 1044-7431/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Expression RPTP In Spinal Cord
trajectories (Saueressig et al., 1999). Secretion of retinoids by early-born lateral motor column (LMC) motorneurons is required for the induction of the lateral LMC (Sockanathan and Jessell, 1998; Berggen et al., 1999). There remains, however, a considerable empirical chasm between the expression patterns of these transcription factors and their ultimate link to expression of cell surface receptors. Such receptors are the essential interface with extracellular signals that control cell migration in the spinal cord as well as axon growth and guidance. There are several families of molecules with the potential to control axon guidance and cell migration (reviewed by Goodman, 1996). Cell adhesion molecules (CAMs), including the calcium-dependent cadherins and the calcium-independent neural cell adhesion molecules (N-CAMs), have been shown to regulate cell movements (Barami et al., 1994) as well as axon fasciculation and outgrowth. However, there is scant evidence that CAMs alone govern changes in growth cone direction and guidance specificity. Such control appears to require signals generated in part through protein tyrosine phosphorylation. Neurite growth and retraction and filopodial movement can all be perturbed by protein tyrosine kinase (PTK) inhibitors (Bixby and Jhabvala, 1992), while axons and growth cones contain numerous PTKs of receptor and nonreceptor (src-like) families (Bixby and Jhabvala, 1993). CAM-driven axonogenesis is dependent on the fibroblast growth factor receptor, which is a tyrosine kinase (Williams et al., 1994) and a null mutation of the Src PTK in mouse cerebellar neurons reduces the rate of axon growth in vitro on the CAM L1 (Ignelzi Jr. et al., 1994). Research into the Eph family of tyrosine kinases suggests axonal receptors directly control fasciculation and guidance based on growth cone repulsion (Drescher et al., 1995). One member of this family, Cek4, is found specifically on Lim3 ⫹ motorneurons and their muscle targets (Kilpatrick et al., 1996). In light of the marked dependence of signalling on phosphorylation, it is unsurprising that recent studies have shown receptor-like protein tyrosine phosphatases (RPTPs) to be crucially involved in neural development (reviewed in Stoker and Dutta, 1998 and Van Vactor, 1998) and in other aspects of development (reviewed in den Hertog et al., 1999). The structure of RPTPs suggests that they are an integral part of the interface between the extracellular environment of a cell and its intracellular signalling pathways. They all possess at least one, usually two, intracellular phosphatase domain. Their extracellular domains are more variable and distinctive but all contain various motifs implicated
471 in cell adhesion (Fig. 1B; reviewed in Brady-Kalnay and Tonks, 1995). The mode of action of these molecules, most of which are essentially orphan receptors, remains largely unknown and there exists a pressing need to characterize their expression patterns and establish functional ligands and roles for these molecules. The discovery of the DPTP99A and DPTP10D RPTP proteins on major nerve tracts of the Drosophila central nervous system (CNS) during axon outgrowth, provided the first strong hint that RPTPs may have a role in axonal signal transduction (Tian et al., 1991). Recent studies have also demonstrated a role for DPTP10D in controlling commissural axon guidance in the fly midline (Sun et al., 2000). Furthermore, loss-of-function of DPTP69D, DPTP99A, and Dlar, another Drosophila RPTP expressed on many CNS axons, produce specific pathfinding defects in which motor axons fail to defasciculate or recognise specific choice points (Desai et al., 1996; Krueger et al., 1996). Ptp69D, Ptp99A, and Dlar mutations affect overlapping subsets of motor axons, even though their protein products are widely distributed. Thus, further RPTPs must also be required to help guide other axons, with the function of each RPTP being tightly regulated, perhaps by restricted localisation of their ligands. In the chick, the type IIA RPTP, CRYP␣, a homologue of Dlar, is highly expressed in motor columns of the embryonic day (E)6 spinal cord (Stoker, 1994). The protein has been localised to growth cones (Stoker, 1995a) and CRYP␣ is also widely expressed in early axons of optic and sensory nerves (Stoker, 1995a,b). Indeed, CRYP␣ has recently been shown to promote growth of neurites from retinal ganglion cells (Ledig et al., 1999a). RPTP␦, a homologue of CRYP␣, is also expressed in mammalian motorneuron pools (Sommer et al., 1997) and this RPTP influences neurite outgrowth from chick forebrain neurons (Wang and Bixby, 1999). These findings support the likelihood that vertebrate RPTPs play roles in motor nerve development, possibly sharing conserved functions with the Drosophila proteins. To gain greater insight into the involvement of this protein family in spinal cord and specifically motorneuron development, we have carried out an extensive spatiotemporal analysis of RPTP expression. We show novel expression patterns of these genes within the embryonic avian spinal cord. Expression is dynamic and restricted to different subsets of motorneurons and other cells within the spinal cord, consistent with these genes having significant roles during neurogenesis and axonogenesis in the spinal cord.
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FIG. 1. (A) Schematic representation of the expression of LIM homeodomain proteins in the brachial motor pools of a stage 34 (E8) chick embryo, and their corresponding nerve projection patterns. (B) Structural representation of the subfamilies of RPTPs. Five RPTP subtypes (I–V) are shown, according to Brady-Kalnay and Tonks 1995; not all isoforms are included. For vertebrate type IIA RPTPs, two major alternative isoforms are shown with either 4 or 8 fibronectin type III repeats; only one form of Drosophila Dlar, with 9 repeats, has been identified. See Stoker and Dutta (1998) for further information.
RESULTS AND DISCUSSION Before designing experiments to address the developmental function of neural RPTPs, it is first necessary to describe their precise spatiotemporal expression in the embryo. Our principal interest here is in their potential roles in spinal motorneurons. The first brachial lateral motor column (LMC) motorneurons are born at Hamburger-Hamilton stage 15 (Hamburger and Hamilton, 1951), with 50% having been born by stage 20 (ca. E3). The columns are complete at stage 27 (ca. E5) but at least 40% of neurons die by stage 38 (ca. E12) due to trimming of synaptic contacts (Hollyday and Hamburger, 1977). Brachial motorneuron death continues until stage 44 (ca. E18) (Laing, 1982). mRNA in situ hybridization
studies were therefore carried out on brachial spinal cord sections from E4, E6, E8, and E10 embryos in order to examine expression at crucial developmental stages of late neurogenesis, axonogenesis, and maturation. To aid with the identification of cell types, immunohistochemistry was carried out on adjacent sections using antibodies to the LIM-HD proteins Isl1, Isl2, Lim1/2, and Lim3 (Tsuchida et al., 1994). At brachial levels, Isl1 marks the medial motor column (MMC) and the medial LMC (LMC m), Isl2 is found throughout the MMC and LMC. Lim1/2 allows distinction of the lateral LMC (LMC 1), while Lim3 identifies the medial MMC (MMC m). This is summarized in Fig. 1A. In addition, groups of interneurons are labeled, although these are yet to be functionally mapped in detail.
Expression RPTP In Spinal Cord
We examined the expression of RPTPs spread across the current structural classification (Fig. 1B; Neel and Tonks, 1997). The type IIa members CRYP␣ and RPTP␦ have already been introduced (Stoker, 1994; Sommer et al., 1997). The ectodomains of type IIB RPTPs, RPTP, and RPTP⌿ display similarity to immunoglobulin (Ig) superfamily members, such as N-CAM. RPTP binds homophilically (Brady-Kalnay et al., 1993) and associates with cadherin– catenin complexes (Brady-Kalnay et al., 1998), therefore possibly influencing the actin cytoskeleton. CRYP2 is a type III RPTP, found exclusively in the brain during the major period of active axon outgrowth in the chick forebrain (Bodden and Bixby, 1996). A close relative, DPTP10D, is CNS-specific (Tian et al., 1991) and has been shown to influence midline guidance of axons in Drosophila (Sun et al., 2000). The type IV RPTP␣ has a short, highly glycosylated extracellular domain and is strongly expressed in the brain (Matthews et al., 1990; Sap et al., 1990). Finally, the type V member, RPTP␥, has been implicated in haematopoiesis (Sorio et al., 1997) and sarcoma formation (Wary et al., 1993) and is developmentally regulated in the rat brain (Barnea et al., 1993) and avian retina (Ledig et al., 1999b).
