Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue 68-230B, Cambridge, MA 02139, USA
Author for correspondence (e-mail:
pgarrity{at}mit.edu)
Accepted 23 September 2004
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SUMMARY |
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Key words: Glia, Neuron, Compartment boundary, Optic lobe, Drosophila
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Introduction |
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Mechanisms that have been proposed to restrain cell mixing between
compartments include preferential adhesion among cells within a compartment,
preferential adhesion between cells of different compartments at the
compartment boundary, and mutual repulsion between cells of different
compartments (Dahmann and Basler,
1999; Irvine and Rauskolb,
2001
; McNeill,
2000
; Milan et al.,
2001
). Members of the Cadherin family of adhesion molecules and
the Eph/Ephrin family of repellant signaling proteins have been implicated in
regulating cell mixing between compartments in the developing vertebrate
nervous system (Cooke and Moens,
2002
; Inoue et al.,
2001
; Redies,
2000
; Xu et al.,
2000
). In-vitro reconstitution experiments have shown that
differential Cadherin expression or Eph/Ephrin signaling is sufficient to
create groups of non-intermingling cells
(Mellitzer et al., 1999
;
Nose et al., 1988
), while
ectopic expression and dominant-negative studies have shown that these
proteins can alter cell sorting in vivo
(Cooke and Moens, 2002
;
Inoue et al., 2001
;
Xu et al., 1999
). However,
loss-of-function analysis has not yet demonstrated a requirement for either
Cadherin expression or Eph/Ephrin signaling in restricting cell movement
between compartments of the developing brain
(Cooke and Moens, 2002
;
Inoue et al., 2001
).
The developing Drosophila melanogaster brain, like the vertebrate
brain, contains many compartments that give rise to multiple, anatomically
distinct processing centers, and recent work has begun to detail the
morphogenetic events of fly brain development comprehensively
(Dumstrei et al., 2003;
Hartenstein et al., 1998
;
Meinertzhagen et al., 1998
;
Nassif et al., 2003
;
Younossi-Hartenstein et al.,
2003
). The visual centers of the fly brain, the optic lobes,
contain four ganglia (the lamina, medulla, lobula and lobula plate), which are
derived from two distinct populations of progenitor cells, the outer and inner
optic anlagen (Hofbauer and Campos-Ortega,
1990
; Meinertzhagen and
Hanson, 1993
;
Younossi-Hartenstein et al.,
1996
). Progeny of the outer optic anlagen contribute to the lamina
and outer medulla, while progeny of the inner optic anlagen contribute to the
inner medulla, lobula and lobula plate. Descendents of these different anlagen
lie adjacent to one another during development without intermingling and act
as distinct developmental compartments within the brain. For example, the
neurons and glia of the developing lamina, derived from the outer optic
anlagen (Dearborn and Kunes,
2004
; Meinertzhagen and
Hanson, 1993
), lie immediately adjacent to the neurons of the
developing lobula cortex, which are derived from the inner optic anlagen
(Hofbauer and Campos-Ortega,
1990
; Meinertzhagen and
Hanson, 1993
), but the two cell populations remain distinct. How
these cell populations are prevented from intermingling is unknown.
The Slit and Robo protein families are essential for axon guidance and cell
migration in worms, flies, fish and mice
(Brose and Tessier-Lavigne,
2000; Wong et al.,
2002
). Slits are secreted proteins that can act as either
attractive or repulsive guidance cues
(Englund et al., 2002
;
Kramer et al., 2001
), while
members of the Robo family encode transmembrane receptors for Slits
(Brose et al., 1999
;
Rajagopalan et al., 2000b
;
Simpson et al., 2000b
).
Drosophila has a single Slit receptor and three Robo receptors [Robo,
Robo2 (Leak FlyBase) and Robo3]
(Kidd et al., 1999
;
Rajagopalan et al., 2000a
;
Rajagopalan et al., 2000b
;
Simpson et al., 2000a
;
Simpson et al., 2000b
). The
recent identification of mutations in human ROBO3 (RIG1) in individuals with
horizontal gaze palsy and progressive scoliosis with hindbrain dysplasia
demonstrates that ROBO-receptor function is also important for human brain
development (Jen et al.,
2004
).
