Why didn't the glia cross the road?

To cross or not to cross the midline? This is the question that pathfinding axons in the developing CNS must answer. In the past few years the molecular mechanisms involved in this decision process have been determined for neurones. A recent paper now shows that glia are also affected by these same mechanisms and that the presence of glia can tip the balance in favour of repulsion or attraction of neurones to the midline.

Neuroligin 3 is a vertebrate gliotactin expressed in the olfactory ensheathing glia, a growth-promoting class of macroglia.

The molecular mechanisms that drive glia-glial interactions and glia-neuronal interactions during the development of the nervous system are poorly understood. A number of membrane-bound cell adhesion molecules have been shown to play a role, although the precise nature of their involvement is unknown. One class of molecules with cell adhesive properties used in the nervous system is the serine-esterase-like family of transmembrane proteins. A member of this class, a glia-specific protein called gliotactin, has been shown to be necessary for the development of the glial sheath in the peripheral nervous system of Drosophila melanogaster. Gliotactin is essential for the development of septate junctions in the glial sheath of individual and neighboring glia. Mutations that remove this protein result in paralysis and eventually death due to a breakdown in the glial-based blood-nerve barrier. To study the role of gliotactin during vertebrate nervous system development, we have isolated a potential vertebrate gliotactin homologue from mice and rat and found that it corresponds to neuroligin 3. Using a combination of RT-PCR and immunohistochemistry, we have found that neuroligin 3 is expressed during the development of the nervous system in many classes of glia. In particular neuroligin 3 is expressed in the olfactory ensheathing glia, retinal astrocytes, Schwann cells, and spinal cord astrocytes in the developing embryo. This expression is developmentally controlled such that in postnatal and adult stages, neuroligin 3 continues to be expressed at high levels in the olfactory ensheathing glia, a highly plastic class of glia that retain many of their developmental characteristics throughout life.

Peripheral glia direct axon guidance across the CNS/PNS transition zone.

CNS glia have integral roles in directing axon migration of both vertebrates and insects. In contrast, very little is known about the roles of PNS glia in axonal pathfinding. In vertebrates and Drosophila, anatomical evidence shows that peripheral glia prefigure the transition zones through which axons migrate into and out of the CNS. Therefore, peripheral glia could guide axons at the transition zone. We used the Drosophila model system to test this hypothesis by ablating peripheral glia early in embryonic neurodevelopment via targeted overexpression of cell death genes grim and ced-3. The effects of peripheral glial loss on sensory and motor neuron development were analyzed. Motor axons initially exit the CNS in abnormal patterns in the absence of peripheral glia. However, they must use other cues within the periphery to find their correct target muscles since early pathfinding errors are largely overcome. When peripheral glia are lost, sensory axons show disrupted migration as they travel centrally. This is not a result of motor neuron defects, as determined by motor/sensory double-labeling experiments. We conclude that peripheral glia prefigure the CNS/PNS transition zone and guide axons as they traverse this region.

Developmental dynamics of peripheral glia in Drosophila melanogaster.

To study the roles of peripheral glia in nervous system development, a thorough characterization of wild type glial development must first be performed. We present a developmental profile of peripheral glia in Drosophila melanogaster that includes glial genesis, developmental morphology, the establishment of transient cellular contacts, migration patterns, and the extent of nerve wrapping in the embryonic and larval stages. In early embryonic development, immature peripheral glia that are born in the CNS seem to be intermediate targets for neurites that are migrating into the periphery. During migration to the PNS, peripheral glia follow the routes of pioneer neurons. The glia preferentially adhere to sensory axonal projections, extending cytoplasmic processes along them such that by the end of embryogenesis peripheral glial coverage of the sensory system is complete. In contrast, significant lengths of motor branch termini are unsheathed in the mature embryo. During larval stages however, peripheral glia further extend and elaborate their cytoplasmic processes until they often reach to the neuromuscular junction. Throughout the embryonic and larval developmental stages, we have also observed a number of similarities of peripheral glia to vertebrate Schwann cells and astrocytes. Peripheral glia seem to have dynamic and diverse roles and their similarities to vertebrate glia suggest that Drosophila may serve as a powerful tool for analysis of glial roles in PNS development in the future.

Glia as mediators of growth cone guidance: studies from insect nervous systems.

Growth cones experience many different cues in their journey to their final target. They can respond to a variety of attractive and repulsive cues that can be secreted or cellular. These cues are generated by a wide range of cell types. One subset of cells that play an important role in growth cone guidance are glial cells. Glia secrete guidance cues and express cellular cues on their surface that guide axonal outgrowth. In doing so, glia can act as intermediate targets in growth cone guidance, a process that is conserved between vertebrate and invertebrate nervous systems. Recent work in grasshopper, Drosophila and moth nervous system development has underscored the importance of the instructive role glia play during axonal outgrowth.

Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster.

Since the development of the enhancer trap technique, many large libraries of nuclear localized lacZ P-element stocks have been generated. These lines can lend themselves to the molecular and biological characterization of new genes. However they are not as useful for the study of development of cellular morphologies. With the advent of the GAL4 expression system, enhancer traps have a far greater potential for utility in biological studies. Yet generation of GAL4 lines by standard random mobilization has been reported to have a low efficiency. To avoid this problem we have employed targeted transposition to generate glial-specific GAL4 lines for the study of glial cellular development. Targeted transposition is the precise exchange of one P element for another. We report the successful and complete replacement of two glial enhancer trap P[lacZ, ry+] elements with the P[GAL4, w+] element. The frequencies of transposition to the target loci were 1.3% and 0.4%. We have thus found it more efficient to generate GAL4 lines from preexisting P-element lines than to obtain tissue-specific expression of GAL4 by random P-element mobilization. It is likely that similar screens can be performed to convert many other P-element lines to the GAL4 system.

Distribution of two GABA receptor-like subunits in the Drosophila CNS.

Previously we have described the distribution of the Rdl GABA receptor subunit in the Drosophila CNS. Knowing that Rdl can coassemble with LCCH3 (a Drosophila GABA receptor-like subunit showing sequence similarity to vertebrate beta subunit GABAA receptors) in baculovirus infected insect cells, we compared the localization of these two receptor subunits in order to identify any potential overlap in their spatial or temporal distribution. The two subunits show very different patterns of localization. Early in development LCCH3 is found in the majority of developing neuroblasts and later is localized to the cell bodies of the embryonic nerve cord and brain, and the neuronal cell bodies surrounding the adult brain. In contrast, Rdl receptor subunits appear confined to the neuropil in all developmental stages. These results have two important implications. Firstly, they suggest that although these two subunits can coassemble in heterologous expression systems, they may not be found in the same tissues in the nervous system. Secondly, production of LCCH3 before neuronal differentiation leads us to speculate on the role of that LCCH3 containing receptors in the developing nervous system.

Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophila.

Peripheral glia help ensure that motor and sensory axons are bathed in the appropriate ionic and biochemical environment. In Drosophila, peripheral glia help shield these axons against the high K+ concentration of the hemolymph, which would largely abolish their excitability. Here, we describe the molecular genetic analysis of gliotactin, a novel transmembrane protein that is transiently expressed on peripheral glia and that is required for the formation of the peripheral blood-nerve barrier. In gliotactin mutant embryos, the peripheral glia develop normally in many respects, except that ultrastructurally and physiologically they do not form a complete blood-nerve barrier. As a result, peripheral motor axons are exposed to the high K+ hemolymph, action potentials fail to propagate, and the embryos are nearly paralyzed.

Targeted neuronal cell ablation in the Drosophila embryo: pathfinding by follower growth cones in the absence of pioneers.

We developed a rapid method that uses diphtheria toxin, the flp recognition target sequences, and the GAL4-UAS activation system, to ablate specific neurons in the Drosophila embryo and to examine the consequences in large numbers of embryos at many time points. We used this method to show that, in the absence of the aCC axon, which pioneers the intersegmental nerve in the PNS, the three U follower axons are delayed and make frequent errors. However, the pathway ultimately forms in most segments. We also ablated the axons that pioneer the first longitudinal pathways within the CNS and observed similar results; the formation of longitudinal pathways is delayed and disorganized in 70% of segments, but these tracts ultimately form in 80% of segments. Thus, pioneers facilitate the development of PNS and CNS axon pathways; in their absence, followers are delayed and make numerous errors. However, pioneers are not absolutely required, as these embryos display a remarkable ability to correct for the loss of the pioneering neurons.

Developmentally regulated alternative RNA splicing of rat brain sodium channel mRNAs.

Two rat brain Na channel alpha-subunit cDNAs, named RII and RIIA, have almost identical coding regions, with a divergence of only 36 nucleotides (0.6%) over a total length of 6015 residues. A cluster of 20 divergent residues occurs within a 90 nucleotide segment of cDNA sequence. We now demonstrate that this 90 nucleotide segment is encoded twice in the RII/RIIA genomic sequence. Furthermore, the mutually exclusive selection of these two exons is developmentally regulated. RII mRNAs are relatively abundant at birth but are gradually replaced by RIIA mRNAs as development proceeds. The two mRNAs also appear to have different regional distributions in the developing rat brain. Strikingly, although 30 amino acids are encoded by each alternative exon, only amino acid position 209 is altered between the two, specifying asparagine in RII and aspartate in RIIA. Alternative RNA splicing may modulate the RII/RIIA sodium channel properties during neuronal development.

