Drosophilatype II neuroblast lineages keep Prospero levels low to generate large clones that contribute to the adult brain central complex
© Bayraktar et al; licensee BioMed Central Ltd. 2010
Received: 10 June 2010
Accepted: 1 October 2010
Published: 1 October 2010
Tissue homeostasis depends on the ability of stem cells to properly regulate self-renewal versus differentiation. Drosophila neural stem cells (neuroblasts) are a model system to study self-renewal and differentiation. Recent work has identified two types of larval neuroblasts that have different self-renewal/differentiation properties. Type I neuroblasts bud off a series of small basal daughter cells (ganglion mother cells) that each generate two neurons. Type II neuroblasts bud off small basal daughter cells called intermediate progenitors (INPs), with each INP generating 6 to 12 neurons. Type I neuroblasts and INPs have nuclear Asense and cytoplasmic Prospero, whereas type II neuroblasts lack both these transcription factors. Here we test whether Prospero distinguishes type I/II neuroblast identity or proliferation profile, using several newly characterized Gal4 lines. We misexpress prospero using the R19H09-Gal4 line (expressed in type II neuroblasts but no adjacent type I neuroblasts) or R9D11-Gal4 line (expressed in INPs but not type II neuroblasts). We find that differential prospero expression does not distinguish type I and type II neuroblast identities, but Prospero regulates proliferation in both type I and type II neuroblast lineages. In addition, we use R9D11 lineage tracing to show that type II lineages generate both small-field and large-field neurons within the adult central complex, a brain region required for locomotion, flight, and visual pattern memory.
Drosophila neural progenitors, called neuroblasts (NBs), are an excellent model system to study progenitor self-renewal and differentiation mechanisms . NBs divide asymmetrically to generate a larger self-renewing NB and a smaller differentiating progeny. Genetic analyses have identified proteins partitioned into the NB that promote self-renewal and proteins partitioned into the smaller progeny that promote differentiation .
Recent work has shown that there are two types of NBs in the Drosophila larval brain: type I and type II [3–5]. There are approximately 90 type I NBs per brain lobe that have nuclear Deadpan (Dpn), nuclear Asense (Ase), and cytoplasmic Prospero transcription factors. They divide asymmetrically to bud off small ganglion mother cells (GMCs) that undergo a terminal symmetric division to produce two neurons . Type I NBs express all known apical/basal polarity markers. Apical markers are segregated into the NB, where they can promote aspects of NB identity ; basal markers such as Miranda, Prospero, Brain tumor (Brat), and Numb are segregated into the GMC, where they promote neuronal differentiation [7–11]. Axons formed by the neuronal progeny of central brain type I lineages fasciculate with each other and generally project within a single stereotyped tract to their targets ; this is different from type I NB lineages in the ventral nerve cord, which exhibit axon branching .
Recently, type II lineages have been shown to be susceptible to tumor formation: loss of the translational repressor Brat or the Notch repressor Numb or the transcription factor Earmuff from the whole brain results in tumor formation only within type II lineages [5, 9, 14]. Tumor formation is due to INPs reverting back to a type II NB-like identity; interestingly, the tumor phenotype can be suppressed by ectopic Prospero [5, 9, 14]. This raises the possibility that Prospero overexpression suppresses brat or numb tumors by transforming type II NBs to a type I NB identity. Consistent with this model, only type I NBs contain detectable levels of Prospero protein - type II NBs lack Prospero protein [3–5]. Alternatively, Prospero could inhibit proliferation in type II NBs without altering their cell fate. Consistent with this model, loss of prospero from embryonic or larval type I NB lineages leads to failure to repress cell cycle genes [15, 16] and 'tumor' formation [5, 9, 11, 14]. Similarly, the Prox1 vertebrate ortholog is expressed in newly differentiating neurons , inhibits neural progenitor proliferation , and is a candidate tumor suppressor gene [19–21].
Here we characterize two Gal4 lines that allow us to manipulate prospero expression within type II NBs and their INP progeny. We use these lines to test whether Prospero controls the difference between type I and type II NB identity, or whether it acts to limit progenitor proliferation without affecting NB identity. In addition, we use these lines to perform heritable lineage tracing to determine, for the first time, the adult brain neurons generated by the type II NB lineages.
