1 Division of Developmental Genetics,
2 National Institute of Genetics,
3 Department of Genetics, Graduate University for Advanced Studies, Mishima, 411-8540, Japan
4 CREST, Japan Science and Technology Corporation, Kawaguchi, 332-0012, Japan
*Author of correspondence at present address: Laboratory for Evolutionary Regeneration Biology, RIKEN Center for Developmental Biology, Kobe, 650-0047, Japan (e-mail: yumesono{at}lab.nig.ac.jp)
Accepted 11 February 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Notch, gcm, Nubbin, Gliogenesis, PNS, Asymmetric cell division, Drosophila melanogaster
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Drosophila, development of CNS as well as PNS glial cells is dependent on the expression of glial cells missing (gcm) (also called glide) (reviewed by Anderson, 1995). gcm encodes a transcription factor that is sufficient to activate the glial fate through regulation of the expression of its downstream target genes (Hosoya et al., 1995
; Jones et al., 1995
; Schreiber et al., 1997
). reversed polarity (repo), a glia-specific homeobox gene (Campbell et al., 1994
; Xiong et al., 1994
; Halter et al., 1995
), is a good candidate for a direct target of gcm, as it contains multiple GCM-binding sites in the 5' upstream region (Akiyama et al., 1996
). In gcm mutants, presumptive glial cells fail to differentiate, and are often transformed toward neurons. Thus, gcm acts as a binary switch between glial and neuronal cell fates and its transcriptional regulation plays a crucial role in their binary decisions.
Within the glial determination pathway, the gcm gene currently occupies the most upstream position (Hosoya et al., 1995; Jones et al., 1995
; Vincent et al., 1996
; Schreiber et al., 1997
). This suggests that gcm transcription is regulated by a combination of factors that themselves are not specific to glia. The gcm promoter may integrate a set of developmental signals that are identical in all gcm-positive glia. Alternatively, each glial subtype may have its own regulatory system, using various developmental cues differently depending on their context. Distinguishing these possibilities requires a comparative analysis of gcm regulation in multiple glial subtypes.
The transmembrane receptor Notch is used in many developmental contexts for determination of binary cell fates, such as asymmetric cell divisions (reviewed by Artavanis-Tsakonas et al., 1999). Dividing cells can receive a cell-intrinsic cue by Numb, a membrane protein containing a phosphotyrosine-binding domain, that binds Notch and represses Notch signaling in one of the daughter cells (Uemura et al., 1989
; Posakony, 1994
; Guo et al., 1995
; Jan and Jan, 1995
). Recently, a role for Notch signaling in glial differentiation was found in the adult PNS; Notch signaling negatively regulates gcm expression and glial cell differentiation during asymmetric division in the bristle lineage (Van De Bor and Giangrande, 2001
). Given the ability of Notch to function in many developmental decisions, Notch activation may be an obligatory signal in repressing gcm transcription in asymmetric divisions that generate glia. However, a previous report suggested an opposite role for Notch signaling in glial differentiation in the embryonic PNS. The dorsal bipolar dendritic (dbd) lineage consists of one neuron and a glial cell, where glial differentiation depends on gcm activity (Bodmer et al., 1989
; Brewster and Bodmer, 1995
; Jones et al., 1995
). In this lineage the numb mutation shows a double-glia phenotype at the expense of the neuron (Brewster and Bodmer, 1995
). If Numb acts by repressing Notch activity in the dbd lineage, this result would imply that Notch promotes, rather than represses, glial development in this lineage.