RPTPs That Are Expressed in Motorneurons CRYP␣ is widely expressed in early axons, including those of the spinal cord and dorsal root ganglia (Stoker, 1994). There are two main isoforms of CRYP␣: CRYP␣1 and CRYP␣2, produced by alternatively spliced mRNAs but which are both recognised by the CRYP␣ riboprobe used here. Both isoforms have two intracellular phosphatase domains but they differ extracellularly in the number of fibronectin type III-like repeats adjacent to the membrane: CRYP␣1 has four while CRYP␣2 has eight (Fig. 1B). The CRYP␣ probe bound most strongly to the ventrolateral neural tube at E4 (Fig. 2E) overlapping closely with Isl1 in early-born motorneurons (Fig. 2A). Binding also occurred along the ventricular/subventricular zone and in a stripe of LIM1/2 ⫹ cells in the intermediate zone (compare site arrowed in Fig. 2E with 2C). From E6, strong expression could be seen in the LMC and MMC (arrowed) as they became distinguishable (Fig. 3E). Expression was maintained in the ventricular zone while the intermediate zone expression was replaced by a more generalized expression in scattered cells throughout the cord (Figs. 3E and 4D). High levels of CRYP␣ in both the MMC and LMC continued at E8 (Fig. 4D) and E10 (Fig. 5A).
473 A probe specific for the CRYP␣2 isoform produced a signal predominantly in the ventricular/subventricular zone from E4 onwards (Figs. 2I, 3I, 4H, and 5D). In addition, the intermediate cells recognized by the CRYP␣ probe at E4 also bound, albeit more weakly, the CRYP␣2 probe, suggesting that both isoforms are transiently expressed in these cells at this stage. The CRYP␣2 probe also bound to a few scattered cells in the dorsal and ventral horns, these being more evident from E8 onwards (Figs. 4H and 5D). This suggests that CRYP␣2 is predominantly involved in the maturation of nascent neurons and/or glia in the ventricular/subventricular region. Furthermore, it can be deduced that the ␣1 isoform is responsible for CRYP␣ action in most motorneurons of the ventral horn. This is the first demonstration of isoform-specific RPTP expression within the spinal cord. Last, the transient expression of CRYP␣ isoforms in Lim1/2 ⫹ cells may be a feature of Lim1/2 also controlling or initiating the expression of cell surface molecules required for axonal projection or cell migration. CRYP2 is expressed by neurons in the ventral neural tube at E4, corresponding closely with Isl1 expression (Fig. 2G). CRYP2 was originally described as being a neural-specific molecule. We have also seen strong expression in the developing kidney (Fig. 2G, inset), although this may be partly the result of cross-reactivity with the GLEPP-1 gene, a closely related RPTP found in rabbit glomeruli (Bodden and Bixby, 1996). As the ventral neuroepithelium matures, CRYP2 expression is refined to the LMC and is absent from the Lim3-expressing MMC (Figs. 3G, 4F, compare Figs. 3C, 4C). At thoracic levels, visceral preganglionic motorneurons also arise from the ventral neural tube and migrate to the column of Terni; CRYP2 expression is also seen in these cells (Fig. 4J). Furthermore, at E10, it remains in the preganglionic Terni neurons (Fig. 5B) but is absent or very weak in ventral motorneurons. Given that neuromuscular synaptogenesis has finished but that the Terni neurons are still forming contacts at this stage, this suggests a role for CRYP2 during the establishment of motorneuron contacts with their targets in both voluntary and involuntary muscle systems. This temporal regulation of expression correlates quite well with the development and synaptogenesis of the two classes of motorneuron, which arise together from the ventral neuroepithelium (Prasad and Hollyday, 1991). RPTP␣ expression is evident at E4 (Fig. 2F) and by E6 is defined in the LMC and around the floorplate and ventral midline (Fig. 3F). This persists at E8 (Fig. 4G), but is increasingly restricted over time to the lateral LMC (Fig. 5E). RPTP␣, being highly glycosylated, may
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FIG. 2. Expression of RPTPs in transverse sections of E4 chick brachial spinal cord. Immunocytochemical staining of (A) Islet1, (B) Islet 2, (C) Lim1/2, and (D) Lim3 protein expression. DIG-labeled mRNA in situ hybridization to (E) CRYP␣ in the ventricular zone, ventral horns, and intermediate zone (arrow); (F) RPTP␣ in the ventral horn and floorplate; (G) CRYP2 in the ventral neural tube and (inset) kidney; (H) RPTP␥ in the intermediate zone; (I) CRYP␣2 in the ventricular zone; (J) RPTP␦ diffusely across the neural tube; (K) RPTP⌿ in the lateral neural tube; (L) RPTP in the vasculature (arrows). Scale bar is 1 mm.
be involved in carbohydrate-lectin interactions (Matthews et al., 1990). It has been implicated in neuronal differentiation of certain cell lines via interaction with the PTK pp60 c-src (den Hertog et al., 1993), and in interactions with the Ras signalling pathway via the GRB-2 adapter protein (den Hertog et al., 1994). Perhaps most interestingly, in light of its expression pattern shown
here, RPTP␣ has been suggested to control movement of migrating granule cells along Bergmann glia fibres (Fang et al., 1996). Migrating Terni column cells interdigitate with the cells of the ventral ventricular epithelium (Prasad and Hollyday, 1991) and the expression of RPTP␣ along this route may provide a necessary substrate.
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FIG. 3. Expression of RPTPs in transverse sections of E6 chick brachial spinal cord. Immunocytochemical staining of (A) Islet1, (B) Lim1/2, and (C) Lim3 protein expression. DIG-labeled mRNA in situ hybridization to (D, H) RPTP␥ in the intermediate zone, above the floorplate (arrow, H) at thoracic levels (D) and in the dorsomedial LMC at brachial levels (H); (E) CRYP␣ in the ventricular zone, LMC and MMC (arrow); (F) RPTP␣ in the LMC and ventral midline; (G) CRYP2 in LMC; (I) CRYP␣2 in the ventricular zone; (J) RPTP␦ in the dorsal neural tube and between the MMC and floorplate (arrow); (K) RPTP⌿ in the lateral neural tube and floorplate (arrow); (L) RPTP in the vasculature. Scale bar is 1 mm.
The RPTP␥ probe reveals a striking, novel pattern of expression in three distinct areas of the neural tube. The first, a stripe running dorsoventrally along the intermediate zone, with strongest expression in the middle, appears at E4 (Fig. 2H) and closely maps to the pattern of Lim1/2 expression in the intermediate and dorsal neural tube (Fig. 2C). More specifically, this band of cells resembles the location of the subset of Lim1/2 ⫹ interneurons that are also PAX2 ⫹ (Burrill et al., 1997). This stripe persists at E6 (Fig. 3H) in a population of
interneurons still overlapping with Lim1/2 expression (Fig. 3B), although more restricted in its mediolateral extent. The second area of expression is evident at this stage. One is a well-delineated region within the dorsomedial LMC, suggesting a role in targeting to ventral limb muscles. The expression in motorneurons is interesting given that at E4, RPTP␥ expression barely overlaps with Isl1, except at the most medial extent of Isl1. The area of expression in motorneurons may be cells derived from this overlap. Alternatively, its expression
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FIG. 4. Expression of RPTPs in transverse sections of E8 chick brachial and thoracic (J) spinal cord. Immunocytochemical staining of (A) Islet1, (B) Lim1/2, and (C) Lim3 protein expression. DIG-labeled mRNA in situ hybridization to (D) CRYP␣ in the ventricular zone, LMC, and MMC (arrow); (E) RPTP␥ in the intermediate zone, above the floorplate and in the dorsomedial LMC (arrow); (F, J) CRYP2 in the LMC at brachial levels (F) and Terni column at thoracic level (arrow, J); (G) RPTP␣ in the LMC and ventral midline; (H) CRYP␣2 in the ventricular zone and scattered cells in the dorsal and ventral horns (arrows); (I) RPTP␦ concentrated in the intermediodorsal spinal cord; (K) RPTP⌿ at very low levels. Scale bar is 1 mm.