Identification of R19H09, a Gal4 line expressed in type II neuroblasts and INPs
To identify Gal4 lines that would allow us to manipulate Prospero expression in type II lineages and INPs, we screened Gal4 lines available from public stock centers and enhancer-Gal4 lines targeted to the attP third chromosomal location (Manning et al., unpublished results) . Here we describe the R19H09-Gal4 line, which is expressed in five to seven out of the eight type II NBs and their INP progeny. The line is also expressed in a few type I NBs (which are ventral and thus easy to exclude from our analyses) and some post-mitotic neurons that project to the mushroom body (Figure 1E,E'; Additional file 1).
Analysis of 19H09 expression in wild-type and prospero misexpression brains
Genotype and stage
Type II NBa
GFP+ type II NBb
GFP+ type I NBc
GFP+ type II progenye
19H09-G4, UAS-nls-GFP @ 25°C
96 h ALH
4.6 ± 0.5
1.9 ± 1.3
18.9 ± 5.9
58.9 ± 17.4
120 h ALH
5.4 ± 0.5
6.6 ± 1.9
62.9 ± 6.4
138.1 ± 14.1
19H09-G4, UAS-mCD8::GFP @ 25°C
96 h ALH
5.5 ± 0.7
1.9 ± 1.4
31.7 ± 5.9
84 ± 16.3
120 h ALHg
6.5 ± 0.8
8.3 ± 1.5
81.9 ± 9.0
232.5 ± 14.2
19H09-G4, UAS-mCD8::GFP @ 30°C
120 h ALHg
5.8 ± 0.4
7.0 ± 1.8
242.7 ± 34.5
19H09-G4, UAS-mCD8::GFP, UAS-pros @ 30°C
120 h ALH
7.3 ± 0.5
4.6 ± 0.7
7.3 ± 1.5
9.8 ± 2.6
108.8 ± 13.5
As further confirmation that R19H09 drives expression in type II NBs and their progeny, we drove expression of a membrane-tethered GFP to trace axon projections (Figure 1C-E). We observe immature and mature INPs adjacent to the type II NBs (Figure 1C) as well as the projections of the earlier-born neurons in the lineages (Figure 1E,E'; Additional file 1). We observed that some of these secondary axon tracts were split and targeted towards different parts of the brain (Figure 1E, white arrows) unlike type I axon projections in the central brain, which generally extended along a single tract (Figure 1E', red arrow) . We also observed commissural projections from type II lineages (Figure 1E, yellow arrow). Since R19H09 is not expressed before 72 h ALH, only a subset of secondary axon projections of type II lineages were labeled. Our observations confirm and extend the findings from clonal analysis of type II lineages . We conclude that R19H09 can be used to drive gene expression in type II NBs and their INP, GMC, and neuronal progeny beginning at late larval stages.
Identification of R9D11, a Gal4 line expressed in INPs and their progeny
Analysis of 9D11 expression in wild-type and prospero misexpression brains
Genotype and stage
GFP+ type II progenyb
9D11-G4, UAS-nls-GFP @ 25°C
24 h ALH
12.4 ± 3.5
27 ± 5.8
48 h ALH
18.1 ± 2.4
52.6 ± 9.7
72 h ALH
51.7 ± 4.4
142.2 ± 6.6
96 h ALH
80.1 ± 6.2
232.7 ± 6.2
9D11-G4, UAS-mCD8::GFP @ 25°C
24 h ALH
11.8 ± 5.8
25.6 ± 5.8
48 h ALH
16.11 ± 5.5
53.44 ± 18.3
72 h ALH
52.1 ± 10.3
527.5 ± 24.4
96 h ALH
83.5 ± 2.5
548.9 ± 14.9
9D11-G4, UAS-mCD8::GFP @ 30°C
96 h ALH
86.6 ± 6.3
619.3 ± 20.1
120 h ALH
710.8 ± 14.1
97.6 ± 2.3
9D11-G4, UAS-mCD8::GFP, UAS-Pros @ 30°C
96 h ALH
14.2 ± 2.8
197.6 ± 20.6
120 h ALH
18.6 ± 2.8
214.8 ± 18.3
As in the case with R19H09, the type II axon projections labeled with R9D11 were split into several branches and targeted towards different parts of the brain (Figure 2G,G'; Additional file 2). These projections included commissural (Figure 2G, arrows) and descending ipsilateral (Figure 2G', arrowheads) bundles; the former were observed from all six medial type II lineages. Type II axonal fibers entered the larval commissure at different sites but a significant portion of labeled projections were targeted to the dorsoposterior commissure (DPC; Figure 2G, yellow arrow), which is a part of the larval precursor to the central complex of the pupal brain . Upon labeling with R9D11 it was difficult to trace trajectories due to dense staining, yet we were still able to individually identify R9D11+ type II lineages by the positional information of cell body clusters (that is, stereotypical anterior-to-posterior arrangement of the medial lineages) and by matching the visible projections to previous data (Figure 2G,G', lineages labeled) [12, 23]. We conclude that the medial type II lineages make complex secondary axon projections and project a subset of their axons to the interhemispheric commissure.