In this study, we present direct evidence that Notch positively regulates glial differentiation in the dbd lineage. Our data indicate that Notch signaling activates gcm expression in one of the two sibling cells and acts postmitotically to specify the glial fate. Thus, the effect of Notch signaling on gcm transcription is reversed in the bristle lineage compared to the dbd lineage. We identified an additional lineage where Notch promotes glial fate, and have shown that a molecular context similar to that of the dbd lineage works during asymmetric cell division. Our data indicates that one of the factors that provides Notch-dependent gliogenic context is likely the POU-domain protein Nubbin/PDM-1.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Embryo staining
Antibody staining was carried out as described previously (Ito et al., 1995). The following primary antibodies were used: rabbit anti-REPO (Halter et al., 1995
) (a gift from G. M. Technau) at 1:500; rat anti-REPO (Yuasa et al., personal communication) (a gift from H. Okano) at 1:500; rabbit anti-Nubbin/PDM-1 (Yeo et al., 1995
) (a gift from W. Chia) at 1:500; mouse anti-ELAV (9F8A9; Developmental Studies Hybridoma Bank) at 1:100; mouse mAb 22C10 (Fujita et al., 1982
) at 1:100-200 and mouse mAb anti-ß-galactosidase (40-1a; Developmental Studies Hybridoma Bank) at 1:100-200. Secondary antibodies used were biotinylated goat anti-rat, anti-rabbit, anti-mouse (Vector Laboratories), FITC-conjugated goat anti-rat and Cy3-conjugated goat anti-rabbit (Jackson) antibodies, all at 1:200. Biotinylated secondary antibodies were detected using the ABC elite kit (Vector Laboratories). Double labeling involving horseradish peroxidase (HRP) histochemistry was performed using diaminobenzidine as a substrate, with NiCl for the first staining, and without NiCl for the second staining. Immunofluorescence was viewed with a BioRad MRC 1024 confocal microscope. Mutant embryos were identified by the lack of anti-ß-galactosidase staining from the balancer chromosome or by their typical phenotypes reported previously. Whole-mount in situ hybridization was performed essentially as described previously (Lehmann and Tautz, 1994
). Digoxigenin-labeled RNA probes were generated from full-length gcm cDNA (Hosoya et al., 1995
) and from full-length repo cDNA (Xiong et al., 1994
) (a gift from H. Okano). Fluorescence-labeled RNA probe was generated from 1.2 kb PCR fragment derived from nubbin/pdm-1 cDNA. TSA system (NEN Life Science Product) was used for the fluorescence-labeled RNA detection. Images were processed using Photoshop software (Adobe).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Notch activity is restricted to the glia in the dbd lineage
To assess the role of Notch in the binary fate decision of the dbd lineage, we first analyzed Notch activity within this lineage. It is known that activation of Notch results in the nuclear translocation of an intracellular domain of Notch together with a transcription factor Suppressor of Hairless [Su(H)] (reviewed by Honjo, 1996; Weinmaster, 1997
; Bray, 1998
). We used transgenic lines containing a lacZ reporter construct driven by the E(spl)m
promoter fused to multimerized Su(H)-binding sites, which we refer to as the Su(H)-reporter. This line allows effective visualization of a direct response to the Su(H)-dependent Notch activity in vivo (Go et al., 1998
); in the sanpodo mutant, the phenotype of which mimicks the loss of Notch activity (Park et al., 1998
; Skeath and Doe, 1998
), the Su(H)-reporter activity was dramatically reduced throughout the embryo, as it was in Notch mutant embryos (Fig. 3A-C). During normal development of the dbd lineage, strong Su(H)-reporter activity was observed in the glial cell but not in the dbd neuron (Fig. 3D). When we expressed a constitutively active form of Notch (UAS-Notchact) in all neurons, the Su(H)-reporter became activated in the presumptive dbd neuron (Fig. 3E). Conversely, in the sanpodo mutation, the reporter expression was undetectable in the presumptive DBDG (Fig. 3F). Since the sanpodo mutation produces a double-neuron phenotype at the expense of the glial cell (Dye et al., 1998
), the reporter expression correlates with the glial fate. In contrast, forced gcm expression in the presumptive neuron failed to activate the Su(H)-reporter even though neuron-to-glia transformation took place (Jones et al., 1995
) (Fig. 3G). Thus Notch activity is likely to be upstream of gcm expression and glial differentiation.
