- Email: [email protected]
Glial cells aid axonal target selection
432
Research Update
TRENDS in Neurosciences Vol.24 No.8 August 2001
Glial cells aid axonal target selection Jan Pielage and Christian Klämbt A key problem in developmental neurobiology is how axons home in on their correct target tissue and establish the correct synaptic contacts. Recent work shows that in the developing Drosophila visual system a population of distinct lamina glial cells ensures correct target layer selection of retinal axons. In the absence of lamina neurons, photoreceptor axons terminate their growth in the correct zone, but when glial cell migration into the lamina is disrupted, as in nonstop mutants, growth cones advance into deeper layers of the brain.
Once neurons are born they send their axons on a long journey to find their targets. On route the navigating growth cones encounter several different molecules secreted by intermediate targets passed along the way to the final destination1. But who tells the growth cone where to stop in order to make the correct synaptic contacts? Using the developing Drosophila compound eye as a model Poeck and collaborators2 recently showed in a series of elegant experiments that glial cells play a vital role in target layer selection of retinal axons. In these studies they utilized the advantages of the Drosophila system – genetic manipulation and the wellcharacterized relatively simple morphology – to show that a ubiquitin-dependent protease, encoded by the gene nonstop, controls glial migration into the lamina. The Drosophila compound eye
The Drosophila compound eye comprises ~750 ommatidia, each containing eight photoreceptor cells belonging to two subgroups: six outer photoreceptor cells (R1–R6) and two inner photoreceptor cells (R7, R8). These subgroups differ not only in their physiological characteristics but also in their final synaptic target zones within the fly brain (Fig. 1). The eight axons of each ommatidium project together as a fascicle through the optic stalk in a topographic fashion into the brain lobes. In the lamina layer the R1–R6 axons stall and form the laminar plexus, whereas R7 and R8 axons travel deeper into the brain and stop in the medulla3. The lamina develops as photoreceptor cell axons invade the optic lobes. By releasing hedgehog, the photoreceptor cell http://tins.trends.com
axons induce a final round of cell division of the lamina precursor cells (LPC). In response to hedgehog signaling the LPC activate Dachshund as well as EGF-receptor expression. Subsequently, the in-growing photoreceptor axons release the TGF-α homolog Spitz, which initiates terminal neuronal differentiation reflected by expression of the Elav protein4–6. Finally, five lamina neurons are found in each cartridge, the number of which matches the number of ommatidia. The growth cones of R1–R6 terminate in the lamina between two distinct glial cell layers, the epithelial glial cells and the marginal glial cells (Fig. 1). A third glial cell sheath, the medulla glial, is found deeper in the brain demarcating the boundary between the lamina and medulla. These glial cells stem from distinct progenitor pools and were proposed to exert an important role during target selection7,8. However, previously, there was no genetic evidence to support this function. Growth cones of R1–R6 pause between the epithelial and the marginal glial cell layers for about four days before they start to form synaptic connections with specific lamina neurons residing in neighboring cartridges3,9. This latter process depends on nitric oxide (NO) signaling. If NO signaling is blocked photoreceptor axons resume growth into deeper layers of the brain10. Genetics of Drosophila photoreceptor connectivity
A few years ago Zipursky and colleagues11 conducted a large histological screen for mutations that affect axonal pathfinding of photoreceptor axons in the developing fly brain11. This, in addition to an elegant mosaic screen developed in the laboratory of Dickson12, identified several genes required in the receptor cells for the correct stopping of R1–R6 growth cones in the lamina (PTP69D, brakeless, limbo, nonstop)12–16. Now the Zipursky laboratory have presented a detailed analysis of the gene nonstop, the first gene known to be required in the target tissue2. Using a Ro-τlacZ reporter that is selectively expressed in R2–R5 cells Poeck et al. have convincingly shown that in nonstop mutants outer photoreceptor cells do not stop their growth in the lamina but instead continue to advance into the medulla.