may arise de novo, once the motorneurons have reached this position. At more thoracic levels this spot of expression is lost, although the remaining expression pattern of RPTP␥ is otherwise unchanged (Fig. 3D), which would further suggest that the expression is in a limb motor pool. The third area of expression is a crescent above the floorplate, the site of oligodendrocyte birth (Ono et al., 1995). The expression pattern at E8 (Fig. 4E) closely resembles that at E6 but by E10 it reproducibly disappears to be replaced by a random, almost fibrous, pattern (Fig. 5C). In the case of the presumed oligodendrocyte expression this would tally with the time course of dispersal of these cells (Richardson et al., 1997). In terms of expression in motorneurons, the downregulation of RPTP␥ may correlate with the termination of a role in synaptogenesis or, less likely, may be due to the death of those cells during trimming of motor pools. There are four known rat isoforms of RPTP␥, RPTP␥A,B,C, and S (a secreted form) (Shintani et al., 1997). The
chick RPTP␥ probe used in this study recognises the membrane proximal phosphatase domain of RPTP␥ and so (if similar isoforms do exist in the chick) would detect transmembrane isoforms A, B, and C. RPTPs That Are Not Expressed in Motorneurons RPTP␦, a homologue of CRYP␣, is expressed in mammalian pyramidal cells (Mizuno et al., 1993) and motorneuron pools (Sommer et al., 1997). In the developing chick spinal cord it is diffusely expressed at E4 (Fig. 2J). The sense probe gave no signal, confirming that this is not a background effect. By E6, the expression pattern becomes organized into a strong dorsal region of expression (Fig. 3J), which correlates with the dorsal expression of Lim1/2 (Fig. 3B). RPTP␦ is also found in a ventromedial population of cells. Comparison with serial sections stained with antibodies against Isl1 (Fig. 3A) or Lim3 (Fig. 3C) suggests that this expression is not in the MMC but is in a population of cells lying
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FIG. 5. Expression of RPTPs in transverse sections of E10 chick brachial (A, C–F) and thoracic (B) spinal cord. DIG-labeled mRNA in situ hybridization to (A) CRYP␣ in the ventricular zone, LMC, and MMC; (B) CRYP2 in the Terni column; (C) RPTP␥ in a diffuse pattern; (D) CRYP␣2 in the ventricular zone and scattered cells in the dorsal and ventral horns (arrows); (E) RPTP␣ in the LMC (arrow) and ventral midline; (F) RPTP␦ concentrated in the intermediodorsal spinal cord. Scale bar is 1 mm.
between it and the floorplate. At E8 RPTP␦ is in a broad intermediodorsal band of cells and also diffusely spread in cells across the rest of the spinal cord (Fig. 4I). The expression is similar at E10 but notably absent from the motor pools and the ventricular zone (Fig. 5F). Four isoforms of murine RPTP␦ have been described and shown to be developmentally regulated as a collective in the central nervous system, but the expression of individual isoforms has not been addressed in the embryo (Mizuno et al., 1993, 1994). Our chick RPTP probe should pick up all the major, predicted mRNA isoforms. Therefore, expression of RPTP␦ in rodent motorneurons (Sommer et al., 1997) and its absence in these cells of the chick, highlights the fact that expression patterns of apparently orthologous RPTP genes are not necessarily conserved between vertebrate species. RPTP⌿ shares 67% amino acid identity with RPTP and is expressed in the floorplate and roofplate of the mouse spinal cord (Sommer et al., 1997). In contrast, it is expressed weakly by scattered chick spinal cord neurons at E4, predominantly in the lateral neural tube (Fig. 2K). The dorsal expression pattern at E6, though still weak (Fig. 3K), resembles that of RPTP␦ (Fig. 3J). In
477 light of the murine data, it is worth noting a focus of floor plate expression. By E8, expression is weak and in no obvious pattern (Fig. 4K). RPTP, a close relative of RPTP⌿ sharing 44% overall amino acid identity (McAndrew et al., 1998), is expressed in a striking pattern of cells in both dorsal and ventral halves of the Xenopus spinal cord (Johnson and Holt, 2000). RPTP, in contrast to its subfamily relatives RPTP⌿ and RPTP, is only expressed in the capillaries of the spinal cord at all stages studied (Figs. 2L, 3L, data not shown) congruent with the murine CNS localization (Fuchs et al., 1998). In summary, a spatiotemporal expression screen of RPTP genes has revealed that several family members, spanning most of the currently defined subtypes of RPTP, are expressed in motorneuron pools of the embryonic chick spinal cord. Such widespread expression in motorneurons indicates that RPTP functions may be conserved from Drosophila through to higher vertebrates. We also present the first evidence for isoformspecific expression in the vertebrate spinal cord, in the case of CRYP␣1 and CRYP␣2. The former is found in motorneurons, the latter appears to be restricted to the subventricular zone and may play a role in maturation of neurons and glia or provide a substrate for cells migrating out from the proliferative zone. These two isoforms are also differentially expressed within the developing chick retinotectal system (Haj et al., 1999), suggesting that the RPTP isoforms perform distinct temporal and spatial roles in the nervous system. Other RPTPs are produced in multiple isoforms, including RPTP␦ and RPTP␥, and it will be of interest to examine their specific developmental expression. The data obtained for RPTP␦ have demonstrated that the expression patterns are by no means always conserved between vertebrate species. This also appears to be the case for RPTP␥, where the striking expression pattern seen in the developing chick spinal cord is apparently not seen in the mouse at approximately equivalent developmental stages to those we have studied in the chick. In fact we have only seen expression in mouse motorneurons at E13.5 when it is expressed in a small population of cells at the ventrolateral edge of the spinal cord (J. Chilton, unpublished data). The data presented here are consistent with RPTPs playing a variety of roles in the patterning and function of the spinal cord. They are expressed at such times and locations as to be potentially involved in processes ranging from neuronal differentiation and maturation, to axon growth and guidance, through to synapse formation and maintenance. All bar one of the RPTPs we examined are expressed within neuronal or glial cells.