|
|
Notch pathway activates glial fate
If Notch activity plays an instructive role in gliogenesis, artificial activation of Notch in the presumptive neuron may cause a neuron-to-glia transformation. When constitutively active Notch was expressed in all neurons, the dbd neuron was replaced by an extra REPO-positive cell that was associated with and resembled a glial cell (83% of hemisegments; n=42, Fig. 5B). In contrast, expression levels of neuronal markers ELAV (data not shown) and Nubbin were dramatically reduced in the dbd lineage (Fig. 5F). Accompanying glial transformation of the presumptive dbd neuron to a glial cell, gcm was ectopically expressed in this cell. gcm responded quickly to Notch activation; at stage 12, gcm mRNA was already detectable in the presumptive neuron, coinciding with the expression of the driver construct (Fig. 5J). Although normal expression of gcm in the glial cell was transient, ectopic gcm that was induced by constitutively active Notch continued to at least stage 16 (Fig. 5L). This is unlikely to be due to autoregulation, because forced expression of gcm in the dbd neuron did not activate transcription of a lacZ reporter gene inserted into the gcm locus (data not shown). These data indicate that within the dbd lineage Notch activation is sufficient for inducing gcm expression.
|
The Notch pathway can activate gcm transcription outside the dbd lineage
While the dbd lineage is the only place in the embryonic PNS where Notch activity is necessary for glial development, we discovered that artificial activation of Notch can initiate gliogenesis outside this lineage. When constitutively active Notch was expressed in all neurons, often two REPO-positive cells formed in the dorsal cluster of sensory organs (Fig. 6A). One of these cells is the transformed dbd neuron, as discussed above. We identified that another cell was derived from the dorsal dendritic arbor (dda) organ (Lloyd and Sakonju, 1991; Brewster et al., 2001
) (Fig. 6A), and focused our analysis on this dda lineage.
|
To analyze the role of Notch signaling within the dda lineage, we examined the effects of removing sanpodo and numb activities. In normal embryos a single dda neuron can be found dorsal to the dbd organ in each abdominal hemisegment at stage 16. In sanpodo mutant embryos, duplication of the dda neuron was frequently observed (71% of hemisegments; n=49, Fig. 5H), whereas in numb mutants they were absent (63% of hemisegments; n=49, Fig. 5G). Thus, as in the dbd lineage, Notch activity represses neuronal development in the dda lineage. In numb mutant embryos, we detected ectopic gcm and REPO expression in the presumptive dda neurons (34% of hemisegments; n=41, Fig. 5D and data not shown), a phenotype mimicking the artificial activation of Notch in neurons (Fig. 5L, Fig. 6A). We conclude that Notch signaling can activate gcm transcription not only in the dbd lineage, but also in the dda lineage.
The specific response of dbd and dda lineages to Notch activation suggests that factors specifically expressed in these lineages may provide the developmental contexts that allow Notch activation to be interpreted as a gliogenic signal. Although the proneural gene amos is specifically expressed in these two lineages, its expression is transient and is absent by the time of the SOP division (Huang et al., 2000). Furthermore, coexpression of AMOS and constitutively active Notch in all neurons did not generate any additional glial cells compared to Notch activation alone (data not shown). Another candidate is the POU-domain protein Nubbin. In both dbd and dda lineages, Nubbin expression is initiated in the SOP and high levels of Nubbin protein are found in both daughter cells after the SOP division (Fig. 2, Fig. 6B). To test whether Nubbin can modify the effect of Notch activation, we coexpressed Nubbin and constitutively active Notch in all neurons. Upon such treatment, a few extra REPO-positive glial cells appeared dorsally to the dda lineage (Fig. 6C). These cells likely correspond to the extra glial cells that have been reported to form upon ectopic expression of GCM in neurons (Jones et al., 1995
). This suggests that some of the presumptive neurons in the dorsal cluster were redirected to the glial differentiation pathway upon Notch activation. Such a phenotype was never observed when constitutively active Notch alone or Nubbin alone was expressed (data not shown). We propose that Nubbin is one of the factors that provide a developmental context for Notch-dependent gcm expression and glial differentiation.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
While our present data demonstrate a positive role for Notch in gliogenesis in the dbd lineage, other embryonic PNS glial cells do not require Notch activity for their formation. For example, in the adult bristle lineage Notch has an opposite function on gliogenesis; that of repressing gcm expression and glial development (Van De Bor and Giangrande, 2001). Thus the role of Notch in the regulation of gcm expression is context-dependent (Fig. 7). Notch has recently been shown to be a component of combinatorial signaling in cell fate determination in the Drosophila eye (Flores et al., 2000
). It is possible that Notch signaling has different consequences depending on other factors that act on the same regulatory element.
