Receptor tyrosine phosphatases regulate birth order-dependent axonal fasciculation and midline repulsion during development of the Drosophila mushroom body

Receptor tyrosine phosphatases (RPTPs) are required for axon guidance during embryonic development in Drosophila. Here we examine the roles of four RPTPs during development of the larval mushroom body (MB). MB neurons extend axons into parallel tracts known as the peduncle and lobes. The temporal order of neuronal birth is reflected in the organization of axons within these tracts. Axons of the youngest neurons, known as core fibers, extend within a single bundle at the center, while those of older neurons fill the outer layers. RPTPs are selectively expressed on the core fibers of the MB. Ptp10D and Ptp69D regulate segregation of the young axons into a single core bundle. Ptp69D signaling is required for axonal extension beyond the peduncle. Lar and Ptp69D are necessary for the axonal branching decisions that create the lobes. Avoidance of the brain midline by extending medial lobe axons involves signaling through Lar.     

The expression of antigens by embryonic neurons and glia in segmental ganglia of the leech Haemopis marmorata

Monoclonal antibodies (mAbs) raised against adult leech nervous systems were screened on embryos of the leech Haemopis marmorata in order to determine when in development specific antigens are first expressed and the order in which they are expressed by different cells or tissues. Three of the mAbs produced by Zipser and McKay (Zipser, B., and R. McKay (1981) Nature 289: 549-554) were screened: Lan3-1, Lan3-5, and Lan3-6. Each mAb shows a different pattern of labeling in the adult leech nerve cord (Zipser, B. (1982) J. Neurosci. 2: 1453-1464). The embryonic stages studied were from 5 days after egg deposition to 30 days (emergence from the cocoon). The pattern of labeling was assayed in whole mounts using horseradish peroxidase-conjugated second antibodies. The principal results are as follows. (1) Antigens recognized by Lan3-5 are first expressed by the glia of the roots of the anterior segmental ganglia at 6 to 7 days, several days later by the interganglionic connective glia, and near the end of embryonic development by ganglionic neurons. An anterior to posterior temporal gradient is observed in the expression of these antigens. In addition, Lan3-5 also labels the protonephridia and nephridia from early development onward. (2) Antigens recognized by Lan3-6 are first expressed by a pair of neurons in each segmental ganglion and later in development by additional neurons. By the time of emergence, however, only about half of the neurons that label in the adult have done so, implying that some neurons express these antigens postembryonically. Labeling with Lan3-6 is first seen in neuronal somata and only later in neuronal processes. (3) Antigens recognized by Lan3-1 and expressed by segmentally specific neurons in ganglia 5 and 6 are not detectable during embryonic development, but are so at early postembryonic stages. Thus, these three mAbs provide an approach to study different aspects of the development of the leech nervous system, specifically the relation between glial and neuronal differentiation and the genesis of segmentally specific phenotypes.     

Identification of leech embryonic neurons that express a Hox gene required for the differentiation of a paired,segment-specific motor neuron

This study investigated the embryonic expression and function of the Hox gene Lox1 in the simple, well-characterized central nervous system (CNS) of the medicinal leech Hirudo medicinalis. Lox1 was expressed in an anterior–posterior domain, extending from the posterior aspect of the fourth segment (rostral neuromere 4, R4) to the seventeenth segment (midbody ganglion 13, M13). Lox1 expression was also found in both sex organ primordia (male and female). Lox1 expression was not detected in every cell of the ganglia included in its domain. It was detected in a specific subset that included several segmentally iterated neurons and segment-specific neurons. Several central neurons (neurons located in the central nervous system – CNS) that coexpressed both Lox1 and FMRFamide-like peptides were identified using antibody staining of leech embryos and epifluorescence and confocal microscopy. RNA interference was used to block the expression of Lox1. The expression pattern and the effect of RNA interference indicate that Lox1 is required for the differentiation of a segment-specific pair of motor neurons, the RPE (rostral penile evertor) neurons, which appear only in midbody ganglion 6 (M6) and innervate the male sex organ.     

Development of neuronal circuits and behaviors in the medicinal leech

We are studying the neuronal mechanisms responsible for establishing circuitry underlying the local bending response in the medicinal leech. Local bending replaces an embryonic behavior, circumferential indentation, during the time of initial chemical synaptogenesis in leech embryos. We found that the electrical connections among the motor neurons are established first, about 5% of embryonic time (almost 2 full days) before chemical connections form. The inhibitory connections from muscle inhibitors to muscle excitors are, we hypothesize, responsible for the emergence of local bending. We have also found that the central processes of the excitors--but not the inhibitors--have much longer central processes when their peripheral processes are kept from contacting their target muscles. This system should allow us to test ideas about how individual neurons find their appropriate targets to form functional neuronal circuits.     

