Two receptor tyrosine phosphatases of the LAR family are expressed in the developing leech by specific central neurons as well as select peripheral neurons, muscles, and other cells

Abstract

Receptor protein tyrosine phosphatases (rPTPs) are thought to play a crucial role in neuronal development, particularly in pathfinding by growing processes. We have cloned and sequenced two Hirudo medicinalis rPTPs that are homologous to the Drosophila and vertebrate rPTPs of the Leukocyte common antigen-related (LAR) subfamily. These Hirudo rPTPs, HmLAR1 and HmLAR2, are products of different, homologous genes, both containing two tandem intracellular phosphatase domains and ectodomains with three tandem Ig domains and different numbers of tandem fibronectin type III (FIII) domains. They are expressed in distinct patterns during embryogenesis. HmLAR1 mRNA is expressed by a subset of central and peripheral neurons and by several peripheral muscular structures, whereas HmLAR2 mRNA is expressed by a different subset of central neurons and by the peripheral, neuron-like Comb cells. HmLAR1 and HmLAR2 proteins are located on the neurites of central neurons. In addition, HmLAR2 is expressed on the cell body, processes, and growth cones of the Comb cells. Because of their CAM-like ectodomains and homology to proteins known to be involved in pathfinding and because they are expressed by different subsets of neurons, we hypothesize that HmLAR1 and HmLAR2 participate in navigational decisions that distinguish the sets of neurons that express them. Furthermore, we hypothesize that HmLAR2 is also involved in setting up the highly regular array of parallel processes established by the Comb cells. Lastly, we propose that the HmLAR1 ectodomain on peripheral muscle cells plays a role in target recognition via interactions with neuronal receptors, which might include HmLAR1 or HmLAR2.

Fig. 1.

The deduced amino acid sequences of HmLAR1 and HmLAR2, aligned with the Bestfit program from the GCG software package.Dashes represent conserved residues, whereasdots represent gaps introduced into the sequence to allow the alignment of corresponding regions. The putative signal sequences of ∼30 amino acids are shown boxed at the start of the ORF. The initiating methionine is the most 5′-methionine in the open reading frame and is preceded by stop codons in all three reading frames. Conserved cysteine sites of the three Ig domains are marked by arrowheads, the 5′-ends of the fibronectin type III repeat domain (FNIII domain) and of the two catalytic phosphatase domains (PTPase 1 andPTPase 2) are indicated by arrows, and the transmembrane domain is bracketed.

Fig. 2.

The number of central neurons that express HmLAR1 mRNA increases with the stage of development. Shown here is labeling using full-length HmLAR1 antisense probes of embryos at E7 and E10, as well as a series of images of individual midbody ganglia taken at three focal planes and from embryos at three different ages. Identical labeling was observed with probes transcribed from either intracellular or extracellular cDNA template, and no labeling was seen with sense RNA probes (data not shown). A, At E7, HmLAR1 is expressed in the CNS and in two flanking, stripe-like domains. Note that expression is highest in the more anterior, more developed segments. Staining of the cryptolarval mouth (MO), a transient structure, is probably artifactual. B, At E10, the general pattern of HmLAR1 expression is preserved, and mature structures such as the heart tubes (HT), nephridia (N), and tail sucker (TS) have developed. C–E, At E7, five pairs of central neurons labeled strongly, two pairs close together in the anterodorsal region of the ganglion (C), one pair near the posterior margin, midway along the dorsoventral axis (D), and two pairs near the ventral surface (E).F–H, By E10, high levels of expression were observed in ∼12 pairs of central neurons distributed throughout the ganglia.I–K, By E17, a few additional cells expressed HmLAR1, although the rate of increase was much less than was that between E7 and E10. Anterior is up. Scale bars, 10 μm.

Fig. 3.