In the present work, we identify members of the Slit and Robo families as key factors that limit cell mixing between two adjacent cell populations in the Drosophila brain, the lamina glia and the distal cell neurons of the lobula cortex. We characterize a set of molecular markers that permit us to examine the behavior of cells at the boundary between the lamina and the lobula cortex. We find that Slit protein surrounds the lamina glia, while the distal cell neurons of the lobula cortex express multiple Robo family receptors. We show that either loss of Slit or the tissue-specific knockdown of multiple Robo family members causes distal cell neurons to intermingle with the lamina glia, disrupting the boundary between the lamina and lobula cortex. We propose that Slit and Robo family proteins prevent cell mixing at the lamina/lobula interface, enforcing a boundary between adjacent compartments of the developing Drosophila brain that is essential for morphogenesis of the visual system.
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Materials and methods |
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Immunohistochemistry and in-situ hybridization
Third-instar whole mounts were performed as described
(Garrity et al., 1996). Distal
cell neuron positioning, glial positioning and photoreceptor axon targeting
defects were observed in all slit and triple Robo family RNA
interference (RNAi) animals examined and more than 20 hemispheres were
examined for each genotype. The following primary antibodies were obtained
from the Developmental Studies Hybridoma Bank and used at the concentrations
indicated: 24B10 mAb (1:200); Slit C555.6D (1:200); Robo mAb 13C9 (1:200);
Robo3 mAb 14C9 (1:200); Fas2 1D4 (1:200); Fas3 7G10 (1:50); Repo 8D12 (1:200);
Elav 7E8A10 (1:20); and ß-galactosidase 40-1A (1:200). Robo2 polyclonal
antisera (1:750) (Rajagopalan et al.,
2000b
; Simpson et al.,
2000a
) were provided by C. Goodman and by B. Dickson, and Repo
polyclonal (1:1000) (Campbell et al.,
1994
) by A. Tomlinson. Anti-phosphohistone H3 (1:200) was
purchased from Upstate Biotechnology. Secondary antibodies were obtained from
Jackson Laboratories and used at the following concentrations: goat-anti-mouse
hrp-conjugated (1:200); goat-anti-mouse Cy3-conjugated (1:500); goat-rat-mouse
Cy5-conjugated (1:400). Fluorescent samples were visualized using a Nikon
PCM2000 confocal microscope. In-situ hybridization was performed as described
(Wolff, 2000
).
Molecular biology
Genomic DNA flanking the slitdui P-element was isolated
by plasmid rescue and sequenced to identify the insertion site as described
(Garrity et al., 1996).
Western blot analysis used the following antibodies: Robo mAb 13C9 (1:2000);
Robo3 mAb 14C9 (1:1000); Elav 7E8A10 (1:1000); anti-hrp-conjugated secondary
antibody (1:5000). Robo family RNAi constructs were generated using the
strategy described (Kalidas and Smith,
2002
). Fragments for creating the RNAi constructs were generated
by PCR (Expand Hi-Fidelity, Roche) and cloned into pUASt
(Brand and Perrimon, 1993
). PCR
primers used to create UAS-RoboRNAi were: genomic fragment
5'-ACCGGGCAGCTGATCCTAGC and
5'-ATACTAGTCTGTCGAATAATAAGAAGATATAAAATGATTC; cDNA fragment
5'-TGTCAGTCGCACCAGCATTAGTC and 5'-ATACTAGTCATCTTCATAGGTGAGGGCTGTC.