Prospero misexpression suppresses proliferation in type II NBs but does not induce type I NB identity
Prospero misexpression suppresses INP proliferation
R9D11 is expressed in a small subset of neurons in the adult brain that project to the fan-shaped body of the central complex
We and others have shown that although there are only 8 type II NBs among the approximately 100 central brain NBs, the type II NBs generate a disproportionately high percentage of the total neurons in the late larval brain [3–5]. We were curious to know if the shared developmental history of the type II neurons directs them to form a specific structure in the adult brain, or whether these neurons are dispersed throughout the adult brain. Recent work has shown that clones generated within type II NBs preferentially contribute to the central complex of the pupal brain , supporting a 'common function' model. The Drosophila central complex is a major neuropil in the adult brain that has been implicated in several behaviors, including locomotion, flight, and visual pattern memory [25–27], and consists of four interconnected substructures located on the midline of the protocerebrum: the protocerebral bridge (PB), the fan-shaped body (FB), the paired noduli (NO) and the ellipsoid body (EB). These neuropils are closely associated with the accessory areas, lateral accessory lobes (LAL; also known as ventral bodies) and bulbs (BUs; also known as lateral triangles) [28–30]. In addition, central complex neurons can be classified as either large-field or small-field. Large-field neurons link a single central complex substructure to regions outside the central complex; most project to one of the accessory areas. Small-field neurons are primarily intrinsic to the central complex, where they innervate a single substructure or link two to three substructures in a columnar fashion [29, 30].
Lineage tracing of R9D11-expressing cells labels the central complex and associated regions in the adult brain
To identify the structures type II lineages contribute to adult brain, we crossed R9D11 to UAS-FLP, actin[FRT-CD2-FRT]gal4, UAS-GFP and induced FLP-out clones at larval stages to permanently express GFP in INPs and their neuronal progeny. We found that type II lineages primarily contribute to the central complex of the adult brain, as well as some optic lobe labeling due to R9D11 expression in this tissue.
A detailed analysis of the adult brain pattern revealed the majority of the labeled cell bodies in the dorsal posterior cortex (Figure 5; Additional file 6), similar to the few neurons that maintain R9D11 expression in the adult brain (previous section). Additional cell bodies were seen in the anterior cortex lateral to the anterior LAL and other areas (Additional file 6).
We next describe the adult brain axon projection patterns for the type II lineages, although the high density of labeling made it difficult to link axon projections to specific cell bodies. We observed labeling of all four central complex neuropils, the two central complex accessory areas and several other regions in the central brain (Figure 5C-G; Additional files 6 and 7).
Central complex: protocerebral bridge neuropil
The PB neuropil is the most posterior of the central complex and is divided into 16 segments . The PB was diffusely labeled with its lateral edges showing slightly denser staining, and the segments were not distinguishable (Figure 5D; Additional file 8A; compare Additional file 6C to 6D for denser labeling of lateral PB). Several types of small-field neurons connect the PB to other central complex neuropils but we could not distinguish them by their dispersed projections in the PB. The projections we observed in other neuropils suggest that small-field types, such as ventral fiber system (VFS) and horizontal fiber system (HFS) neurons, which connect the PB to the FB, and pontine, pb-eb-no, and eb-pb-lal neurons are labeled (see sections below) .