Inactivation of cloned Na channels expressed in Xenopus oocytes.

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J., A. L. Goldin, D. S. Krafte, J. Marshall, J. M. Dunn, W. A. Catterall, H. A. Lester, N. Davidson, and R. J. Dunn. 1988. Neuron. 1:449-461), their inactivation properties resemble each other strongly but differ markedly from channels induced by poly(A+) rat brain RNA. Rat IIA currents inactivate more slowly, recover from inactivation more slowly, and display a steady-state voltage dependence that is shifted to more positive potentials. The macroscopic inactivation process for poly(A+) Na channels is defined by a single exponential time course; that for rat IIA channels displays two exponential components. At the single-channel level these differences in inactivation occur because rat IIA channels reopen several times during a depolarizing pulse; poly(A+) channels do not. Repetitive stimulation (greater than 1 Hz) produces a marked decrement in the rat IIA peak current and changes the waveform of the currents. When low molecular weight RNA is coinjected with rat IIA RNA, these inactivation properties are restored to those that characterize poly(A+) channels. Slow inactivation is similar for rat IIA and poly(A+) channels, however. The data suggest that activation and inactivation involve at least partially distinct regions of the channel protein.

Functional properties of rat brain sodium channels expressed in a somatic cell line.

Transfection of Chinese hamster ovary cells with complementary DNA encoding the RIIA sodium channel alpha subunit from rat brain led to expression of functional sodium channels with the rapid, voltage-dependent activation and inactivation characteristic of sodium channels in brain neurons. The sodium currents mediated by these transfected channels were inhibited by tetrodotoxin, persistently activated by veratridine, and prolonged by Leiurus alpha-scorpion toxin, indicating that neurotoxin receptor sites 1 through 3 were present in functional form. The RIIA sodium channel alpha subunit cDNA alone is sufficient for stable expression of functional sodium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.

A neutral amino acid change in segment IIS4 dramatically alters the gating properties of the voltage-dependent sodium channel.

Sodium channels encoded by the rat IIA cDNA clone [Auld, V. J., Goldin, A. L., Krafte, D. S., Marshall, J., Dunn, J., Catterall, W. A., Lester, H. A., Davidson, N. & Dunn, R. J. (1988) Neuron 1, 449-461] differ at seven amino acid residues from those encoded by the rat II cDNA [Noda, M., Ikeda, T., Kayano, T., Suzuki, H., Takeshima, H., Kurasaki, M., Takahashi, H. & Numa, S. (1986) Nature (London) 320, 188-192]. When expressed in Xenopus oocytes, rat IIA channels display a current-voltage relationship that is shifted 20-25 mV in the depolarizing direction relative to channels expressed from rat II cDNA or rat brain poly(A)+ mRNA. By modifying each variant residue in rat IIA to the corresponding residue in rat II, we demonstrate that a single Phe----Leu substitution at position 860 in the S4 segment of domain II is sufficient to shift the current-voltage relationship to that observed for channels expressed from rat brain poly(A)+ RNA or rat II cDNA. Rat genomic DNA encodes leucine but not phenylalanine at position 860, indicating that the phenylalanine at this position in rat IIA cDNA likely results from reverse transcriptase error.

Developmental regulation of sodium channel expression in the rat forebrain.