PCR primers used to create UAS-Robo2RNAi were: genomic fragment
5'-GTTCCCTCTGAGGCACCATATG and 5'-ATACTAGTGTGTGATTGCCTGCAGGTGAG;
cDNA fragment 5'-GTTCCCTCTGAGGCACCATATG and
5'-ATACTAGTCCACGCATTGTATTTAGGGCCG. PCR primers used to create
UAS-Robo3RNAi were: genomic fragment 5'-TATATCGCAGTGGCGGCTGCC and
5'-ATAGATCTCTGCAATTGGAGGGGATGAAATCAG; cDNA fragment
5'-TATATCGCAGTGGCGGCTGCC and 5'-ATAGATCTCTCTCGTAATCGGGTAGCAGC.
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Results |
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As the ingrowth of photoreceptor axons induces many developmental events in the optic lobe, we tested whether Slit production depended upon photoreceptor axon innervation. Slit protein was still present in the optic lobe of eyes absent (eya) mutant animals that had no photoreceptor neurons, indicating that photoreceptor axon innervation was not essential for Slit production (Fig. 4I).
To begin to further characterize the identity of the cells producing Slit protein in the optic lobe, we examined optic lobe expression of an enhancer trap transposon insertion in the Slit locus. The slitl(2)k05248 insertion is located 30,258 bases upstream of the Slit mRNA start site, 1853 bases from the slitdui insertion site, and behaves as a loss-of-function slit allele in the visual system (see Fig. 2E). Expression of the lacZ enhancer trap in slitl(2)k05248 resembled the Slit RNA in-situ pattern, with strong expression in the optic lobe and in the midline of the ventral ganglion, and is referred to here as Slit:lacZ. Slit:lacZ was expressed at the base of the lamina by the medulla glia, the most basal of the three layers of lamina glia (Fig. 4J). Slit:lacZ was also expressed by cells in the medulla cortex (Fig. 4J). These cells lay immediately adjacent to the glia that surround the medulla neuropil (Fig. 4K) and appear to be differentiating neurons of the medulla cortex as they express varying levels of the neuronal marker Elav (Fig. 4L). Medulla cortex neurons are known to project axons into the medulla neuropil and could thus deliver Slit protein to the medulla neuropil region. These Slit:lacZ enhancer trap data combine with the Slit protein and RNA in-situ data to provide a consistent picture, in which expression of Slit, a diffusible protein, by cells at the base of the lamina and at the periphery of the medulla generate a region of Slit expression extending from the lamina into the medulla.
Distal cell neurons express Robo family proteins
Robo family receptors commonly mediate responses to Slit proteins, so we
characterized the distribution of the three Drosophila Robo proteins
in the developing visual system. Robo, Robo2 and Robo3 were all expressed
within the developing optic lobes (Fig.
5A,D,G). More detailed analysis of Robo and Robo2 expression
showed that both proteins were expressed by IPC neuroblasts and distal cell
neurons (Fig. 5B,C,E,F). Robo3
protein was not detected on IPC neuroblasts, but was present on distal cell
neurons (Fig. 5H,I). Thus, all
three Robo receptors were expressed within the developing lobula cortex in
partially overlapping patterns, consistent with Robo family receptors
mediating responses to Slit in this region of the visual system.
|
As robo, robo2 and robo3 have partially redundant
functions in the embyronic central nervous system
(Rajagopalan et al., 2000a;
Rajagopalan et al., 2000b
;
Simpson et al., 2000a
;
Simpson et al., 2000b
), we
wanted to examine the effect of simultaneous disruption of multiple Robo
family proteins in the visual system. However, analysis of Robo family
function using existing alleles proved insufficient. First, marked clones of
robo5 mutant tissue were generated in a homozygous
robo31 background, but no defects were observed (T.D.T.
and P.A.G., unpublished). Second, robo,robo2 double mutant mosaic
analysis could not be performed because the necessary animals did not survive
to form adult visual systems, and the proximity of the robo2 and
robo3 genes [87 kb (Simpson et
al., 2000b
)] prevented the creation of a robo2,robo3
recombinant. Third, we determined that the only existing mutant allele of
robo3 (robo31), characterized as a strong
loss-of-function or null allele in the embryo
(Rajagopalan et al., 2000b
),
produced substantial quantities of full-length Robo3 protein and increased
levels of a lower molecular weight form of Robo3 in the adult head
(Fig. 4A). Significant amounts
of Robo3 immunostaining were also observed in the developing visual system of
robo31 animals (T.D.T. and P.A.G., unpublished),
suggesting that robo31 is not a null in the visual system.