HmLAR1 mRNA can be detected in specific peripheral neurons and muscles by in situ hybridization.A, Approximately one and a half segments of an E10 preparation are shown. HmLAR1 expression was observed in two bilateral lines of cells (arrows) in the body wall, one of the groups aligned ventral to the heart tubes (HT) and the other more lateral. From their positions, these cells are likely to be Lan 3–6-expressing peripheral neurons (see Results). Out-of-focus ganglia of the ventral nerve cord are noted byCNS. B, Labeled neurons are easily discernible (arrows) in the small anterior root ganglia (ARG) in this image from an E17 embryo. Nomarski optics were used to enhance the visualization of the root nerves of the midbody ganglion at the center as well as at the ARG. C, HmLAR1 was also expressed by the bilateral, segmentally iterated nephridial neuron (white arrowhead). In this image from an E25 embryo, the urinary bladder (Bl) was visualized using Nomarski optics. D, Low power view of the fifth and sixth body segments of an E17 embryo is shown. HmLAR1 was found to be expressed by two lateral bands of cells (arrowheads) corresponding to the dorsoventral muscles (see G,H), by the lateral HT and the muscular portion of the secondary vasculature that connects to them (arrow), and by cells in the muscular portion of the male genitalia, probably the penile sheath (male symbol). In contrast, no HmLAR1 expression by cells of the female genitalia (female symbol) was detected. E, Image of approximately one and a half segments of an E25 preparation is shown. The number of HmLAR1-expressing cells in the body wall (white arrow) had increased significantly relative to that observed at E10 (compare with A). HmLAR1 was expressed late in embryogenesis by a regular array of cells on each annulus that appear to correspond to Lan 3–6-expressing sensory neurons. HmLAR1 expression by the dorsoventral muscles (black arrowheads), by the nephridial (N) neurons (white arrowheads), and by the HT and secondary vasculature (black arrows), as well as by theCNS (out of focal plane), persisted at this late stage.F, The tail and posterior-most midbody segment of an E11 embryo are shown. HmLAR1 expression is evident in the muscle cells of the tail sucker (TS). G,H, High magnification views of a lateral region of the body wall of an E17 embryo are shown. The HmLAR1-expressing cells of the medial (G) orlateral (H) groups of dorsoventral muscles are in focus and show a strong signal in the perinuclear domain and a weaker one more distally along the muscle fibers. The HT is also clearly visible to theright; a muscular tributary (G,arrow) is also visible. Anterior is up. Scale bars, 10 μm.

Fig. 4.

HmLAR1 immunoreactivity in the CNS and periphery.A, Immunofluorescent labeling of HmLAR1 in the four most anterior ganglia forming in an E7 animal. Note that HmLAR1 antisera did not label somata but strongly labeled fibers in the anterior and posterior commissures (arrowheads) and in the interganglionic connective nerves (arrows).B, HmLAR1 staining of an E11 segmental ganglion. Most staining was concentrated at the intersections of commissural and longitudinal fiber tracts. Labeled fibers, however, can also be clearly seen to enter or exit the ganglia through the anterior and posterior nerve roots (arrowheads). C, An E11 segmental ganglion after 24 hr of treatment with brefeldin-A. Two bilateral neuronal cell bodies were prominently labeled in the dorsal anterior cell packet (arrows). D, HmLAR1 immunolabeling of two segments of an E14 embryo, visualized with peroxidase staining. Labeling can be seen in the fiber tracts of the two segmental ganglia, in the interganglionic and peripheral nerves, and on the lateral heart tubes (HT), dorsoventral muscle fibers (arrowheads), and bilateral nephridial ducts (arrows). Anterior isup. Scale bars: A–C, 10 μm;D, 200 μm.

Fig. 5.

HmLAR2 mRNA is detectable by in situ hybridization in a small number of central neurons and in the Comb cells (no signal was observed from sense HmLAR2 probes; data not shown). A, B, At E7, HmLAR2 mRNA was expressed in three small clusters of central neurons in anterior midbody ganglia. A shows a more ventral focal plane of this ganglion than does B, Dotted lineshighlight the margins of the ganglion.C–E, At E14, HmLAR2 continued to be expressed by the same clusters of neurons at high levels, but at least one more cluster, as well as some cells expressing lower levels, could be detected. Thepanels progress from a more ventral (C) to a more dorsal (E) plane of focus through this midbody ganglion. F, HmLAR2 mRNA probes also labeled the paired Comb cells (white arrows) in the ventral body wall. Shown here are several anterior segments of an E14 embryo. Anterior isup. Scale bars: A–E, 10 μm;F, 100 μm.