478 The early expression of CRYP␣2 implies that it is generically expressed by progenitor cells, possibly to regulate signalling between these cells before their progeny move out into the spinal cord. RPTP␣ may have a similar role, though the ventral restriction of its expression may reflect an interaction between certain subsets of progenitors or migrating cells. This would parallel the proposed role for RPTP␣ in influencing neuronal migration in the brain and retinotectal system (Fang et al., 1996; Ledig et al., 1999a). RPTP␥ may also play a role in cell migration or differentiation as it is expressed at the site of oligodendrocyte birth in the floor plate. However, in contrast to its possible role there, at a site of cell production, it is also expressed in a persistent stripe within the intermediate zone. This seems likely to result from a wave of expression by cells moving through that area, perhaps being necessary for the initial lateral migration from the ventricular zone before a choice to move along the dorsoventral axis is made. Gene expression appears to be switched off again as the neurons mature and move to their more lateral locations. Four of the RPTPs in this paper were found to be expressed in the ventral horn. CRYP␣ appears to be common to all motorneurons. Bearing in mind also its persistent expression in these cells from E4 right through to E10, it could initially act as a molecule utilised by motor axons for outgrowth and then later for synaptic maintenance. In contrast to CRYP␣, the CRYP2, RPTP␥, and RPTP␣ genes are expressed in specific subsets of motor pools. This raises a number of interesting possibilities regarding function. The localization of CRYP2, RPTP␥, and RPTP␣ in specific regions of the ventral horn suggests a role in guidance to the muscles innervated by these particular motor pools. We have not seen any complementary expression in the limb to suggest any homophilic interaction is occurring at axonal target sites. Also of note is the fact that none of these three molecules are found in the MMC at the brachial level examined. Therefore their expression appears to be linked to other factors specifying the LMC and thus neurons targeted to limb muscles. This specification could be either in response to locally secreted molecules or more directly by transcription factors that determine the limb field and the brachial segments of the spinal cord. Since their expression is maintained during the period of neuromuscular synaptogenesis, they could also be involved in regulating the site or event of synapse formation or in modulating synaptic strength. In conclusion, we have shown that there is appropriate spatiotemporal and isoform-specific distribution of
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RPTPs for them to play roles in the migration and synaptogenesis of embryonic chick spinal cord cells and in motor axonogenesis. These proteins would therefore form part of the combinatorial code of surface receptors on motorneuron subpopulations, acting in concert with CAMs and RPTKs. This provides the necessary groundwork for ongoing studies to elucidate the functional significance of this intriguing class of molecules.
EXPERIMENTAL METHODS In Situ mRNA Hybridization In situ mRNA hybridization was carried out based on previously published protocols (Ledig et al., 1999b). Details of the riboprobes for CRYP␣, CRYP␣2, CRYP2, RPTP␥, RPTP␣, RPTP⌿, and RPTP can be found in Ledig et al. (1999b). The probe for RPTP␦ was constructed from cDNA coding for amino acids 240 to 440 of chick RPTP␦ (gift from J. Bixby). This was subcloned into the HindIII and XbaI restriction sites of the pBluescript vector (Stratagene) and transcribed with T7 RNA polymerase. Immunohistochemistry The Isl1, Isl2, Lim1/2, Lim3 antibodies were obtained from the Developmental Studies Hybridoma Bank, University of Iowa, in supernatant form and were all used at a dilution of 1:5 in PBS with 3% BSA and 0.05% Triton X-100, with the exception of Isl1 which was diluted 1:100. Briefly, 10-m cryostat sections were rinsed in PBS, fixed in 4% paraformaldehyde and then preblocked with 1% BSA in PBS with 0.25% Triton X-100. The primary antibodies were added and incubated with the sections for 1 h, before being washed in PBS with 0.1% BSA and 0.05% Triton X-100. HRP-conjugated anti-mouse secondary antibodies (Dako) were added for 1 h, these were then washed off and the staining visualized with DAB.
ACKNOWLEDGMENTS This work was supported by a Wellcome Trust Studentship (046122/D/95/Z) for J. Chilton and by a Royal Society University Research Fellowship (A.W.S.). We thank John Bixby for kindly providing the PTP␦ and CRYP-2 probes, and Ivor Mason for providing the PTP␣, ␥, and probes. We are also grateful to R. Aricescu, I. McKinnell, and C. Paternotte for their critical reading of the manuscript.
Expression RPTP In Spinal Cord
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480 HPTP␦, is expressed in specialised regions of the brain and in the B-cell lineage. Mol. Cell Biol. 13: 5513–5523. Mizuno, K., Hasegawa, K., Ogimoto, M., Katagiri, T., and Yakura, H. (1994). Developmental regulation of gene expression for the MPTP␦ isoforms in the central nervous system and the immune system. FEBS Lett. 355: 223–228. Neel, B. G., and Tonks, N. K. (1997). Protein tyrosine phosphatases in signal transduction. Curr. Opin. Cell Biol. 9: 193–204. Ono, K., Bansal, R., Payne, J., Rutishauser, U., and Miller, R. H. (1995). Early development and dispersal of oligodendrocyte precursors in the embryonic chick spinal cord. Development 121: 1743–1754. Pfaff, S. L., Mendelsohn, M., Stewart, C. L., Edlund, T., and Jessell, T. M. (1996). Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell 84: 309 –320. Prasad, A., and Hollyday, M. (1991). Development and migration of avian sympathetic preganglionic neurons. J. Comp. Neurol. 307: 237–258. Richardson, W. D., Pringle, N. P., Yu, W-P., and Hall, A. C. (1997). Origins of spinal cord oligodendrocytes: Possible developmental and evolutionary relationships with motor neurons. Dev. Neurosci. 19: 58 – 68. Sap, J., D’Eustachio, P., Givol, D., and Schlessinger, J. (1990). Cloning and expression of a widely expressed receptor tyrosine phosphatase. Proc. Natl. Acad. Sci. USA 87: 6112– 6116. Saueressig, H., Burrill, J., and Goulding, M. (1999). Engrailed-1 and Netrin-1 regulate axon pathfinding by association interneurons that project to motor neurons. Development 126: 4201– 4212. Sharma, K., Sheng, H. Z., Lettieri, K., Li, H., Karavanov, A., Potter, S., Westphal, H., and Pfaff, S. L. (1998). LIM homeodomain factors Lhx3 and Lhx4 assign subtype identities for motorneurons. Cell 95: 817– 828. Shintani, T., Maeda, N., Nishiwaki, T., and Noda, M. (1997). Characterization of rat receptor-like protein tyrosine phosphatase gamma isoforms. Biochem. Biophys. Res. Commun. 230: 419 – 425. Sockanathan, S., and Jessell, T. M. (1998). Motor neuron-derived retinoid signalling specifies the subtype identity of spinal motor neurons. Cell 94: 503–514. Sommer, L., Rao, M., and Anderson, D. J. (1997). RPTP␦ and the novel protein tyrosine phosphatase RPTP⌿ are expressed in restricted regions of the developing central nervous system. Dev. Dyn. 208: 48 – 61. Sorio, C., Melotti, P., D’Arcangelo, D., Mendrola, J., Calabretta, B., Croce, C. M., and Huebner, K. (1997). Receptor protein tyrosine
Chilton and Stoker phosphatase gamma, Ptp␥, regulates haematopoietic differentiation. Blood 90: 49 –57. Stoker, A. W. (1994). Isoforms of a novel cell adhesion molecule-like protein tyrosine phosphatase are implicated in neural development. Mech. Dev. 46: 201–217. Stoker, A. W., Gehrig, B., Haj, F., and Bay, B-H. (1995a). Axonal localisation of the CAM-like tyrosine phosphatase CRYP␣: A signalling molecule of embryonic growth cones. Development 121: 1833–1844. Stoker, A. W., Gehrig, B., Newton, M. R., and Bay, B. H. (1995b). Comparative localisation of CRYP␣, a CAM-like tyrosine phosphatase and NgCAM in the developing chick visual system. Dev. Brain Res. 90: 129 –140. Stoker, A. W., and Dutta, R. (1998). Protein tyrosine phosphatases and neural development. Bioessays 20: 463– 472. Sun, Q., Bahri, S., Schmid, A., Chia, W., and Zinn, K. (2000). Receptor tyrosine phosphatases regulate axon guidance across the midline of the Drosophila embryo. Development 127: 801– 812. Tanabe, Y., and Jessell, T. M. (1996). Diversity and pattern in the developing spinal cord. Science 274: 1115–1122. Tian, S-S., Tsoulfas, P., and Zinn, K. (1991). Three receptor-linked protein-tyrosine phosphatases are selectively expressed on central nervous system axons in the Drosophila embryo. Cell 67: 675– 685. Tsuchida, T., Ensini, M., Morton, S. B., Baldassare, M., Edlund, T., Jessell, T. M., and Pfaff, S. L. (1994). Topographic organisation of embryonic motorneurons defined by expression of LIM homeobox genes. Cell 79: 957–970. Van Vactor, D. (1998). Protein tyrosine phosphatases in the developing nervous system. Curr. Opin. Cell Biol. 10: 174 –181. Wang, J., and Bixby, J. L. (1999). Receptor tyrosine phosphatase-delta is a homophilic, neurite-promoting cell adhesion molecule for CNS neurons. Mol. Cell Neurosci. 14: 370 –384. Wary, K. K., Lou, Z., Buchberg, A. M., Siracusa, L. D., Druck, T., LaForgia, S., and Huebner, K. (1993). A homozygous deletion within the carbonic anhydrase-like domain of the Ptprg gene in murine L-cells. Cancer Res. 53: 1498 –1502. Williams, E. J., Furness, J., Walsh, F. S., and Doherty, P. (1994). Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. Neuron 13: 583–594. Yang, X., Seow, K. T., Bahri, S. M., Oon, S. H., and Chia, W. (1991). Two Drosophila receptor-like tyrosine phosphatase genes are expressed in a subset of developing axons and pioneer neurons in the embryonic CNS. Cell 67: 661– 673. Received May 15, 2000 Revised June 22, 2000 Accepted June 27, 2000