Therefore, a different strategy was used to achieve simultaneous inhibition of
robo, robo2 and robo3 in the visual system.
Tissue-specific transgenic RNAi was used to inhibit expression of each of the Robos. UAS-RoboRNAi, UAS-Robo2RNAi and UAS-Robo3RNAi transgenic flies were generated and the transgenes proved effective inhibitors of their targets as assessed using a combination of Western blot analysis and tissue staining (Fig. 6B-M). As shown in Fig. 6E-M, expression of UAS-RoboRNAi, UAS-Robo2RNAi or UAS-Robo3RNAi under the control of Gal4 substantially reduced expression of the corresponding Robo family protein without detectably affecting expression of other Robo family members. Thus, these transgenic RNAi constructs permitted inducible knockdown of each Robo family protein.
|
|
As the role of Slit in visual system development was initially identified through its effect on photoreceptor axon targeting, we examined whether photoreceptor axon targeting was similarly dependent upon Robo family receptors. Indeed, generalized inhibition of all three Robo receptors under the control of Tubulin-Gal4 in Tubulin-Gal4,UAS-RoboRNAi,UAS-Robo2RNAi,UAS-Robo3RNAi animals disrupted photoreceptor axon targeting in a fashion similar to that observed in slit mutants (Fig. 8A,B). Interestingly, simultaneous expression of UAS-RoboRNAi,UAS-Robo2RNAi and UAS-Robo3RNAi under the control of the eye-specific Gal4 source GMR-Gal4 generated no defects in photoreceptor axon targeting (Fig. 8C), while inhibition of Robo family expression using Sca-Gal4 did disrupt photoreceptor axon targeting (Fig. 8D,E,F). In fact, regions of photoreceptor mistargeting corresponded to regions where Sca-Gal4 cells (distal cell neurons) entered the lamina (Fig. 8D,E,F). While these knockdown experiments do not preclude a role for Robo family receptors in the photoreceptors, they nonetheless consistent with the misplacement of distal cell neurons contributes to photoreceptor axon mistargeting (Fig. 8G). These data also further emphasize the similarity of the effects of knockdown of Robo family receptors and reductions in Slit expression on optic lobe morphogenesis.
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Discussion |
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Slit and Robo family proteins are regulators of boundary maintenance
Compartmentalization is important throughout nervous system development
(Pasini and Wilkinson, 2002),
and structural compartmentalization underlies functional compartmentalization
in the adult brain. The adult vertebrate brain contains many distinct
compartments, such as Brodmann's areas of the cerebral cortex and the
brainstem nuclei, and anatomical studies point to similar compartmentalization
in the Drosophila brain
(Younossi-Hartenstein et al.,
2003
). As noted above, several molecules that regulate cell
adhesion or cell repulsion have been implicated in restricting cell mixing
between compartments in the developing nervous system, but loss of these
proteins has not been shown to cause intermingling between compartments. Here
we have shown that Slit and the Robos are required to prevent cell
intermingling across a boundary in the optic lobe.
We determined that knockdown of Robo family protein expression in the optic lobe using the Sca-Gal4 driver caused robust defects in distal cell neuron positioning. In addition to driving gene expression in the inner proliferation center neuroblasts and distal cell neurons, Sca-Gal4 also drives expression in R8 photoreceptor axons and neuroblasts of the outer proliferation center and neurons of the medulla cortex. As noted above, inhibition of Robo family expression only in the photoreceptors caused no detectable defects. In addition, knockdown of all three Robo family proteins in the medulla cortex using apterous-Gal4 had no effect on distal cell neuron behavior, and no defects in medulla neuron movement or axon targeting were identified in either slit mutants or Robo family knockdowns (T.D.T. and P.A.G., unpublished). Taken together with Robo family protein expression data, the Robo family knockdown analysis strongly supports a requirement for Robo family receptors in distal cell neurons in preventing them from invading the lamina neuropil.