nonstop encodes a ubiquitin-dependent protease that functions in the developing brain
nonstop was found to encode a protein homologous to ubiquitin-specific proteases (UBPs). Two lines of evidence support the function of nonstop as an UBP. On the one hand, ubiquitinated proteins accumulate in nonstop mutant tissue, and on the other nonstop genetically interacts with a mutation in a proteasome subunit, that acts within the ubiquitin pathway2. nonstop is required for correct target layer selection in the brain. But where does it have to be expressed in order for correct navigation of the R1–R6 axons? To address this question Poeck et al. analyzed the projection pattern of mutant nonstop photoreceptor axons in heterozygous brain tissue. They found that, in spite of a subtle ommatidial phenotype, nonstop mutant R1–R6 axons terminated in the lamina. Thus, nonstop is probably required in the target tissue in the brain to mediate normal termination of the photoreceptor axons. nonstop is required for the development of the intermediate target for R1–R6 axons
To first assess the morphology of the different glial cells Poeck and colleagues used glial cell specific GAL4 driver strains to express cytoplasmic β-galactosidase. On route into the lamina photoreceptor axons are enwrapped by the eye disc and satellite glial cells. Similarly, the medulla glial cells that R7 and R8 axons encounter en route to the medulla form long, axon ensheathing, cell processes. By contrast, the epithelial and marginal glial cells adopt a distinct cuboidal shape with many fine processes especially into the developing lamina plexus2. This distinct morphology is in good agreement with a function of the epithelial and marginal glial cells as intermediate target cells. Using several markers, Poeck and colleagues showed that, in contrast with the development of the lamina neurons, the development of the epithelial, marginal and medulla glial cells is severely affected by the nonstop mutation. Compared to wild-type larval brains less than one third of the normal complement of glial cells can be detected in nonstop mutants. Glial cells originate from two glial cell precursor (GPC)
0166-2236/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0166-2236(00)01861-0
Research Update
TRENDS in Neurosciences Vol.24 No.8 August 2001
433
Photoreceptor cells MF 4
Optic stalk
Eye disc
5
L1–5 LPC
6
Epithelia glia Marginal glia Medulla glia
7
GPC
Brain
8
TRENDS in Neurosciences
Fig. 1. The developing Drosophila visual system. The adult Drosophila visual system develops at the end of larval life. In the eye imaginal discs the morphogenetic furrow (MF) sweeps across the epithelia cell sheath. In its wake photoreceptor cells begin to differentiate. Photoreceptor axons of one ommatidium travel together as one fascicle through the optic stalk into the brain where they trigger lamina precursor cell (LPC) division and subsequent maturation of the LPC progeny by releasing hedgehog and spitz. Five lamina neurons form one cartridge (L1–5). Growth cones of the outer photoreceptor cells R1–R6 stop in-between the epithelial and marginal glial cell layers that originate from distinct glial precursor cell (GPC) pools. R7 and R8 growth cones advance into the medulla.
9
10
11
areas and subsequently migrate to their final destination in the optic ganglia7,8 . In nonstop mutants proliferation of the GPCs appears normal but subsequently epithelial, marginal and medulla glial cells fail to migrate into the lamina. In situ hybridization and expression of a myctagged Nonstop protein suggests that it is expressed in both the LPCs and the lamina glial cells. Clonal analysis indicated that the glial cell migration phenotype is caused by a cell-autonomous requirement of nonstop in the glia. To finally link the nonstop mutant phenotype to defective glial cell migration, lamina neurons were removed by an elegant genetic trick utilizing a weak hedgehog allele5. In hedgehog mutant larvae just enough hedgehog function is left to allow the formation of 12 rows of photoreceptor cells, but lamina neurons are never induced in these mutants. By contrast, both development of the lamina glial cells and termination of the few outer photoreceptor cell axons in the lamina plexus appear to be normal. Future directions
The present study emphasizes the importance of glial cells as an intermediate target before the establishment of the final synaptic contacts. Studies in other systems indicate that this finding could be of more general relevance. In the developing olfactory http://tins.trends.com