|
One common feature that distinguishes dbd and dda lineages from other PNS lineages is the cell division pattern of their SOP. In dbd and dda lineages, SOPs divide to generate a neuron and a glial cell through an asymmetric division. In other gliogenic PNS lineages, the sibling cells of glial cells are not postmitotic neurons, but tertiary precursors that undergo further division to generate neurons and associated cells (Fig. 7). These observations suggest that an interaction with the neuronal sibling may play a crucial part in promoting the Notch-dependent gcm activation during asymmetric cell division. Recently, Notch was shown to positively regulate gcm expression in the Neuroblast 1-1A lineage of the CNS, where the sibling pattern is identical to that of the dbd lineage (Udolph et al., 2001) (Fig. 7C). This also supports the idea that the cell division pattern provides a context that determines the effect of Notch activity.
We showed that coexpression of constitutively active Notch with Nubbin also generates ectopic glia outside dbd and dda lineages. This raises the possibility that Nubbin may be a part of the developmental context that allows Notch to promote gliogenesis. Within the embryonic PNS, dbd and dda neurons are the only two neurons that express Nubbin. In both lineages, Nubbin is present in both SOP daughter cells, at the time of glia versus neuron cell fate choice. Furthermore, we detected temporal activation of Nubbin in presumptive glial cells derived from the NB1-1A lineage (data not shown). Nubbin thus might create a permissive environment for the activation of gcm expression by the Notch signal (Fig. 7A-C). Since coexpression of Nubbin and constitutively active Notch does not cause glial transformation of all neurons, additional factors must exist that create a Notch-dependent gliogenic context.
Nubbin is a POU-domain transcription factor with sequence-specific DNA-binding activity (Neumann and Cohen, 1998). The contextual role of Nubbin in Notch-dependent expression of gcm could employ a similar mechanism to the modulation of Notch activity in wing development, where Nubbin and Su(H) bind on the same enhancer element of Notch target genes (Neumann and Cohen, 1998
). It will be interesting to further analyze the role of Nubbin in gliogenic lineages.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akiyama, Y., Hosoya, T., Poole, A. M. and Hotta, Y. (1996). The gcm-motif: a novel DNA-binding motif conserved in Drosophila and mammals. Proc. Natl. Acad. Sci. USA 93, 14912-14916.
Anderson, D. J. (1995). A molecular switch for the neuron-glia developmental decision. Neuron 15, 1219-1222.
Artavanis-Tsakonas, S., Matsuno, K. and Fortini, M. E. (1995). Notch signaling. Science 268, 225-232.[Medline]
Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J. (1999). Notch signaling: cell fate control and signal integration in development. Science 284, 770-776.[Medline]
Bailey, A. M. and Posakony, J. W. (1995). Suppressor of Hairless directly activates transcription of Enhancer of split complex genes in response to Notch receptor activity. Genes Dev. 9, 2609-2622.
Bier, E., Ackerman, L., Baebel, S., Jan, L. and Jan, Y. N. (1988). Identification and characterization of a neuron-specific nuclear antigen in Drosophila. Science 240, 913-915.[Abstract]
Bodmer, R., Carretto, R. and Jan, Y. N. (1989). Neurogenesis of the peripheral nervous system in Drosophila embryos: DNA replication patterns and cell lineages. Neuron 3, 21-32.[Medline]
Bray, S. (1998). A Notch Affair. Cell 93, 499-503.[Medline]
Brewster, R. and Bodmer, R. (1995). Origin and specification of type II sensory neurons in Drosophila. Development 121, 2923-2936.[Medline]
Brewster, R., Hardiman, K., Deo, M., Khan, S. and Bodmer, R. (2001). The selector gene cut represses a neural cell fate that is specified independently of the Achaete-Scute-Complex and atonal. Mech. Dev. 105, 57-68.