Slit and Robo family protein expression in the optic lobe
In the Drosophila visual system, Slit protein is present in a
continuous zone from the base of the lamina into the underlying medulla
neuropil. Although Slit mRNA is detected within the optic lobe, and
Slit:lacZ expression is detected in medulla glia at the base of the
lamina and in medulla cortex neurons, the optic lobe does not appear highly
sensitive to the precise source or concentration of Slit. Attempts to use
mosaic analysis to further define the cells in which slit function
was required were unsuccessful, as no phenotypes were observed, despite the
generation of large marked patches of slit2 mutant tissue
in the visual system and the use of the Minute technique to maximize mutant
clone size (T.D.T. and P.A.G., unpublished). We suspect that the diffusibility
of Slit protein combined with the large number of Slit-expressing cells in the
optic lobe permitted the remaining heterozygous and wild-type cells in the
mosaic animals to provide sufficient Slit to support proper optic lobe
development. In addition, expression of Slit in photoreceptors under the
control of GMR-Gal4 rescued the photoreceptor projection phenotype of
slit mutants as effectively as more general expression of Slit in the
optic lobe using Omb-Gal4. Thus, delivery of Slit to these neuropil
regions may be sufficient to restore the boundary between the lobula cortex
and the lamina.
We also examined the effects of overexpression and ectopic expression of Slit and Robo proteins in the optic lobe. Overexpression of Slit in the optic lobe using GMR-Gal4, Sca-Gal4, Omb-Gal4 or the more ubiquitously expressed Tubulin-Gal4 did not generate detectable phenotypes in the optic lobe (T.D.T. and P.A.G., unpublished). The failure to generate strong overexpression phenotypes could reflect the increased Slit expression within the lamina that accompanied overexpression in other regions using these Gal4 drivers. However, overexpression of Robo2 under the control of Sca-Gal4 dramatically distorted the shape of the lobula cortex, causing the distal cell neurons to move around the ventral and dorsal edges of the lamina (T.D.T. and P.A.G., unpublished). As distal cell neurons normally encounter Slit protein at the posterior face of the lamina, this redistribution could reflect repulsion from regions of Slit expression. Overexpression of Robo or Robo3 caused no detectable defects.
Robo family proteins appear to localize around the cell body periphery of
newly differentiated distal cell neurons. This cell-body-associated expression
contrasts with the predominantly axonal expression of Robo family proteins by
more mature lobula cortex neurons. Whether this reflects a regulated shift in
the subcellular localization of Robo proteins, or simply the availability of
axonal processes in more mature neurons, is unknown. However, as Slit and Robo
family proteins control both neuronal migration and axon navigation
(Wong et al., 2002), such a
change in Robo family protein distribution could alter the response of a
neuron to Slit from one involving the cell body to one preferentially
involving the axon. We have not detected obvious misprojections of the axons
of the distal cell neurons in our mutants (T.D.T. and P.A.G., unpublished),
although subtle defects in targeting of these axons would not be detected
using available markers.
Regulation of cell mixing at boundaries in the developing brain
Boundaries are commonly encountered during development, and several
mechanisms have been proposed for preventing mixing between compartments. Our
observations provide evidence for a signal associated with one cell population
preventing the invasion of a neighboring cell population expressing receptors
for that signal. Interestingly, even when the distal cell neurons invade the
lamina in slit mutants or Robo family knockdown animals, they do not
disperse evenly among the lamina glia. Rather, the distal cell neurons remain
preferentially associated with one another, suggesting the persistence of
differential adhesion when the Slit signal is absent. Thus, multiple parallel
mechanisms, possibly involving both repulsion and differential adhesion, are
potentially involved in maintaining the normally precise distinction between
lamina and lobula cortex. Combinations of adhesion and repulsion may act at
other boundaries, providing robustness as well as functional redundancy to the
molecular mechanisms of compartment maintenance.
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ACKNOWLEDGMENTS |
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Footnotes |
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