system of the moth Manduca sexta a glial sorting zone has been described that is required for the convergence of axons in distinct glomeruli in the antennal lobes17. In addition, in the vertebrate visual system, glial cells probably provide important signals that control pathfinding at the optic chiasm18. Here, a member of the Slit protein family has been identified as a candidate for controlling RGC axon divergence19. In the Drosophila nervous system the midline glial cells also express slit and act as an important intermediate target for contralateral projecting axons. In the Drosophila visual system, however, the target-derived molecules that stop R1–R6 growth cones in the lamina are still elusive. Another interesting question that deserves future attention is how glial migration is regulated by the ubiquitindependent protease Nonstop. Because migration of the border cells during Drosophila oogenesis also requires ubiquitin-dependent proteolysis20, it will be interesting to determine the function of the human nonstop ortholog. References 1 Tessier-Lavigne, M. and Goodman, C.S. (1996) The molecular biology of axon guidance. Science 274, 1123–1133 2 Poeck, B. et al. (2001) Glial cells mediate target layer selection of retinal axons in the developing visual system of Drosophila. Neuron 29, 99–113 3 Meinertzhagen, I.A. and Hanson, T.E. (1993) The development of the optic lobe. In Development of
12
13
14
15
16
17
18
19
20
Drosophila melanogaster (Vol. 2) (Bate, C.M. and Martinez-Arias, A., eds), pp. 1363–1491, Cold Spring Harbor Laboratory Press Huang, Z. and Kunes, S. (1996) Hedgehog, transmitted along retinal axons, triggers neurogenesis in the developing visual centers of the Drosophila brain. Cell 86, 411–422 Huang, Z. and Kunes, S. (1998) Signals transmitted along retinal axons in Drosophila: Hedgehog signal reception and the cell circuitry of lamina cartridge assembly. Development 125, 3753–3764 Huang, Z. et al. (1998) A retinal axon fascicle uses spitz, an EGF receptor ligand, to construct a synaptic cartridge in the brain of Drosophila. Cell 95, 693–703 Perez, S.E. and Steller, H. (1996) Migration of glial cells into retinal axon target field in Drosophila melanogaster. J. Neurobiol. 30, 359–373 Winberg, M.L. et al. (1992). Generation and early differentiation of glial cells in the first optic ganglion of Drosophila melanogaster. Development 115, 903–911 Clandinin, T.R. and Zipursky, S.L. (2000) Afferent growth cone interactions control synaptic specificity in the Drosophila visual system. Neuron 28, 427–436 Gibbs, S.M. and Truman, J.W. (1998) Nitric oxide and cyclic GMP regulate retinal patterning in the optic lobe of Drosophila. Neuron 20, 83–93 Martin, K.A. et al. (1995). Mutations disrupting neuronal connectivity in the Drosophila visual system. Neuron 14, 229–240 Newsome, T.P. et al. (2000) Analysis of Drosophila photoreceptor axon guidance in eye-specific mosaics. Development 127, 851–860 Garrity, P.A. et al. (1999) Retinal axon target selection in Drosophila is regulated by a receptor protein tyrosine phosphatase. Neuron 22, 707–717 Rao, Y. et al. (2000) Brakeless is required for photoreceptor growth-cone targeting in Drosophila. Proc. Natl. Acad. Sci. U. S. A. 97, 5966–5971 Ruan, W. et al. (1999) The SH2/SH3 adaptor protein dock interacts with the Ste20-like kinase misshapen in controlling growth cone motility. Neuron 24, 595–605 Senti, K. et al. (2000) Brakeless is required for lamina targeting of R1–R6 axons in the Drosophila visual system. Development 127, 2291–2301 Rossler, W. et al. (1999) Development of a glia-rich axon-sorting zone in the olfactory pathway of the moth Manduca sexta. J. Neurosci. 19, 9865–9877 Mason, C.A. and Sretavan, D.W. (1997) Glia, neurons, and axon pathfinding during optic chiasm development. Curr. Opin. Neurobiol. 7, 647–653 Erskine, L. et al. (2000) Retinal ganglion cell axon guidance in the mouse optic chiasm: expression and function of robos and slits. J. Neurosci. 20, 4975–4982 Rorth, P. et al. (2000) The level of C/EBP protein is critical for cell migration during Drosophila oogenesis and is tightly controlled by regulated degradation. Mol. Cell. 6, 23–30
Jan Pielage Christian Klämbt* Institut für Neurobiologie, Universität Münster, Badestr. 9, 48149 Münster, Germany. *e-mail: [email protected]
Research Update
TRENDS in Neurosciences Vol.24 No.8 August 2001
Glial cells aid axonal target selection Jan Pielage and Christian Klämbt A key problem in developmental neurobiology is how axons home in on their correct target tissue and establish the correct synaptic contacts. Recent work shows that in the developing Drosophila visual system a population of distinct lamina glial cells ensures correct target layer selection of retinal axons. In the absence of lamina neurons, photoreceptor axons terminate their growth in the correct zone, but when glial cell migration into the lamina is disrupted, as in nonstop mutants, growth cones advance into deeper layers of the brain.