Antibody staining reveals novel aspects of segmentation within the leech central nervous system

The leech is a segmented annelid with a well characterized central nervous system. In this report, we use antibodies to map the distribution of neurons confined to selected segmental ganglia in the mud leech Haemopis marmorata. The distribution of these neurons suggest 3 novel aspects of segmentation in the leech nervous system: (1) neurons are assigned to even-numbered ganglia through a mechanism which effectively counts through the leech segmental body plan by units of 2, (2) neurons are assigned to ganglia 7 and 14 through a mechanism which effectively counts in units of 7 and (3) neurons are assigned to the 2nd and 4 fused head ganglia and to the 2nd of 21 unfused midbody ganglia through a mechanism which effectively counts units from the origin of these 2 ganglionic series. These 3 hypothetical counting mechanisms divide the central nervous system (CNS) into supersegmental units. Neurons used to define these supersegmental units have been injected with tracer and identified as interganglionic interneurons. Competitive interactions among embryonic precursors of these neurons may directly eliminate their homologs from intervening ganglia, and thus sculpture supersegmental patterns into the mature nervous system.     

Expression of surface glycoproteins early in leech neural development.

Cell migration and axon growth during neural development rely upon cell-cell and cell-matrix interactions mediated by surface glycoproteins. The surface glycoprotein recognized on leech neurons by monoclonal antibody Lan3-2 has previously been implicated in the process of axon fasciculation during regeneration in adults. In adult leeches, Lan3-2 binds to a carbohydrate epitope of a 130 kD protein. The present study demonstrates that in embryos the antibody binds to the same carbohydrate epitope of glycoproteins with molecular weights of 130 kD and higher. As a first step in evaluating a possible role of the Lan3-2 glycoprotein or the cells that express it during neural development, we determined its distribution in the developing nervous system of the leech Hirudo medicinalis. In embryos, Lan3-2 epitope is expressed on fasciculated sensory afferents and it appears on the cell bodies before neurite outgrowth. The sensory fibers appear rostrally by embryonic day 10, less than halfway through development. Earlier, by 7 days of development at 20 degrees C, Lan3-2 binds to previously undocumented cell types: (1) cells appearing along the embryonic midline and (2) a cluster of cells located at the rostral edge of the germinal plate. These cells only transiently express this antigen and are present at critical left-right and rostrocaudal boundaries during a period of cell proliferation, movement, and migration that produces the nervous system. Thus the Lan3-2 surface glycoprotein or the cells expressing it are candidates for involvement in axon fasciculation, cell migration, and directed axonal growth.     

Receptor tyrosine phosphatases in axon growth and guidance

The last 5 years has seen an explosion of evidence linking RPTPs to the regulation of axon growth and guidance. Important questions to be addressed include the ligand-receptor interactions involved in axon growth regulation, the signaling pathways controlled by RPTPs in neurons, and the manner in which different RPTPs within a class, and different classes of RPTPs, coordinate their functions to ensure appropriate axon growth. Are RPTPs signaling ligands, signaling receptors, or both? Do RPTPs function mainly by modifying adhesive preferences, or are they instructive in guidance decisions? Do specific types of RPTPs send specific signals to neurons, or do they work together to fine-tune levels of tyrosine phosphorylation? Whatever the outcome, it seems certain that the answers to these questions will come only from a combination of the powerful genetic approaches possible in Drosophila (and in mice) with the biochemical and cell biological approaches possible in the vertebrate systems.     

Developmental co-expression and functional redundancy of tyrosine phosphatases with neurotrophin receptors in developing sensory neurons

Receptor-type protein tyrosine phosphatases (RPTPs) have been implicated as direct or indirect regulators of neurotrophin receptors (TRKs). It remains less clear if and how such RPTPs might regulate TRK proteins in vivo during development. Here we present a comparative expression profile of RPTP genes and Trk genes during early stages of murine, dorsal root ganglion maturation. We find little if any specific, temporal mRNA co-regulation between individual RPTP and Ntrk genes between E12.5 and E14.5. Moreover, a double fluorescent in-situ hybridization and immunofluorescence study of seven Rptp genes with Ntrks revealed widespread co-expression of RPTPs in individual neurons, but no tight correlation with Trk expression profiles. No Rptp is expressed in 100% of Ntrk1-expressing neurons, whereas at least 6 RPTPs are expressed in 100% of Ntrk2- and Ntrk3-expressing neurons. An exception is Ptpro, which showed very selective expression. Short hairpin RNA suppression of Ptprf, Ptprs or Ptpro in primary, E13.5 DRG neurons did not alter TRK signalling. We therefore propose that TRK signalling may not be simply dependent on rate-limiting regulation by individual RPTP subtypes during sensory neuron development. Instead, TRK signalling has the potential to be buffered by concurrent inputs from several RPTPs in individual neurons.     