Molecular and Cellular Neuroscience 16, 470 – 480 (2000) doi:10.1006/mcne.2000.0887, available online at http://www.idealibrary.com on
Expression of Receptor Protein Tyrosine Phosphatases in Embryonic Chick Spinal Cord John K. Chilton* ,† and Andrew W. Stoker* ,1 *Neural Development Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, United Kingdom; and †Department of Human Anatomy and Genetics, Oxford University, South Parks Road, Oxford OX1 3QX, United Kingdom
Receptor-like protein tyrosine phosphatases potentially play a crucial role in axon growth and targeting. We focus here on their role within the embryonic avian spinal cord, in particular the development and outgrowth of motorneurons. We have used in situ mRNA hybridization to examine the spatiotemporal expression of eight receptorlike protein tyrosine phosphatases and find that it is both dynamic and highly varied, including novel, isoform-specific expression patterns. CRYP␣1 is expressed in all of the ventral motorneuron pools, whereas CRYP2, RPTP␥, and RPTP␣ are only expressed in specific subsets of these neurons. CRYP␣2, RPTP⌿, and RPTP␦ are neuronally expressed elsewhere in the cord, but not in ventral motorneurons, whereas RPTP is unique in being restricted to capillaries. The developmentally regulated expression of these genes strongly suggests that the encoded phosphatases play numerous roles during neurogenesis and axonogenesis in the vertebrate spinal cord.
INTRODUCTION During spinal cord development many cell types are born in a defined spatial and temporal order, followed by migration of these cells to appropriate positions and the extension of axons and dendrites. Fine control is therefore needed over the birth of adequate numbers of given cell types, their subsequent migration, and, finally, the extension of neurites to appropriate synaptic partners. Recent research into spinal cord patterning during development has identified many transcription factors characteristic of its cells in terms of both general spa-
1 To whom correspondence and reprint requests should be addressed. Fax: 0207 831 4366. E-mail: [email protected].
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tiotemporal qualities and specific subtypes (reviewed in Lumsden and Krumlauf, 1996; Tanabe and Jessell, 1996). The initial dorsoventral distinctions within the neural tube are regulated to a large degree by the action of Sonic hedgehog (Shh) (reviewed in Briscoe and Ericson, 1999). Regional expression of other transcription factors subsequently builds upon this specification, producing progressively more complex patterns of cell types. Members of the LIM homeodomain (LIM-HD) family of transcription factors are expressed in motorneurons where their combinatorial expression correlates with motorneuron subtype (Tsuchida et al., 1994; Fig. 1A). One member, Islet-1 (Isl1), is required for motorneuron generation and, consequently, associated interneurons (Pfaff et al., 1996). Other members serve to refine the subtype identity of motorneurons and their projection patterns (Sharma et al., 1998). Control of interneuron identity is less well established. The combinatorial expression of PAX2 by interneurons in various combinations with the LIM1/2 proteins, Engrailed-1 (EN-1) and EVX-1 defines distinct populations (Burrill et al., 1997). Additionally, GDF-7, a member of the BMP family of proteins is necessary for the generation of specific dorsal interneurons (Lee et al., 1998). The control of cell movement is also vitally important in development of the spinal cord. Cells initially migrate laterally from the ventricular zone along the midline and later turn along the dorsoventral axis as the neural tube matures (Leber et al., 1990; Leber and Sanes, 1995). Therefore, transcription factor combinations must maintain positional and functional identity during the cell mixing that occurs during cell migration. For example, EN-1 appears not to be required for initial specification of identity (Matise and Joyner, 1997) but instead regulates later interactions with the secreted factor netrin-1 and the formation of appropriate axonal 1044-7431/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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trajectories (Saueressig et al., 1999). Secretion of retinoids by early-born lateral motor column (LMC) motorneurons is required for the induction of the lateral LMC (Sockanathan and Jessell, 1998; Berggen et al., 1999). There remains, however, a considerable empirical chasm between the expression patterns of these transcription factors and their ultimate link to expression of cell surface receptors. Such receptors are the essential interface with extracellular signals that control cell migration in the spinal cord as well as axon growth and guidance. There are several families of molecules with the potential to control axon guidance and cell migration (reviewed by Goodman, 1996). Cell adhesion molecules (CAMs), including the calcium-dependent cadherins and the calcium-independent neural cell adhesion molecules (N-CAMs), have been shown to regulate cell movements (Barami et al., 1994) as well as axon fasciculation and outgrowth. However, there is scant evidence that CAMs alone govern changes in growth cone direction and guidance specificity. Such control appears to require signals generated in part through protein tyrosine phosphorylation. Neurite growth and retraction and filopodial movement can all be perturbed by protein tyrosine kinase (PTK) inhibitors (Bixby and Jhabvala, 1992), while axons and growth cones contain numerous PTKs of receptor and nonreceptor (src-like) families (Bixby and Jhabvala, 1993). CAM-driven axonogenesis is dependent on the fibroblast growth factor receptor, which is a tyrosine kinase (Williams et al., 1994) and a null mutation of the Src PTK in mouse cerebellar neurons reduces the rate of axon growth in vitro on the CAM L1 (Ignelzi Jr. et al., 1994). Research into the Eph family of tyrosine kinases suggests axonal receptors directly control fasciculation and guidance based on growth cone repulsion (Drescher et al., 1995). One member of this family, Cek4, is found specifically on Lim3 ⫹ motorneurons and their muscle targets (Kilpatrick et al., 1996). In light of the marked dependence of signalling on phosphorylation, it is unsurprising that recent studies have shown receptor-like protein tyrosine phosphatases (RPTPs) to be crucially involved in neural development (reviewed in Stoker and Dutta, 1998 and Van Vactor, 1998) and in other aspects of development (reviewed in den Hertog et al., 1999). The structure of RPTPs suggests that they are an integral part of the interface between the extracellular environment of a cell and its intracellular signalling pathways. They all possess at least one, usually two, intracellular phosphatase domain. Their extracellular domains are more variable and distinctive but all contain various motifs implicated
471 in cell adhesion (Fig. 1B; reviewed in Brady-Kalnay and Tonks, 1995). The mode of action of these molecules, most of which are essentially orphan receptors, remains largely unknown and there exists a pressing need to characterize their expression patterns and establish functional ligands and roles for these molecules. The discovery of the DPTP99A and DPTP10D RPTP proteins on major nerve tracts of the Drosophila central nervous system (CNS) during axon outgrowth, provided the first strong hint that RPTPs may have a role in axonal signal transduction (Tian et al., 1991). Recent studies have also demonstrated a role for DPTP10D in controlling commissural axon guidance in the fly midline (Sun et al., 2000). Furthermore, loss-of-function of DPTP69D, DPTP99A, and Dlar, another Drosophila RPTP expressed on many CNS axons, produce specific pathfinding defects in which motor axons fail to defasciculate or recognise specific choice points (Desai et al., 1996; Krueger et al., 1996). Ptp69D, Ptp99A, and Dlar mutations affect overlapping subsets of motor axons, even though their protein products are widely distributed. Thus, further RPTPs must also be required to help guide other axons, with the function of each RPTP being tightly regulated, perhaps by restricted localisation of their ligands. In the chick, the type IIA RPTP, CRYP␣, a homologue of Dlar, is highly expressed in motor columns of the embryonic day (E)6 spinal cord (Stoker, 1994). The protein has been localised to growth cones (Stoker, 1995a) and CRYP␣ is also widely expressed in early axons of optic and sensory nerves (Stoker, 1995a,b). Indeed, CRYP␣ has recently been shown to promote growth of neurites from retinal ganglion cells (Ledig et al., 1999a). RPTP␦, a homologue of CRYP␣, is also expressed in mammalian motorneuron pools (Sommer et al., 1997) and this RPTP influences neurite outgrowth from chick forebrain neurons (Wang and Bixby, 1999). These findings support the likelihood that vertebrate RPTPs play roles in motor nerve development, possibly sharing conserved functions with the Drosophila proteins. To gain greater insight into the involvement of this protein family in spinal cord and specifically motorneuron development, we have carried out an extensive spatiotemporal analysis of RPTP expression. We show novel expression patterns of these genes within the embryonic avian spinal cord. Expression is dynamic and restricted to different subsets of motorneurons and other cells within the spinal cord, consistent with these genes having significant roles during neurogenesis and axonogenesis in the spinal cord.