Once neurons are born they send their axons on a long journey to find their targets. On route the navigating growth cones encounter several different molecules secreted by intermediate targets passed along the way to the final destination1. But who tells the growth cone where to stop in order to make the correct synaptic contacts? Using the developing Drosophila compound eye as a model Poeck and collaborators2 recently showed in a series of elegant experiments that glial cells play a vital role in target layer selection of retinal axons. In these studies they utilized the advantages of the Drosophila system – genetic manipulation and the wellcharacterized relatively simple morphology – to show that a ubiquitin-dependent protease, encoded by the gene nonstop, controls glial migration into the lamina. The Drosophila compound eye
The Drosophila compound eye comprises ~750 ommatidia, each containing eight photoreceptor cells belonging to two subgroups: six outer photoreceptor cells (R1–R6) and two inner photoreceptor cells (R7, R8). These subgroups differ not only in their physiological characteristics but also in their final synaptic target zones within the fly brain (Fig. 1). The eight axons of each ommatidium project together as a fascicle through the optic stalk in a topographic fashion into the brain lobes. In the lamina layer the R1–R6 axons stall and form the laminar plexus, whereas R7 and R8 axons travel deeper into the brain and stop in the medulla3. The lamina develops as photoreceptor cell axons invade the optic lobes. By releasing hedgehog, the photoreceptor cell http://tins.trends.com
axons induce a final round of cell division of the lamina precursor cells (LPC). In response to hedgehog signaling the LPC activate Dachshund as well as EGF-receptor expression. Subsequently, the in-growing photoreceptor axons release the TGF-α homolog Spitz, which initiates terminal neuronal differentiation reflected by expression of the Elav protein4–6. Finally, five lamina neurons are found in each cartridge, the number of which matches the number of ommatidia. The growth cones of R1–R6 terminate in the lamina between two distinct glial cell layers, the epithelial glial cells and the marginal glial cells (Fig. 1). A third glial cell sheath, the medulla glial, is found deeper in the brain demarcating the boundary between the lamina and medulla. These glial cells stem from distinct progenitor pools and were proposed to exert an important role during target selection7,8. However, previously, there was no genetic evidence to support this function. Growth cones of R1–R6 pause between the epithelial and the marginal glial cell layers for about four days before they start to form synaptic connections with specific lamina neurons residing in neighboring cartridges3,9. This latter process depends on nitric oxide (NO) signaling. If NO signaling is blocked photoreceptor axons resume growth into deeper layers of the brain10. Genetics of Drosophila photoreceptor connectivity
A few years ago Zipursky and colleagues11 conducted a large histological screen for mutations that affect axonal pathfinding of photoreceptor axons in the developing fly brain11. This, in addition to an elegant mosaic screen developed in the laboratory of Dickson12, identified several genes required in the receptor cells for the correct stopping of R1–R6 growth cones in the lamina (PTP69D, brakeless, limbo, nonstop)12–16. Now the Zipursky laboratory have presented a detailed analysis of the gene nonstop, the first gene known to be required in the target tissue2. Using a Ro-τlacZ reporter that is selectively expressed in R2–R5 cells Poeck et al. have convincingly shown that in nonstop mutants outer photoreceptor cells do not stop their growth in the lamina but instead continue to advance into the medulla.