Central complex: fan-shaped body neuropil
The FB is the largest structure in the central complex and is divided into several vertical staves and horizontal stratifications . Small-field neurons, which typically have their cell bodies in the DPC, contribute largely to the vertical staves while large-field neurons, which are found in both the posterior and anterior cortex, form most of the horizontal strata . The FB was heavily innervated throughout, revealing its vertical and horizontal layers (Figure 5E,F; Additional files 6D-H and 8B-D). A single horizontal layer in the dorsal FB was more heavily innervated than other sections (Figure 5E; indicated with yellow dashed lines in Additional file 8B-D). We also observed dense staining in tracts dorsal to the anterior FB that are connected to arborizations in the posterior superior medial protocerebrum (psmpr) and middle superior medial protocerebrum (msmpr) regions; these projections appear to connect to the LALs as well (Figure 5F; Additional file 6E-I). We propose that these tracts are part of the anterior commisure of the FB .
The cell bodies and the projection pattern of several small-field types match our observations in the FB and other central complex neuropils. These include VFS and HFS neurons that project along the vertical staves (Image 5 and 6 in ), pontine neurons that innervate all parts of the FB (Image 9 in ), fb-eb neurons that innervate two horizontal layers in the FB (Image 7 in ) and fb-no neurons that are restricted to few staves and horizontal layers (Image 11c,d in ). The cell bodies and the projection pattern of some large-field F neurons (fan-shaped neurons) also match our observations in the FB. The Fm1 and Fm3 subtypes (fan-shaped medial neurons) have cell bodies in the DPC, and Fl subtypes (fan-shaped lateral neurons) are primarily in the anterior cortex ventrolateral to LALs. The Fm1 and Fm3 neurons project anterior to the FB then posterior through the EB canal to form arbors in the second ventral layer of FB, whereas Fl neurons project to all layers of the FB . Some Fl neurons project through the anterior commisure and innervate the msmpr (Image 22 g in ). Another type of Fl neuron, ExFl2 (an extrinsic fan-shaped neuron), has its cell body located in the DPC lateral to mushroom body calyces and forms arbors at psmpr before innervating a dorsal horizontal FB layer in a segmented fashion (Image 13 in ). These projections are remarkably similar to those made by type II-derived neurons, especially the tracts dorsal to anterior FB that are connected to arbors in the psmpr and msmpr (Additional file 6) and the dense segmented dorsal layer of innervations at the FB (Additional file 8, indicated with yellow dashed lines).
Central complex: ellipsoid body neuropil
The EB neuropil is anterior to the FB and can be divided into a posterior and anterior ring. The posterior EB is innervated by small-field neurons while large-field R neurons (ring neurons), which have cell bodies ventrolateral to the LALs in the anterior cortex, fill the anterior and the median parts of the EB in concentric rings [29, 31]. However, certain R neurons are known to innervate only fragments of the EB, and ExR2, a rare extrinsic type of R neuron, is known to innervate the posterior EB only . Parts of the EB were also innervated (Figure 5F; Additional file 6G,I). The posterior ring of EB was innervated in a ring-like fashion (dorsoposterior part in Additional file 8C and the middle ring in Additional file 8D); however, the more dorsoanterior parts were less innervated (Additional file 2E,F). The innervation of the anterior ring of the EB was weaker and found in a radial, evenly spaced fashion rather than a continuous ring (Additional file 8E,F). Projections through the EB canal were also observed (Additional file 8D-F, circle inside the anterior ring).
While the R neurons that project to fragments of EB could contribute to the staining of the posterior ring of EB, it is more likely generated by the small-field types such as fb-eb and pb-eb-no neurons or the rare ExR2 neuron , which mostly innervate the posterior ring of EB .
Central complex: noduli neuropil
The NO neuropil is ventral to the FB and is divided into three horizontal layers. Several small-field types innervate the NO [29, 30]. The NO was also heavily innervated (Figure 5E; Additional file 6F,G). The three horizontal layers of the NO were revealed (Figure 2C) and the top layers were heavily innervated (Additional file 8C, arrows) . This pattern matches the projections of fb-no and pb-eb-no small-field neurons, which innervate only the dorsal segments of the NO .
Central complex: accessory areas
Few small-field neurons project to small regions of LALs, while many large-field neurons innervate the whole LAL neuropil . BUs are also innervated by both small-field and large-field neurons and they are connected to the contralateral LALs . In addition to the four central complex neuropils, the LAL and BU accessory areas were also labeled (Figure 5F,G; Additional file 6G-K). There were widespread arborizations in the LALs, including the ventral body commissure that connects LALs across the midline (Figure 5F). Small regions in the lateral sides of the dorsoanterior LALs, bound dorsally by the mushroom body medial lobes and ventrally by the antennal lobes (ALs), were innervated heavily (Figure 5G; Additional file 8J,K). We also observed labeling of BUs and connections between BUs and ipsilateral LALs (Figure 3F). The extensive labeling of LALs accompanied with dense staining of small regions and the labeling in BUs is consistent with the notion that large-field types, like Fl neurons, and small-field types, such as eb-pb-lal, HFS, and pb-eb-bu neurons, are derived from type II lineages . We conclude that type II lineages contribute to all central complex neuropils and accessory areas in the adult brain.