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FIG. 1. (A) Schematic representation of the expression of LIM homeodomain proteins in the brachial motor pools of a stage 34 (E8) chick embryo, and their corresponding nerve projection patterns. (B) Structural representation of the subfamilies of RPTPs. Five RPTP subtypes (I–V) are shown, according to Brady-Kalnay and Tonks 1995; not all isoforms are included. For vertebrate type IIA RPTPs, two major alternative isoforms are shown with either 4 or 8 fibronectin type III repeats; only one form of Drosophila Dlar, with 9 repeats, has been identified. See Stoker and Dutta (1998) for further information.
RESULTS AND DISCUSSION Before designing experiments to address the developmental function of neural RPTPs, it is first necessary to describe their precise spatiotemporal expression in the embryo. Our principal interest here is in their potential roles in spinal motorneurons. The first brachial lateral motor column (LMC) motorneurons are born at Hamburger-Hamilton stage 15 (Hamburger and Hamilton, 1951), with 50% having been born by stage 20 (ca. E3). The columns are complete at stage 27 (ca. E5) but at least 40% of neurons die by stage 38 (ca. E12) due to trimming of synaptic contacts (Hollyday and Hamburger, 1977). Brachial motorneuron death continues until stage 44 (ca. E18) (Laing, 1982). mRNA in situ hybridization
studies were therefore carried out on brachial spinal cord sections from E4, E6, E8, and E10 embryos in order to examine expression at crucial developmental stages of late neurogenesis, axonogenesis, and maturation. To aid with the identification of cell types, immunohistochemistry was carried out on adjacent sections using antibodies to the LIM-HD proteins Isl1, Isl2, Lim1/2, and Lim3 (Tsuchida et al., 1994). At brachial levels, Isl1 marks the medial motor column (MMC) and the medial LMC (LMC m), Isl2 is found throughout the MMC and LMC. Lim1/2 allows distinction of the lateral LMC (LMC 1), while Lim3 identifies the medial MMC (MMC m). This is summarized in Fig. 1A. In addition, groups of interneurons are labeled, although these are yet to be functionally mapped in detail.
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We examined the expression of RPTPs spread across the current structural classification (Fig. 1B; Neel and Tonks, 1997). The type IIa members CRYP␣ and RPTP␦ have already been introduced (Stoker, 1994; Sommer et al., 1997). The ectodomains of type IIB RPTPs, RPTP, and RPTP⌿ display similarity to immunoglobulin (Ig) superfamily members, such as N-CAM. RPTP binds homophilically (Brady-Kalnay et al., 1993) and associates with cadherin– catenin complexes (Brady-Kalnay et al., 1998), therefore possibly influencing the actin cytoskeleton. CRYP2 is a type III RPTP, found exclusively in the brain during the major period of active axon outgrowth in the chick forebrain (Bodden and Bixby, 1996). A close relative, DPTP10D, is CNS-specific (Tian et al., 1991) and has been shown to influence midline guidance of axons in Drosophila (Sun et al., 2000). The type IV RPTP␣ has a short, highly glycosylated extracellular domain and is strongly expressed in the brain (Matthews et al., 1990; Sap et al., 1990). Finally, the type V member, RPTP␥, has been implicated in haematopoiesis (Sorio et al., 1997) and sarcoma formation (Wary et al., 1993) and is developmentally regulated in the rat brain (Barnea et al., 1993) and avian retina (Ledig et al., 1999b).
RPTPs That Are Expressed in Motorneurons CRYP␣ is widely expressed in early axons, including those of the spinal cord and dorsal root ganglia (Stoker, 1994). There are two main isoforms of CRYP␣: CRYP␣1 and CRYP␣2, produced by alternatively spliced mRNAs but which are both recognised by the CRYP␣ riboprobe used here. Both isoforms have two intracellular phosphatase domains but they differ extracellularly in the number of fibronectin type III-like repeats adjacent to the membrane: CRYP␣1 has four while CRYP␣2 has eight (Fig. 1B). The CRYP␣ probe bound most strongly to the ventrolateral neural tube at E4 (Fig. 2E) overlapping closely with Isl1 in early-born motorneurons (Fig. 2A). Binding also occurred along the ventricular/subventricular zone and in a stripe of LIM1/2 ⫹ cells in the intermediate zone (compare site arrowed in Fig. 2E with 2C). From E6, strong expression could be seen in the LMC and MMC (arrowed) as they became distinguishable (Fig. 3E). Expression was maintained in the ventricular zone while the intermediate zone expression was replaced by a more generalized expression in scattered cells throughout the cord (Figs. 3E and 4D). High levels of CRYP␣ in both the MMC and LMC continued at E8 (Fig. 4D) and E10 (Fig. 5A).