Na+ channels in adult rat brain are heterotrimeric complexes consisting of alpha subunits (260 kDa) noncovalently associated with a beta 1 subunit (36 kDa) and disulfide-linked to a beta 2 subunit (33 kDa). The time course of developmental accumulation of the 9-kilobase mRNA encoding sodium channel alpha subunits in the rat forebrain was measured by RNA blotting. These transcripts were present at low levels until birth, increased rapidly in abundance to peak by postnatal day 7, and subsequently declined to 50% of this maximum value in adult animals. Sodium channel gene transcription measured by a nuclear run-on assay was first detectable on embryonic day 16, increased to a maximum on postnatal days 1 through 7, and declined in adulthood. The level of gene transcription was highest during the period of rapid rise of Na+ channel alpha subunit mRNA levels and decreased during the period of Na+ channel mRNA decline. The levels of Na+ channel alpha subunit protein measured by immunoblotting increased from postnatal day 1 to postnatal day 21, with the greatest rate of increase falling between days 7 and 21. The number of high affinity saxitoxin binding sites increased in parallel to the increase in alpha subunit protein. The period of most rapid rise in Na+ channel alpha subunit levels corresponded to the period of greatest Na+ channel mRNA abundance. Na+ channel alpha subunits were resolved into free alpha subunits and alpha subunits disulfide-linked to beta 2 subunits. On postnatal day 1, virtually all Na+ channel alpha subunits were in the free alpha form. The fraction of disulfide-linked alpha subunits increased to 60% by postnatal day 21 and 90% by postnatal day 90. The concentration of free alpha subunits was maximum on postnatal days 7 to 14 and declined to less than 10% in adulthood. We conclude from these data that the formation of mature heterotrimeric sodium channel complexes is regulated by at least two processes in developing rat forebrain. Activation of Na+ channel alpha subunit gene transcription and the subsequent increase in Na+ channel mRNA are responsible for the major increases in alpha subunit protein and functional Na+ channels in the neonatal brain. However, changes in alpha subunit mRNA abundance alone are not sufficient to explain the kinetics of alpha subunit protein accumulation. Kinetic analysis suggests a requirement for a developmentally regulated translational or post-translational step in brain sodium channel expression.

A rat brain Na+ channel alpha subunit with novel gating properties.

We have constructed a full-length rat brain Na+ channel alpha subunit cDNA that differs from the previously reported alpha subunit of Noda et al. at 6 amino acid positions. Transcription of the cDNA in vitro and injection into Xenopus oocytes resulted in the synthesis of functional Na+ channels. Although the single-channel conductance of the channels resulting from cloned cDNA was the same as that of channels resulting from injection of rat brain RNA, we observed two significant differences in the gating properties of the channels. The Na+ currents from cloned cDNA displayed much slower macroscopic inactivation compared with those from rat brain mRNA. In addition, the current-voltage relationship for currents from cloned cDNA was shifted 20-25 mV in the depolarizing direction compared with currents from rat brain RNA. Coinjection of low MW rat brain RNA restored normal inactivation of the channels indicating the presence of a component, either a structural subunit of the channel complex or a modifying enzyme, necessary for normal gating of the channel.

Tissue-specific expression of the RI and RII sodium channel subtypes.

Anti-peptide antibodies that distinguish between the rat brain sodium channel subtypes referred to as RI and RII were prepared and used to determine their relative expression in nerve and muscle tissues. Sodium channels purified from rat brain are approximately 18% RI and 80% RII. In brain, the RII subtype is preferentially expressed with RI/RII ratios ranging from 0.07 in the hippocampus to 0.17 in the cerebral cortex. The RI subtype is preferentially expressed in more caudal areas of the central nervous system with values of RI/RII of 0.98 for medulla oblongata and 2.2 for spinal cord. Expression of additional unidentified sodium channel subtype(s) is detected in midbrain, medulla, and spinal cord, and expression of unidentified sodium channel subtypes predominates over expression of RI and RII in retina and optic nerve. The RI and RII subtypes are primarily expressed in the central nervous system and are not detected in significant numbers in skeletal or cardiac muscle, sympathetic ganglia, adrenal medulla, sciatic nerve, or cauda equina. The RII subtype appears first in development of both brain and spinal cord but declines in adult spinal cord as the RI subtype increases. The strict regional expression of these two sodium channel subtypes suggests that they may have distinct functional properties or physiological roles.

Messenger RNA coding for only the alpha subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes.

Several cDNA clones coding for the high molecular weight (alpha) subunit of the voltage-sensitive Na channel have been selected by immunoscreening a rat brain cDNA library constructed in the expression vector lambda gt11. As will be reported elsewhere, the amino acid sequence translated from the DNA sequence shows considerable homology to that reported for the Electrophorus electricus electroplax Na channel. Several of the cDNA inserts hybridized with a low-abundance 9-kilobase RNA species from rat brain, muscle, and heart. Sucrose-gradient fractionation of rat brain poly(A) RNA yielded a high molecular weight fraction containing this mRNA, which resulted in functional Na channels when injected into oocytes. This fraction contained undetectable amounts of low molecular weight RNA. The high molecular weight Na channel RNA was selected from rat brain poly(A) RNA by hybridization to a single-strand antisense cDNA clone. Translation of this RNA in Xenopus oocytes resulted in the appearance of tetrodotoxin-sensitive voltage-sensitive Na channels in the oocyte membrane. These results demonstrate that mRNA encoding the alpha subunit of the rat brain Na channel, in the absence of any beta-subunit mRNA, is sufficient for translation to give functional channels in oocytes.