Acetylcholinesterase expression in NTera 2 human neuronal cells: A model for developmental expression in the nervous system

Acetylcholinesterase (AChE; EC 3.1.1.7) is expressed in the central nervous system in multiple molecular forms that may subserve multiple functions and may be selectively lost in neurodegenerative illnesses such as Alzheimer's disease. AChE expression has been studied in primary cultures of developing vertebrate nervous system, but investigation has been limited by the lack of a suitable human CNS surrogate cell model system for in vitro studies and the inability of primary brain cultures to provide large numbers of pure neurons. To develop an in vitro model for studies of neuronal AChE expression and function, we utilized a neuronally committed human teratocarcinoma cell line, NTera 2, that can be induced to differentiate to a post-mitotic CNS neuronal phenotype. We found that NTera 2 cells express multiple molecular forms of AChE that are similar to CNS-derived AChE isoforms in velocity sedimentation profile, anion exchange elution profile, and sensitivity to inhibitors. At least two forms of ACNE are expressed (G1 and G4), similar to human and rodent brain, and induction of NTera 2 cell differentiation results in an increased G4/G1 ratio, which is characteristic of mature neurons. As in primary CNS neurons, AChE is present in NTera 2 cells in both the cytosolic fraction and in the outer membrane, and is also released in a soluble form. These observations indicate that NTera 2 cells provide a useful human model system for studies of expression of cell-associated and soluble cell-free AChE in developing and mature human neurons and for elucidating the potential role(s) of acetylcholinesterase metabolism in both normal development and neurodegenerative disease states. ©1995 Wiley-Liss, Inc.     

A clonal line of mesencephalic progenitor cells converted to dopamine neurons by hematopoietic cytokines: a source of cells for transplantation in Parkinson's disease

Neural progenitor cells potentially provide a limitless, on-demand source of cells for grafting into patients with Parkinson's disease (PD) if the signals needed to control their conversion into dopamine (DA) neurons could be identified. We have recently shown that cytokines which instruct cell division and differentiation within the hematopoeitic system may provide similar functions in the central nervous system. We have shown that mitotic progenitor cells can be isolated from embryonic rat mesencephalon and that these cells respond to a combination of interleukin-1, interleukin-11, leukemia inhibitory factor, and glial cell line-derived neurotrophic factor yielding a tyrosine hydroxylase-immunoreactive (THir) phenotype in 20-25% of total cells. In the present study, 24 clonal cell lines derived from single cells of mesencephalic proliferation spheres were examined for their response to the cytokine mixture. The clone yielding the highest percentage of THir neurons (98%) was selected for further study. This clone expressed several phenotypic characteristics of DA neurons and expression of Nurr1. The response to cytokines was stable for several passages and after cryopreservation for several months. When grafted into the striatum of DA-depleted rats, these cells attenuated rotational asymmetry to the same extent as freshly harvested embryonic DA neurons. These data demonstrate that mesencephalic progenitor cells can be clonally expanded in culture and differentiated in the presence of hematopoietic cytokines to yield enriched populations of DA neurons. When transplanted, these cells provide significant functional benefit in the rat model of PD.     

The receptor protein tyrosine phosphatase HmLAR1 is up-regulated in the CNS of the adult medicinal leech following injury and is required for neuronal sprouting and regeneration