473 A probe specific for the CRYP␣2 isoform produced a signal predominantly in the ventricular/subventricular zone from E4 onwards (Figs. 2I, 3I, 4H, and 5D). In addition, the intermediate cells recognized by the CRYP␣ probe at E4 also bound, albeit more weakly, the CRYP␣2 probe, suggesting that both isoforms are transiently expressed in these cells at this stage. The CRYP␣2 probe also bound to a few scattered cells in the dorsal and ventral horns, these being more evident from E8 onwards (Figs. 4H and 5D). This suggests that CRYP␣2 is predominantly involved in the maturation of nascent neurons and/or glia in the ventricular/subventricular region. Furthermore, it can be deduced that the ␣1 isoform is responsible for CRYP␣ action in most motorneurons of the ventral horn. This is the first demonstration of isoform-specific RPTP expression within the spinal cord. Last, the transient expression of CRYP␣ isoforms in Lim1/2 ⫹ cells may be a feature of Lim1/2 also controlling or initiating the expression of cell surface molecules required for axonal projection or cell migration. CRYP2 is expressed by neurons in the ventral neural tube at E4, corresponding closely with Isl1 expression (Fig. 2G). CRYP2 was originally described as being a neural-specific molecule. We have also seen strong expression in the developing kidney (Fig. 2G, inset), although this may be partly the result of cross-reactivity with the GLEPP-1 gene, a closely related RPTP found in rabbit glomeruli (Bodden and Bixby, 1996). As the ventral neuroepithelium matures, CRYP2 expression is refined to the LMC and is absent from the Lim3-expressing MMC (Figs. 3G, 4F, compare Figs. 3C, 4C). At thoracic levels, visceral preganglionic motorneurons also arise from the ventral neural tube and migrate to the column of Terni; CRYP2 expression is also seen in these cells (Fig. 4J). Furthermore, at E10, it remains in the preganglionic Terni neurons (Fig. 5B) but is absent or very weak in ventral motorneurons. Given that neuromuscular synaptogenesis has finished but that the Terni neurons are still forming contacts at this stage, this suggests a role for CRYP2 during the establishment of motorneuron contacts with their targets in both voluntary and involuntary muscle systems. This temporal regulation of expression correlates quite well with the development and synaptogenesis of the two classes of motorneuron, which arise together from the ventral neuroepithelium (Prasad and Hollyday, 1991). RPTP␣ expression is evident at E4 (Fig. 2F) and by E6 is defined in the LMC and around the floorplate and ventral midline (Fig. 3F). This persists at E8 (Fig. 4G), but is increasingly restricted over time to the lateral LMC (Fig. 5E). RPTP␣, being highly glycosylated, may
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FIG. 2. Expression of RPTPs in transverse sections of E4 chick brachial spinal cord. Immunocytochemical staining of (A) Islet1, (B) Islet 2, (C) Lim1/2, and (D) Lim3 protein expression. DIG-labeled mRNA in situ hybridization to (E) CRYP␣ in the ventricular zone, ventral horns, and intermediate zone (arrow); (F) RPTP␣ in the ventral horn and floorplate; (G) CRYP2 in the ventral neural tube and (inset) kidney; (H) RPTP␥ in the intermediate zone; (I) CRYP␣2 in the ventricular zone; (J) RPTP␦ diffusely across the neural tube; (K) RPTP⌿ in the lateral neural tube; (L) RPTP in the vasculature (arrows). Scale bar is 1 mm.
be involved in carbohydrate-lectin interactions (Matthews et al., 1990). It has been implicated in neuronal differentiation of certain cell lines via interaction with the PTK pp60 c-src (den Hertog et al., 1993), and in interactions with the Ras signalling pathway via the GRB-2 adapter protein (den Hertog et al., 1994). Perhaps most interestingly, in light of its expression pattern shown
here, RPTP␣ has been suggested to control movement of migrating granule cells along Bergmann glia fibres (Fang et al., 1996). Migrating Terni column cells interdigitate with the cells of the ventral ventricular epithelium (Prasad and Hollyday, 1991) and the expression of RPTP␣ along this route may provide a necessary substrate.
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FIG. 3. Expression of RPTPs in transverse sections of E6 chick brachial spinal cord. Immunocytochemical staining of (A) Islet1, (B) Lim1/2, and (C) Lim3 protein expression. DIG-labeled mRNA in situ hybridization to (D, H) RPTP␥ in the intermediate zone, above the floorplate (arrow, H) at thoracic levels (D) and in the dorsomedial LMC at brachial levels (H); (E) CRYP␣ in the ventricular zone, LMC and MMC (arrow); (F) RPTP␣ in the LMC and ventral midline; (G) CRYP2 in LMC; (I) CRYP␣2 in the ventricular zone; (J) RPTP␦ in the dorsal neural tube and between the MMC and floorplate (arrow); (K) RPTP⌿ in the lateral neural tube and floorplate (arrow); (L) RPTP in the vasculature. Scale bar is 1 mm.
The RPTP␥ probe reveals a striking, novel pattern of expression in three distinct areas of the neural tube. The first, a stripe running dorsoventrally along the intermediate zone, with strongest expression in the middle, appears at E4 (Fig. 2H) and closely maps to the pattern of Lim1/2 expression in the intermediate and dorsal neural tube (Fig. 2C). More specifically, this band of cells resembles the location of the subset of Lim1/2 ⫹ interneurons that are also PAX2 ⫹ (Burrill et al., 1997). This stripe persists at E6 (Fig. 3H) in a population of
interneurons still overlapping with Lim1/2 expression (Fig. 3B), although more restricted in its mediolateral extent. The second area of expression is evident at this stage. One is a well-delineated region within the dorsomedial LMC, suggesting a role in targeting to ventral limb muscles. The expression in motorneurons is interesting given that at E4, RPTP␥ expression barely overlaps with Isl1, except at the most medial extent of Isl1. The area of expression in motorneurons may be cells derived from this overlap. Alternatively, its expression
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FIG. 4. Expression of RPTPs in transverse sections of E8 chick brachial and thoracic (J) spinal cord. Immunocytochemical staining of (A) Islet1, (B) Lim1/2, and (C) Lim3 protein expression. DIG-labeled mRNA in situ hybridization to (D) CRYP␣ in the ventricular zone, LMC, and MMC (arrow); (E) RPTP␥ in the intermediate zone, above the floorplate and in the dorsomedial LMC (arrow); (F, J) CRYP2 in the LMC at brachial levels (F) and Terni column at thoracic level (arrow, J); (G) RPTP␣ in the LMC and ventral midline; (H) CRYP␣2 in the ventricular zone and scattered cells in the dorsal and ventral horns (arrows); (I) RPTP␦ concentrated in the intermediodorsal spinal cord; (K) RPTP⌿ at very low levels. Scale bar is 1 mm.
may arise de novo, once the motorneurons have reached this position. At more thoracic levels this spot of expression is lost, although the remaining expression pattern of RPTP␥ is otherwise unchanged (Fig. 3D), which would further suggest that the expression is in a limb motor pool. The third area of expression is a crescent above the floorplate, the site of oligodendrocyte birth (Ono et al., 1995). The expression pattern at E8 (Fig. 4E) closely resembles that at E6 but by E10 it reproducibly disappears to be replaced by a random, almost fibrous, pattern (Fig. 5C). In the case of the presumed oligodendrocyte expression this would tally with the time course of dispersal of these cells (Richardson et al., 1997). In terms of expression in motorneurons, the downregulation of RPTP␥ may correlate with the termination of a role in synaptogenesis or, less likely, may be due to the death of those cells during trimming of motor pools. There are four known rat isoforms of RPTP␥, RPTP␥A,B,C, and S (a secreted form) (Shintani et al., 1997). The
chick RPTP␥ probe used in this study recognises the membrane proximal phosphatase domain of RPTP␥ and so (if similar isoforms do exist in the chick) would detect transmembrane isoforms A, B, and C. RPTPs That Are Not Expressed in Motorneurons RPTP␦, a homologue of CRYP␣, is expressed in mammalian pyramidal cells (Mizuno et al., 1993) and motorneuron pools (Sommer et al., 1997). In the developing chick spinal cord it is diffusely expressed at E4 (Fig. 2J). The sense probe gave no signal, confirming that this is not a background effect. By E6, the expression pattern becomes organized into a strong dorsal region of expression (Fig. 3J), which correlates with the dorsal expression of Lim1/2 (Fig. 3B). RPTP␦ is also found in a ventromedial population of cells. Comparison with serial sections stained with antibodies against Isl1 (Fig. 3A) or Lim3 (Fig. 3C) suggests that this expression is not in the MMC but is in a population of cells lying
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FIG. 5. Expression of RPTPs in transverse sections of E10 chick brachial (A, C–F) and thoracic (B) spinal cord. DIG-labeled mRNA in situ hybridization to (A) CRYP␣ in the ventricular zone, LMC, and MMC; (B) CRYP2 in the Terni column; (C) RPTP␥ in a diffuse pattern; (D) CRYP␣2 in the ventricular zone and scattered cells in the dorsal and ventral horns (arrows); (E) RPTP␣ in the LMC (arrow) and ventral midline; (F) RPTP␦ concentrated in the intermediodorsal spinal cord. Scale bar is 1 mm.