Outside the central complex, we observed dense innervation in a region that lies dorsal to the LALs, posterior to the mushroom body medial lobes, and lateral to the anterior EB (Figure 5G; Additional file 6). The central and anterior parts of medial protocerebrum were also labeled (Additional file 6F-M). Interestingly, projections were observed in the mushroom body vertical and medial lobes (Additional file 6L,M) as well as specific glomeruli in the AL (Additional file 6J-M). The labeling we observe outside the central complex could be connections between the central complex and other brain regions or non-central complex neurons made in type II lineages.
The recent identification of the type II lineages containing transit amplifying intermediate progenitors provides an important new model for investigating progenitor self-renewal and differentiation [3–5, 14]. However, we know little about their development, cell biology, gene expression, and functional importance in the Drosophila central nervous system. This is primarily due to a lack of genetic tools and markers that are specifically expressed in type II NBs and/or INPs. Here we characterize the R19H09-Gal4 line expressed in type II NBs, and the R9D11-Gal4 line expressed in INPs but not their parental type II NBs. Using R19H09 we show that Ase is upregulated before Dpn during INP maturation. Using both lines, we show that Prospero misexpression regulates proliferation but not identity within type II lineages. And using R9D11 we permanently label the majority of type II-derived neurons to show they are major contributors to the adult central complex brain region.
R19H09 and R9D11 as tools to understand brain development and function
The R19H09-Gal4 and R9D11-Gal4 lines can also be used to monitor the development of type II NBs and INPs in different mutant backgrounds to help clarify the origin of a mutant phenotype. For example, early studies on tumor suppressor genes showed increases in global brain NB numbers; for some of these mutants (for example, brat, numb) we know now that the phenotype arises specifically within the type II lineages . The R19H09-Gal4 and R9D11-Gal4 lines can also be used to drive UAS-RNAi, UAS-GFP constructs to test the role of any gene within these lineages. In addition, because these lines are made from defined enhancer fragments driving Gal4 placed into a specific attP site in the genome, it is easy to generate different transgenes with precisely the same expression pattern. Some future uses would be: using R19H09-FLPase to generate mutant clones or MARCM genetic screens in type II lineages; using R9D11 to drive expression of uracil phosphoribosyltranferase  to isolate RNA from INP sublineages; or using R9D11-grim to ablate specifically type II neurons to determine their role in larval or adult behavior.
The role of Prospero in type I and type II NB lineages
We have used the R19H09 and R9D11 lines to show that misexpression of Prospero can suppress proliferation within type II NBs and INPs without altering NB identity. As R19H09 is expressed only during the late larval stages, Prospero misexpression with R19H09 clearly distinguishes the effects of Prospero on NB proliferation from its effects on NB fate specification, which occurs in the embryonic stages. Misexpression with both R19H09 and R9D11 lead to a reduction in the number of INPs and neurons made by each type II NB. This reduction is unlikely to be due to an effect on the parental type II NBs, such as slowed down cell cycle or compromised NB survival, for the following reasons: first, low levels of ectopic Prospero are cytoplasmic in type II NBs, where Prospero has no known function; second, ectopic Prospero does not transform type II lineages to a type I identity based on the failure to upregulate ase expression; and third, misexpression of Prospero with both R9D11 and R19H09 give similar phenotypes, yet R9D11 is not expressed in type II NBs. We suggest that the reduction of clone size is due to an effect in the INP cell type. Possible mechanisms include INP apoptosis, INP cell cycle lengthening, premature cell cycle exit, or transforming INPs into central brain type II GMCs, which generate lineages with bifurcated axon fascicles. While we could not distinguish between these possibilities, we can tentatively exclude the mechanism of a transformation of INP to central brain type I GMC identity because the neurons still retained their ability to form bifurcated axon fascicles (Figure 4F; Additional file 4), which are not a feature of central brain type I GMCs.