Campbell, G., Göring, H., Lin, T., Spana, E., Andersson, S., Doe, C. Q. and Tomlinson, A. (1994). RK2, a glial-specific homeodomain protein required for embryonic nerve cord condensation and viability in Drosophila. Development 120, 2957-2966.[Medline]
Campos-Ortega, J. A. (1995). Genetic mechanisms of early neurogenesis in Drosophila melanogaster. Mol. Neurobiol. 10, 75-89.
Campuzano, S. and Modolell, J. (1992). Patterning of the Drosophila nervous system: the achaete-scute gene complex. Trends Genet. 8, 202-208.[Medline]
Condron, B. G. and Zinn, K. (1994). The grasshopper median neuroblast is a multipotent progenitor cell that generates glia and neurons in distinct temporal phases. J. Neurosci. 14. 5766-5777.[Medline]
Dick, T., Yang, X., Yeo, S and Chia, W. (1991). Two closely linked Drosophila POU domain genes are expressed in neuroblasts and sensory elements. Proc. Natl. Acad. Sci. USA 88, 7645-7649.[Abstract]
Doherty, D., Feger, G., Younger-Shepherd, S., Jan, L. Y. and Jan, Y. N. (1996). Delta is a ventral to dorsal signal complementary to Serrate, another Notch ligand, in Drosophila wing formation. Genes Dev. 10, 421-434.[Abstract]
Dye, C. A., Lee, J., Atkinson, R. C. Brewster, R., Han, P. and Bellen, H. J. (1998). The Drosophila sanpodo gene controls sibling cell fate and encodes a tropomodulin homolog, an actin/tropomyosin-associated protein. Development 125, 1845-1856.[Abstract]
Flores, G. V., Duan, H., Yan, H., Nagaraj, R., Fu, W., Zou, Y., Noll, M. and Banerjee, U. (2000). Combinatorial signaling in the specification of unique cell fates. Cell 103, 75-85.
Fujita, S. C., Zipursky, S. L., Benzer, S., Ferrus, A. and Shotwell, S. L. (1982). Monoclonal antibodies against the Drosophila nervous system. Proc. Natl. Acad. Sci. USA 79, 7929-7933.[Medline]
Go, J. M., Eastman, S. D. and Artavanis-Tsakonas, S. (1998). Cell proliferation control by Notch signaling in Drosophila development. Development 125, 2031-2040.[Abstract]
Guo, M., Bier, E., Jan, L. Y. and Jan, Y. N. (1995). tramtrack acts downstream of numb to specify distinct daughter cell fates during asymmetric cell divisions in the Drosophila PNS. Neuron 14, 913-925.
Halter, D. A., Urban, J., Rickert, C., Ner, S. S., Ito, K., Travers, A. A. and Technau, G. M. (1995). The homeobox gene repo is required for the differentiation and maintenance of glia function in the embryonic nervous system of Drosophila melanogaster. Development 121, 317-332.[Medline]
Honjo, T. (1996). The shortest path from the surface to the nucleus: RBP-Jk/Su(H) transcription factor. Genes Cells 1, 1-9.
Hosoya, T., Takizawa, K., Nitta, K. and Hotta, Y. (1995). glial cells missing: a binary switch between neuronal and glial determination in Drosophila. Cell 82, 1025-1036.
Huang, M., Hsu, C. and Chien, C. (2000). The proneural gene amos promotes multiple dendritic neuron formation in the Drosophila peripheral nervous system. Neuron 25, 57-67.[Medline]
Ito, K., Urban, J. and Technau, G. M. (1995). Distribution, classification, and development of Drosophila glial cells in the late embryonic and early larval ventral nerve cord. Rouxs Arch. Dev. Biol. 204, 284-307.[Medline]
Jan, Y. N. and Jan, L. Y. (1995). Maggots hair and bugs eye: role of cell interactions and intrinsic factors in cell fate specification. Neuron 14, 1-5.