Fig. 6.

HmLAR2 protein localization in the CNS as revealed by immunohistochemistry. A, Antibodies to HmLAR2-labeled cell bodies of two pairs of neurons (arrowheads) in this midbody ganglion from an E7 embryo are shown. These neurons correspond positionally with the most anterior and posterior pairs of neurons labeled by in situ hybridization (see Fig. 5).B, A more dorsal view of the same ganglion reveals more labeled processes. C, In E7 preparations processed for anti-HmLAR2 immunohistochemistry without Triton X-100 permeabilization, tracts of fibers were labeled, but somata were not. Shown here is an optical section through the neuropil of a midbody ganglion at approximately the same level shown in A.D, A more dorsal view of the ganglion shown inC is given at approximately the same level shown inB. E, By E9, HmLAR2 protein was detected on neuronal processes exiting the CNS through the nerve roots (arrowheads) as well as on tracts in the neuropil and interganglionic connective nerves. This midbody ganglion was processed without Triton X-100 permeabilization. F–H, At E12, a subset of somata labeled by in situ hybridization with HmLAR2 probes was also labeled by anti-HmLAR2 immunohistochemistry (arrowheads). Panels progress from ventral (F) to dorsal (H). There was no time point in which somata of all neurons expressing HmLAR2 at high levels were simultaneously immunolabeled. To label somata, we permeabilized tissue with Triton X-100. Note that the longitudinal tracts in the neuropil are strongly labeled and that somatic labeling, as expected, is cytoplasmic but not nuclear. Tracts in the roots were also strongly labeled but not in the focal planes shown. Anterior isup. Scale bars, 10 μm.

Fig. 7.

HmLAR2 antisera labeling peripheral processes of central neurons. A, Labeled by antisera raised against HmLAR2, an axon (arrowhead) from the 10th midbody ganglion of an E14 embryo could be followed from the CNS(data not shown) to the heart tube (HT). HmLAR1 antisera similarly labeled axons innervating the heart tubes.B, Fluorescence image of the preparation shown inA, which was also processed with antisera for the neuropeptide FMRFamide and a fluorescent secondary, is shown. Note that the axon labeled in A is labeled here as well (arrowheads). Anterior is up. Scale bars, 10 μm.

Fig. 8.

HmLAR2 protein can be observed in the Comb cells, in the Comb cell processes, and in their growth cones.A, Comb cells (CC) from the 15th midbody segment of an E7 embryo were labeled by antisera raised against HmLAR2. Labeling was perinuclear, and no fine processes can be seen.B, More developed CC from the fourth midbody segment of the same preparation shown in A were also labeled by HmLAR2 antisera. These more mature cells have elaborated short, fine processes in which HmLAR2 was detected (arrowheads). C, At E9, HmLAR2 antisera continued to label the CC body, and fine processes (arrowheads) were more easily discerned. Growth cones are labeled lightly, at approximately the same intensity that processes are labeled. D, In an unpermeabilized E9 preparation, HmLAR2 antibodies can be observed to label the surface of a Comb cell. Labeling is excluded from the cytoplasm, indicating that only extracellular proteins were available to the primary antibody. Both processes (black arrowheads) and growth cones (white arrowhead) were also labeled. E, At E11, HmLAR2 antisera labeled somata (CC), processes (black arrowheads), and growth cones (white arrowheads) of the Comb cells. Growth cones marked bywhite arrowheads are on processes extending posteriorly and across the ventral midline from contralateral Comb cells. Note that at this stage the growth cones label more strongly than do the processes they lead. F, At E14, HmLAR2 antibody labeling could be resolved on filopodia (arrowheads) extending from Comb cell growth cones (black arrowheads). Additionally, HmLAR2 antibodies also labeled fine, filopodia-like extensions that emanated from the shafts of the Comb cell processes. Anterior is up. Scale bars, 10 μm.