Type II NBs lack both Ase and Prospero, whereas type I NBs contain both proteins. Yet only misexpression of Ase can transform type II into type I NBs ( and this work), suggesting that Ase is sufficient to upregulate prospero expression in NBs. However, loss of Ase does not transform type I NBs into type II NBs , so there must be additional factors promoting the expression of Prospero in type I NBs. The analysis of gene expression differences between type I and II NBs would be one way of uncovering genes that control the difference between them.
The contribution of type II lineages to the adult brain
Lineage-tracing of INP-derived neurons shows that type II lineages make major contributions to all aspects of the central complex of the adult brain, as well as the BU and LAL accessory structures, including both small-field and large-field neurons . Central complex neurons derived from type II lineages likely include several small-field types, such as VFS, pontine, fb-eb, fb-no, and pb-eb-no neurons, and, to a lesser extent, large-field types, such as F neurons, including Fm, Fl and ExFl subtypes and some extrinsic R neurons. A recent study found that type II NB clones in the pupal brain projected to the PB, FB and NO regions, with some projections forming restricted arbors at the PB and innervating domains of the FB and NO, while others made widespread arborizations outside the central complex . Our data showing labeling of the majority of type II neuronal progeny are consistent with those of , and complementary to these data: while we do not have the resolution to link cell bodies with axon projections, we are able to provide a more comprehensive view showing that type II lineages contribute to all central complex neuropils and accessory areas in the adult brain. Future studies that selectively ablate different spatial or temporal cohorts of type II neurons will be necessary to determine if all type II-derived neurons share a common function.
Although a large subset of central complex neurons derive from type II lineages, there are clearly some central complex neurons that originate from type I NBs or embryonic type II lineages. For example, we do not see projections that match those of the well-characterized large-field R neurons (R1 to R4) [29, 31]. It is not clear which small-field types are not derived from type II lineages as they are difficult to distinguish. However, it is clear that the type II lineages do not make up the entire central complex so there must be contributions from type I lineages as well.
Outside the central complex, we observed labeling of the region-specific staining of both the mushroom body and ALs; staining in the ALs was restricted to a subset of glomeruli. These could be novel connections from the central complex to the mushroom body and ALs formed by large-field or poorly understood extrinsic small-field neurons , or the projections of non-central complex neurons labeled by R9D11. Previous studies have revealed no direct connection between central complex and mushroom bodies or between LALs and ALs, and very few connections from LALs to mushroom bodies [29, 33]. The type II projection patterns from larval and pupal brains suggest that the lineages are not dedicated to a single neuropile center, which is consistent with type II lineages giving rise to non-central complex neurons as well. We also observed labeling of large regions in the protocerebrum outside the central complex. However, it was not possible to distinguish whether they were connected to the central complex or its accessory areas. Another caveat to our analysis is that R9D11 is also expressed in the larval optic lobes, and indeed we observed labeling in the adult optic lobes (Additional file 6, R-R'''). We could not distinguish the projections from these cells from those of the central brain cell bodies due to dense staining. Analysis of 1,200 Golgi-impregnated brains revealed direct connections between optic lobes and the BU neuropil, but not to the other central complex neuropils that we find labeled . This suggests that most if not all central complex labeling is due to type II-derived neurons.
In addition to using R9D11 to lineage trace the contribution of larval-derived type II neurons to the adult brain, we also detected maintained expression of R9D11 in a small subset of adult neurons, which are likely to be P3 or P4 small-field pontine neurons, which are also detected by the Gal4 line NP2320 . Thus, the R9D11 line, and others with similarly specific adult expression patterns, should be useful for future studies using TU-tagging to transcriptionally profile neuronal subsets , GRASP to identify pre/post-synaptic partners , or for expression of optogenetic modulators of neuronal activity to determine the role of specific neurons in behavior .
Our characterization of type II lineages suggests that as a group the type II NBs produce a wide variety of neuronal subtypes. This neural diversity can be achieved spatially if each type II NB generates just one or two types of neurons; this model is supported by clonal data showing that each type II NB produces neurons with distinct axon projection patterns . In addition, temporal identity could generate further neuronal diversity as seen in type I NB lineages . This model is supported by clonal analysis of a small central complex sublineage in the adult brain, which has revealed temporally distinct neuronal fates . Finally, hemilineages could provide a final doubling of neuronal diversity, in which each sibling neuron derived from a single GMC takes either an 'A' or a 'B' cell fate . The fact that bifurcating axon projections are seen even in the highly sparse type II lineages following Prospero overexpression is consistent with GMCs producing A/B neurons that have different fasciculation patterns. In the future, it will be important to determine the birth-order and identities of neurons in each type II lineage and the mechanisms that regulate spatial and temporal neural fate specification in these lineages.