nonstop encodes a ubiquitin-dependent protease that functions in the developing brain
nonstop was found to encode a protein homologous to ubiquitin-specific proteases (UBPs). Two lines of evidence support the function of nonstop as an UBP. On the one hand, ubiquitinated proteins accumulate in nonstop mutant tissue, and on the other nonstop genetically interacts with a mutation in a proteasome subunit, that acts within the ubiquitin pathway2. nonstop is required for correct target layer selection in the brain. But where does it have to be expressed in order for correct navigation of the R1–R6 axons? To address this question Poeck et al. analyzed the projection pattern of mutant nonstop photoreceptor axons in heterozygous brain tissue. They found that, in spite of a subtle ommatidial phenotype, nonstop mutant R1–R6 axons terminated in the lamina. Thus, nonstop is probably required in the target tissue in the brain to mediate normal termination of the photoreceptor axons. nonstop is required for the development of the intermediate target for R1–R6 axons
To first assess the morphology of the different glial cells Poeck and colleagues used glial cell specific GAL4 driver strains to express cytoplasmic β-galactosidase. On route into the lamina photoreceptor axons are enwrapped by the eye disc and satellite glial cells. Similarly, the medulla glial cells that R7 and R8 axons encounter en route to the medulla form long, axon ensheathing, cell processes. By contrast, the epithelial and marginal glial cells adopt a distinct cuboidal shape with many fine processes especially into the developing lamina plexus2. This distinct morphology is in good agreement with a function of the epithelial and marginal glial cells as intermediate target cells. Using several markers, Poeck and colleagues showed that, in contrast with the development of the lamina neurons, the development of the epithelial, marginal and medulla glial cells is severely affected by the nonstop mutation. Compared to wild-type larval brains less than one third of the normal complement of glial cells can be detected in nonstop mutants. Glial cells originate from two glial cell precursor (GPC)
0166-2236/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0166-2236(00)01861-0
Research Update
TRENDS in Neurosciences Vol.24 No.8 August 2001
433
Photoreceptor cells MF 4
Optic stalk
Eye disc
5
L1–5 LPC
6
Epithelia glia Marginal glia Medulla glia
7
GPC
Brain
8
TRENDS in Neurosciences
Fig. 1. The developing Drosophila visual system. The adult Drosophila visual system develops at the end of larval life. In the eye imaginal discs the morphogenetic furrow (MF) sweeps across the epithelia cell sheath. In its wake photoreceptor cells begin to differentiate. Photoreceptor axons of one ommatidium travel together as one fascicle through the optic stalk into the brain where they trigger lamina precursor cell (LPC) division and subsequent maturation of the LPC progeny by releasing hedgehog and spitz. Five lamina neurons form one cartridge (L1–5). Growth cones of the outer photoreceptor cells R1–R6 stop in-between the epithelial and marginal glial cell layers that originate from distinct glial precursor cell (GPC) pools. R7 and R8 growth cones advance into the medulla.
9
10
11
areas and subsequently migrate to their final destination in the optic ganglia7,8 . In nonstop mutants proliferation of the GPCs appears normal but subsequently epithelial, marginal and medulla glial cells fail to migrate into the lamina. In situ hybridization and expression of a myctagged Nonstop protein suggests that it is expressed in both the LPCs and the lamina glial cells. Clonal analysis indicated that the glial cell migration phenotype is caused by a cell-autonomous requirement of nonstop in the glia. To finally link the nonstop mutant phenotype to defective glial cell migration, lamina neurons were removed by an elegant genetic trick utilizing a weak hedgehog allele5. In hedgehog mutant larvae just enough hedgehog function is left to allow the formation of 12 rows of photoreceptor cells, but lamina neurons are never induced in these mutants. By contrast, both development of the lamina glial cells and termination of the few outer photoreceptor cell axons in the lamina plexus appear to be normal. Future directions
The present study emphasizes the importance of glial cells as an intermediate target before the establishment of the final synaptic contacts. Studies in other systems indicate that this finding could be of more general relevance. In the developing olfactory http://tins.trends.com