LAR-like receptor protein tyrosine phosphatases (RPTPs), which are abundantly expressed in the nervous systems of most if not all bilaterian animals thus far examined, have been implicated in regulating a variety of critical neuronal processes. These include neuronal pathfinding, adhesion and synaptogenesis during development and, in adult mammals, neuronal regeneration. Here we explored a possible role of a LAR-like RPTP (HmLAR1) in response to mechanical trauma in the adult nervous system of the medicinal leech. In situ hybridization and QPCR analyses of HmLAR1 expression in individual segmental ganglia revealed a significant up-regulation in receptor expression following CNS injury, both in situ and following a period in vitro. Furthermore, we observed up-regulation in the expression of the leech homologue of the Abelson tyrosine kinase, a putative signaling partner to LAR receptors, but not among other tyrosine kinases. The effects on neuronal regeneration were assayed by comparing growth across a nerve crush by projections of individual dorsal P neurons (P_D) following single-cell injection of interfering RNAs against the receptor or control RNAs. Receptor RNAi led to a significant reduction in HmLAR1 expression by the injected cells and resulted in a significant decrease in sprouting and regenerative growth at the crush site relative to controls. These studies extend the role of the HmLARs from leech neuronal development to adult neuronal regeneration and provide a platform to investigate neuronal regeneration and gene regulation at the single cell level.     

Subcellular Distribution of Patched and Smoothened in the Cerebellar Neurons

The Sonic hedgehog (Shh) signaling pathway carries out a wide range of biological functions such as patterning of the embryonic neural tube and expansion of cerebellar granule cell precursors. We previously have found that the Shh signaling receptors, Patched1 (Ptch1) and Smoothened (Smo), are expressed in hippocampal neurons of developing and adult rats, suggesting the continued presence of Shh signaling in postmitotic, differentiated neurons. Here, we report that Ptch1 and Smo are present in the processes and growth cones of immature neurons in the developing cerebellum, and that, in the mature cerebellum, Ptch1 and Smo are expressed by several types of neurons including Purkinje cells, granule cells, and interneurons. Within these neurons, Ptch1 and Smo are predominantly localized in the postsynaptic side of the synapses, a distribution pattern similar to that found in hippocampal neurons. Our findings provide morphological evidence that Shh signaling events are not confined to neuronal precursors and are likely to have ongoing roles within the postmitotic neurons of the developing and adult cerebellum.     

Distribution and developmental expression of octopamine-immunoreactive neurons in the central nervous system of the leech

Octopamine, a biogenic amine analogous to norepinephrine, plays an important role in the orchestration and modulation of invertebrate behavior. In the leech, the behavioral actions of octopamine have been demonstrated; however, identification of octopaminergic neurons had not been determined by using immunohistochemical techniques. Thus, we used an antibody highly specific to octopamine to examine the distribution of octopamine-immunoreactive neurons in the segmental ganglia of American and European medicinal leeches (Macrobdella decora and Hirudo medicinalis). One pair of octopamine-immunoreactive neurons was located in the dorsolateral ganglionic region of anterior ganglia 1–6 and posterior ganglia 15–21. No corresponding octopamine-immunoreactive neurons were found in midbody ganglia 7–14. Using Neutral Red staining in combination with intracellular Neurobiotin injections and octopamine immunostaining, we determined the identity of the dorsolateral octopamineimmunoreactive cells. The dorsolateral octopamine-immunoreactive neuron (the DLO) was not cell 21, the only previously reported Neutral Red staining neuron in the dorsolateral position. We also determined that the Leydig neuron was not octopamine immunoreactive in either of the two medicinal leech species. Octopamine immunostaining in the sex ganglia revealed hundreds of immunoreactive neurons in sexually mature leeches. Such neurons were not observed in juvenile leeches. The developmental time course of octopamine immunoreactivity in the dorsolateral octopamine-immunoreactive neurons was also investigated by staining embryonic Hirudo medicinalis, Octopamine expression occurred relatively late as compared with the detectable onset of serotonin expression. Octopamine expression in the dorsolateral octopamine-immunoreactive cells was not detectable at early to mid-embryonic stages, and must commence during late embryonic to early juvenile stages. The identification of octopamineimmunoreactive cells now sets the stage for further investigations into the functional role of octopamine in leech behavior and the development of behavior. © 1995 Wiley-Liss, Inc.     