Jones, B. W., Fetter, R. D., Tear, G. and Goodman, C. S. (1995). glial cells missing: a genetic switch that controls glial versus neuronal fate. Cell 82, 1013-1023.[Medline]
Lecourtois, M. and Schweisguth, F. (1995). The neurogenic Suppressor of Hairless DNA-binding protein mediates the transcriptional activation of the Enhancer of split complex genes triggered by Notch signaling. Genes Dev. 9, 2598-2608.[Medline]
Lehmann, R. and Tautz, D. (1994). In situ hybridization to RNA. In Methods in Cell Biology vol. 44: Drosophila melanogaster (eds Goldstein and Fyrberg), pp. 575-598. San Diego: Academic Press.[Abstract]
Lin, D. M. and Goodman, C. S. (1994). Ectopic and increased expression of fasciclin II alters motoneuron growth cone guidance. Neuron 13, 507-523.[Medline]
Lindsley, D. L. and Zimm, G. G. (1992). The genome of Drosophila melanogaster. San Diego: Academic Press.[Medline]
Lloyd, A. and Sakonju, S. (1991). Characterization of two Drosophila POU domains genes, related to oct-1 and oct-2, and regulation of their expression patterns. Mech. Dev. 36, 87-102.[Medline]
Luskin, M. B., Pearlman, A. L. and Sanes, J. R. (1988). Cell lineage in the cerebral cortex of the mouse studied in vivo and in vitro with a recombinant retrovirus. Neuron 1, 635-647.[Medline]
Neumann, C. J. and Cohen, S. M. (1998). Boundary formation in Drosophila wing: Notch activity attenuated by the POU protein Nubbin. Science 281, 409-413.[Medline]
Park, M., Yaich, L. E. and Bodmer, R. (1998). Mesodermal cell fate decisions in Drosophila are under the control of the lineage genes numb, Notch, and sanpodo. Mech. Dev. 75, 117-126.
Posakony, J. W. (1994). Nature versus nurture: asymmetric cell divisions in Drosophila bristle development. Cell 76, 415-418.[Medline]
Schreiber, J., Sock, E. and Wegner, M. (1997). The regulator of early gliogenesis glial cells missing is a transcription factor with a novel type of DNA-binding domain. Proc. Natl. Acad. Sci. USA 94, 4739-4744.[Medline]
Simpson, P. (1994). The Notch Receptors. Austin, Texas: R. G. Landes Company.
Skeath, J. B. and Doe, C. Q. (1998). Sanpodo and Notch act in opposition to Numb to distinguish sibling neuron fates in the Drosophila CNS. Development 125, 1857-1865.
Turner, D. L. and Cepko, C. L. (1987). A common progenitor for neurons and glia persists in rat retina late in development. Nature 328, 131-136.
Udolph, G., Prokop, A., Bossing, T. and Technau, G. M. (1993). A common precursor for glia and neurons in the embryonic CNS of Drosophila gives rise to segment-specific lineage variants. Development 118, 765-775.[Medline]
Udolph, G., Rath, P. and Chia, W. (2001). A requirement for Notch in the genesis of a subset of glial cells in the Drosophila embryonic central nervous system which arise through asymmetric cell divisions. Development 128, 1457-1466.
Uemura, T., Sheperd, S., Ackerman, L., Jan, L. Y. and Jan, Y. N. (1989). numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos. Cell 58, 349-360.
Van De Bor, V. and Giangrande, A. (2001). Notch signaling represses the glial fate in fly PNS. Development 128, 1381-1390.[Medline]
Vincent, S., Vonesch, J.-L. and Giangrande, A. (1996). glide directs glial fate commitment and cell fate switch between neurones and glia. Development 122, 131-139.
Weinmaster, G. (1997). The ins and outs of Notch signaling. Mol. Cell. Neurosci. 9, 91-102.
Xiong, W. C., Okano, H., Patel, N. H., Blendy, J. A. and Montell, C. (1994). repo encodes a glial-specific homeo domain protein required in the Drosophila nervous system. Genes Dev. 8, 981-994.[Medline]
Yeo, S. L., Lloyd, A., Kozak, K., Dinh, A., Dick, T., Yang, X., Sakonju, S. and Chia, W. (1995). On the functional overlap between two Drosophila POU homeo domain genes and the cell fate specification of a CNS neural precursor. Genes Dev. 9, 1223-1236.[Abstract]