system of the moth Manduca sexta a glial sorting zone has been described that is required for the convergence of axons in distinct glomeruli in the antennal lobes17. In addition, in the vertebrate visual system, glial cells probably provide important signals that control pathfinding at the optic chiasm18. Here, a member of the Slit protein family has been identified as a candidate for controlling RGC axon divergence19. In the Drosophila nervous system the midline glial cells also express slit and act as an important intermediate target for contralateral projecting axons. In the Drosophila visual system, however, the target-derived molecules that stop R1–R6 growth cones in the lamina are still elusive. Another interesting question that deserves future attention is how glial migration is regulated by the ubiquitindependent protease Nonstop. Because migration of the border cells during Drosophila oogenesis also requires ubiquitin-dependent proteolysis20, it will be interesting to determine the function of the human nonstop ortholog. References 1 Tessier-Lavigne, M. and Goodman, C.S. (1996) The molecular biology of axon guidance. Science 274, 1123–1133 2 Poeck, B. et al. (2001) Glial cells mediate target layer selection of retinal axons in the developing visual system of Drosophila. Neuron 29, 99–113 3 Meinertzhagen, I.A. and Hanson, T.E. (1993) The development of the optic lobe. In Development of
12
13
14
15
16
17
18
19
20
Drosophila melanogaster (Vol. 2) (Bate, C.M. and Martinez-Arias, A., eds), pp. 1363–1491, Cold Spring Harbor Laboratory Press Huang, Z. and Kunes, S. (1996) Hedgehog, transmitted along retinal axons, triggers neurogenesis in the developing visual centers of the Drosophila brain. Cell 86, 411–422 Huang, Z. and Kunes, S. (1998) Signals transmitted along retinal axons in Drosophila: Hedgehog signal reception and the cell circuitry of lamina cartridge assembly. Development 125, 3753–3764 Huang, Z. et al. (1998) A retinal axon fascicle uses spitz, an EGF receptor ligand, to construct a synaptic cartridge in the brain of Drosophila. Cell 95, 693–703 Perez, S.E. and Steller, H. (1996) Migration of glial cells into retinal axon target field in Drosophila melanogaster. J. Neurobiol. 30, 359–373 Winberg, M.L. et al. (1992). Generation and early differentiation of glial cells in the first optic ganglion of Drosophila melanogaster. Development 115, 903–911 Clandinin, T.R. and Zipursky, S.L. (2000) Afferent growth cone interactions control synaptic specificity in the Drosophila visual system. Neuron 28, 427–436 Gibbs, S.M. and Truman, J.W. (1998) Nitric oxide and cyclic GMP regulate retinal patterning in the optic lobe of Drosophila. Neuron 20, 83–93 Martin, K.A. et al. (1995). Mutations disrupting neuronal connectivity in the Drosophila visual system. Neuron 14, 229–240 Newsome, T.P. et al. (2000) Analysis of Drosophila photoreceptor axon guidance in eye-specific mosaics. Development 127, 851–860 Garrity, P.A. et al. (1999) Retinal axon target selection in Drosophila is regulated by a receptor protein tyrosine phosphatase. Neuron 22, 707–717 Rao, Y. et al. (2000) Brakeless is required for photoreceptor growth-cone targeting in Drosophila. Proc. Natl. Acad. Sci. U. S. A. 97, 5966–5971 Ruan, W. et al. (1999) The SH2/SH3 adaptor protein dock interacts with the Ste20-like kinase misshapen in controlling growth cone motility. Neuron 24, 595–605 Senti, K. et al. (2000) Brakeless is required for lamina targeting of R1–R6 axons in the Drosophila visual system. Development 127, 2291–2301 Rossler, W. et al. (1999) Development of a glia-rich axon-sorting zone in the olfactory pathway of the moth Manduca sexta. J. Neurosci. 19, 9865–9877 Mason, C.A. and Sretavan, D.W. (1997) Glia, neurons, and axon pathfinding during optic chiasm development. Curr. Opin. Neurobiol. 7, 647–653 Erskine, L. et al. (2000) Retinal ganglion cell axon guidance in the mouse optic chiasm: expression and function of robos and slits. J. Neurosci. 20, 4975–4982 Rorth, P. et al. (2000) The level of C/EBP protein is critical for cell migration during Drosophila oogenesis and is tightly controlled by regulated degradation. Mol. Cell. 6, 23–30
Jan Pielage Christian Klämbt* Institut für Neurobiologie, Universität Münster, Badestr. 9, 48149 Münster, Germany. *e-mail: [email protected]