Plexin-B family members demonstrate non-redundant expression patterns in the developing mouse nervous system: an anatomical basis for morphogenetic effects of Sema4D during development

Semaphorins and their receptors play important roles in patterning the connectivity of the developing nervous system and recent data suggest that members of the plexin-B family of semaphorin receptors may be involved in axonal guidance. Here we show that the mRNAs of the three plexin-B genes, plxnb1, plxnb2 and plxnb3 (plexin-B1, plexin-B2 and plexin-B3), respectively, are expressed in highly specific and non-redundant patterns in peripheral and central components of the nervous system over defined periods during murine development. Whereas plexin-B1 and plexin-B2 are strongly expressed in the neuroepithelium and developing neurons, plexin-B3 mRNA is selectively localized to the white matter. Moreover, plexin-B1 and its ligand Sema4D are expressed in complementary patterns in several regions such as the developing neopallial cortex, the dorsal root ganglia and the spinal cord over embryonic stages. The Sema4d gene demonstrates a dramatic switch from prenatal expression in neuronal populations to a postnatal expression in oligodendrocytes. In contrast to its collapsing activity on growth cones of embryonic retinal ganglion cells and hippocampal neurons, soluble Sema4D enhances axonal outgrowth in embryonic cortical explants cultured in collagen matrices. Thus, plexin-B family members and Sema4D are likely to play complex and non-redundant roles during the development of the nervous system.     

Regeneration of a distinctive set of axosomatic contacts in the leech central nervous system

The nociceptive sensory neurons (N cells) in the leech Hirudo medicinalis contact other neurons through conventional synapses in the neuropile and through baskets of processes that wrap the somata of particular cells. These axosomatic contacts are made with the pressure (P) and N sensory neurons in the adjacent segmental ganglia, but not with cells within the same ganglion as the wrapping cell. Physiological evidence indicates that these contacts may be synaptic, although conventional synapses have not been observed with electron microscopy. After they have been injured, lateral N cell processes can grow into the anterior adjacent ganglion and regenerate somatic contacts. In general, regenerated N cell processes wrap the same somata as do intact N cells, but they often wrap fewer somata. However, six of 14 regenerated N cells also wrapped the soma of a small posterior cell that was contacted in only one of 120 normal ganglia examined. It thus appears that the growing processes of an injured N cell select certain cell somata to wrap, but that the selection is somewhat broader than that in the embryonic leech.     

Phenotypic plasticity of avian embryonic sympathetic neurons grown in a chemically defined medium: direct evidence for noradrenergic and cholinergic properties in the same neurons.

Avian embryonic sympathetic ganglia possess both catecholaminergic and cholinergic features and can synthesize noradrenaline (NAd) and acetylcholine (ACh) simultaneously. In the present study we sought to determine (1) whether or not this coproduction of NAd and ACh corresponds to the existence of two non-overlapping populations, and (2) to what extent the levels of synthesis are influenced by non-neuronal ganglion cells. We have focused on the correlation between the immunocytochemically demonstrable presence of the noradrenergic and cholinergic enzymes tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), respectively, and the synthesis of the corresponding neurotransmitters in embryonic quail sympathetic neuronal and non-neuronal cells purified by fluorescence-activated cell sorting. We show that (1) freshly sorted neurons synthesize both NAd and ACh, whereas non-neuronal cells produce neither; (2) the overwhelming majority of the sympathetic neurons display TH immunoreactivity; (3) about half of the TH-positive neurons are recognized by an anti-ChAT antibody in an artificial medium that selectively enhances synthesis and/or accumulation of ACh; (4) the non-neuronal cells are important for survival of the neurons and potentiate their synthesis of ACh in this medium, and (5) finally, we present evidence that expression of TH in noradrenergic neurons and in small intensely fluorescent cells of sympathetic ganglia is differentially regulated.     

Expression of tyrosine hydroxylase in neurons of cultured cerebral cortex: evidence for phenotypic plasticity in neurons of the CNS

In vivo, neurons of the cerebral cortex of rat embryos did not stain with antibodies to the catecholamine (CA) biosynthetic enzyme tyrosine hydroxylase (TH) even when examined using a highly sensitive technique for radioimmunocytochemistry. However, when embryonic day (E) 13 cortex was grown 1 d in culture, several thousand cells expressed immunoreactive and catalytically active TH. All TH cells simultaneously labeled with the neuronal enzyme, neuronal specific enolase, indicating that the TH was exclusively localized in neurons. Moreover, all TH neurons were postmitotic since they did not incorporate 3H-thymidine. With time in culture, the number of TH cells selectively declined from nearly 3000 cells at 2 d to several cells at 14 d. Similarly, the number of neurons competent to express TH in culture declined with advancing age of the donor embryo. Thus, by E18, very few cortical neurons had the capacity to express TH. We conclude that during a critical period of development, postmitotic cerebral cortical neurons can express catecholamine traits in vitro but not in vivo. Thus, the neurotransmitter phenotype of certain classes of central neurons is not fixed but can be influenced by epigenetic factors found in their environment, thereby providing evidence of phenotypic plasticity in the central nervous system (CNS).