between it and the floorplate. At E8 RPTP␦ is in a broad intermediodorsal band of cells and also diffusely spread in cells across the rest of the spinal cord (Fig. 4I). The expression is similar at E10 but notably absent from the motor pools and the ventricular zone (Fig. 5F). Four isoforms of murine RPTP␦ have been described and shown to be developmentally regulated as a collective in the central nervous system, but the expression of individual isoforms has not been addressed in the embryo (Mizuno et al., 1993, 1994). Our chick RPTP probe should pick up all the major, predicted mRNA isoforms. Therefore, expression of RPTP␦ in rodent motorneurons (Sommer et al., 1997) and its absence in these cells of the chick, highlights the fact that expression patterns of apparently orthologous RPTP genes are not necessarily conserved between vertebrate species. RPTP⌿ shares 67% amino acid identity with RPTP and is expressed in the floorplate and roofplate of the mouse spinal cord (Sommer et al., 1997). In contrast, it is expressed weakly by scattered chick spinal cord neurons at E4, predominantly in the lateral neural tube (Fig. 2K). The dorsal expression pattern at E6, though still weak (Fig. 3K), resembles that of RPTP␦ (Fig. 3J). In
477 light of the murine data, it is worth noting a focus of floor plate expression. By E8, expression is weak and in no obvious pattern (Fig. 4K). RPTP, a close relative of RPTP⌿ sharing 44% overall amino acid identity (McAndrew et al., 1998), is expressed in a striking pattern of cells in both dorsal and ventral halves of the Xenopus spinal cord (Johnson and Holt, 2000). RPTP, in contrast to its subfamily relatives RPTP⌿ and RPTP, is only expressed in the capillaries of the spinal cord at all stages studied (Figs. 2L, 3L, data not shown) congruent with the murine CNS localization (Fuchs et al., 1998). In summary, a spatiotemporal expression screen of RPTP genes has revealed that several family members, spanning most of the currently defined subtypes of RPTP, are expressed in motorneuron pools of the embryonic chick spinal cord. Such widespread expression in motorneurons indicates that RPTP functions may be conserved from Drosophila through to higher vertebrates. We also present the first evidence for isoformspecific expression in the vertebrate spinal cord, in the case of CRYP␣1 and CRYP␣2. The former is found in motorneurons, the latter appears to be restricted to the subventricular zone and may play a role in maturation of neurons and glia or provide a substrate for cells migrating out from the proliferative zone. These two isoforms are also differentially expressed within the developing chick retinotectal system (Haj et al., 1999), suggesting that the RPTP isoforms perform distinct temporal and spatial roles in the nervous system. Other RPTPs are produced in multiple isoforms, including RPTP␦ and RPTP␥, and it will be of interest to examine their specific developmental expression. The data obtained for RPTP␦ have demonstrated that the expression patterns are by no means always conserved between vertebrate species. This also appears to be the case for RPTP␥, where the striking expression pattern seen in the developing chick spinal cord is apparently not seen in the mouse at approximately equivalent developmental stages to those we have studied in the chick. In fact we have only seen expression in mouse motorneurons at E13.5 when it is expressed in a small population of cells at the ventrolateral edge of the spinal cord (J. Chilton, unpublished data). The data presented here are consistent with RPTPs playing a variety of roles in the patterning and function of the spinal cord. They are expressed at such times and locations as to be potentially involved in processes ranging from neuronal differentiation and maturation, to axon growth and guidance, through to synapse formation and maintenance. All bar one of the RPTPs we examined are expressed within neuronal or glial cells.
478 The early expression of CRYP␣2 implies that it is generically expressed by progenitor cells, possibly to regulate signalling between these cells before their progeny move out into the spinal cord. RPTP␣ may have a similar role, though the ventral restriction of its expression may reflect an interaction between certain subsets of progenitors or migrating cells. This would parallel the proposed role for RPTP␣ in influencing neuronal migration in the brain and retinotectal system (Fang et al., 1996; Ledig et al., 1999a). RPTP␥ may also play a role in cell migration or differentiation as it is expressed at the site of oligodendrocyte birth in the floor plate. However, in contrast to its possible role there, at a site of cell production, it is also expressed in a persistent stripe within the intermediate zone. This seems likely to result from a wave of expression by cells moving through that area, perhaps being necessary for the initial lateral migration from the ventricular zone before a choice to move along the dorsoventral axis is made. Gene expression appears to be switched off again as the neurons mature and move to their more lateral locations. Four of the RPTPs in this paper were found to be expressed in the ventral horn. CRYP␣ appears to be common to all motorneurons. Bearing in mind also its persistent expression in these cells from E4 right through to E10, it could initially act as a molecule utilised by motor axons for outgrowth and then later for synaptic maintenance. In contrast to CRYP␣, the CRYP2, RPTP␥, and RPTP␣ genes are expressed in specific subsets of motor pools. This raises a number of interesting possibilities regarding function. The localization of CRYP2, RPTP␥, and RPTP␣ in specific regions of the ventral horn suggests a role in guidance to the muscles innervated by these particular motor pools. We have not seen any complementary expression in the limb to suggest any homophilic interaction is occurring at axonal target sites. Also of note is the fact that none of these three molecules are found in the MMC at the brachial level examined. Therefore their expression appears to be linked to other factors specifying the LMC and thus neurons targeted to limb muscles. This specification could be either in response to locally secreted molecules or more directly by transcription factors that determine the limb field and the brachial segments of the spinal cord. Since their expression is maintained during the period of neuromuscular synaptogenesis, they could also be involved in regulating the site or event of synapse formation or in modulating synaptic strength. In conclusion, we have shown that there is appropriate spatiotemporal and isoform-specific distribution of
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RPTPs for them to play roles in the migration and synaptogenesis of embryonic chick spinal cord cells and in motor axonogenesis. These proteins would therefore form part of the combinatorial code of surface receptors on motorneuron subpopulations, acting in concert with CAMs and RPTKs. This provides the necessary groundwork for ongoing studies to elucidate the functional significance of this intriguing class of molecules.
EXPERIMENTAL METHODS In Situ mRNA Hybridization In situ mRNA hybridization was carried out based on previously published protocols (Ledig et al., 1999b). Details of the riboprobes for CRYP␣, CRYP␣2, CRYP2, RPTP␥, RPTP␣, RPTP⌿, and RPTP can be found in Ledig et al. (1999b). The probe for RPTP␦ was constructed from cDNA coding for amino acids 240 to 440 of chick RPTP␦ (gift from J. Bixby). This was subcloned into the HindIII and XbaI restriction sites of the pBluescript vector (Stratagene) and transcribed with T7 RNA polymerase. Immunohistochemistry The Isl1, Isl2, Lim1/2, Lim3 antibodies were obtained from the Developmental Studies Hybridoma Bank, University of Iowa, in supernatant form and were all used at a dilution of 1:5 in PBS with 3% BSA and 0.05% Triton X-100, with the exception of Isl1 which was diluted 1:100. Briefly, 10-m cryostat sections were rinsed in PBS, fixed in 4% paraformaldehyde and then preblocked with 1% BSA in PBS with 0.25% Triton X-100. The primary antibodies were added and incubated with the sections for 1 h, before being washed in PBS with 0.1% BSA and 0.05% Triton X-100. HRP-conjugated anti-mouse secondary antibodies (Dako) were added for 1 h, these were then washed off and the staining visualized with DAB.
ACKNOWLEDGMENTS This work was supported by a Wellcome Trust Studentship (046122/D/95/Z) for J. Chilton and by a Royal Society University Research Fellowship (A.W.S.). We thank John Bixby for kindly providing the PTP␦ and CRYP-2 probes, and Ivor Mason for providing the PTP␣, ␥, and probes. We are also grateful to R. Aricescu, I. McKinnell, and C. Paternotte for their critical reading of the manuscript.
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