Materials and methods
Fly stocks were: FRTG13, UAS-mcd8::GFP (Bloomington Stock Center); UAS-nls::GFP (Bloomington Stock Center); worniu-Gal4 ; R9D11-Gal4 ; R19H09-Gal4 (G Rubin, unpublished); UAS-prosL  (F Matsuzaki, unpublished); Act[FRT-CD2-FRT]-Gal4, UAS-GFP (gift from Bruce Edgar) crossed to UAS-FLP/CyO (Bloomington Stock Center).
Tissue preparation and immunohistochemistry
Larval brains were dissected in Schneider's medium (Sigma, St Louis, MO, USA); fixed in 100 mM Pipes (pH 6.9), 1 mM EGTA, 0.3% Triton X-100, and 1 mM MgSO4 containing 4% formaldehyde for 25 minutes; washed 30 minutes in phosphate-buffered saline (PBS) containing 0.3% Triton X-100 (PBS-T); washed 30 minutes in PBS-T with 1% bovine serum albumin (PBS-BT); and incubated with primary antibodies in PBS-BT overnight at 4°C. Afterwards, brains were washed 1 h in PBS-BT, incubated with secondary antibodies for 2 h and washed 1 h in PBS-T.
Adult females 3 to 10 days old were anesthetized on ice and dissected immediately in ice-cold PBS (dissection time per brain approximately 4 minutes). Brains were fixed in PBS with 4% formaldehyde for 25 minutes; washed 10 minutes in PBS containing 1% Triton X-100 (PBT) three times and blocked with PBT containing 5% normal-goat serum (Vector Laboratories, Burlingame, CA, USA) prior to incubation with primary antibodies in PBT overnight at 4°C. Afterwards, brains were washed 10 minutes in PBT three times, incubated with secondary antibodies for 2 h and washed 10 minutes in PBT three times.
Primary antibodies were rat Dpn monoclonal (1:1), rabbit Ase (1:2,000), mouse Prospero monoclonal (purified MR1A, 1:1,000), rabbit GFP (1:500; Molecular Probes, Eugene, OR, USA), mouse GFP (1:500; Molecular Probes), chicken GFP (1:500; Aves Laboratories, Tigard, OR, USA), rat mCD8 (1:150; Invitrogen, Eugene, OR, USA), mouse Fasciclin II (1:100; Developmental Studies Hybridoma Bank), mouse nc82 (1:10; Developmental Studies Hybridoma Bank), and mouse Dlg (1:100). Secondary antibodies were from Molecular Probes (Eugene, OR, USA) and diluted at 1:500 in PBS-BT or PBT for larval and adult brains respectively.
Histology and imaging
Brains were mounted in Vectashield mounting medium (Vector Laboratories). Images were captured with a Biorad Radiance or Zeiss700 confocal microscope with a z-resolution of 1.0 (for three-dimensional reconstructions) or 1.5 microns and processed in ImageJ (NIH, Bethesda, MD, SUA) and Photoshop CS3 (Adobe, San Jose, CA, USA). Figures were made in Illustrator CS3 (Adobe). Three-dimensional brain reconstructions and movies were generated using Imaris software (Bitplane, Zurich, Switzerland).
anterior ring of EB
anterior inferior medial protocerebrum
after larval hatching
anterior superior medial protocerebrum
dorsomedial type II lineage
neuron connecting EB to PB to LAL
extrinsic fan-shaped neuron
extrinsic ring neuron
neuron connecting FB to EB
neuron connecting FB to NO
fan-shaped lateral neuron
fan-shaped medial neuron
green fluorescent protein
ganglion mother cell
horizontal fiber system
intermediate neural progenitor
lateral accessory lobe
middle inferior lateral protocerebrum
middle inferior medial protocerebrum
middle superior medial protocerebrum
neuron connecting PB to EB to BU
neuron connecting PB to EB to NO
posterior ring of EB
posterior superior medial protocerebrum
ventral body commisure
ventral fiber system
We thank Gerry Rubin (JFRC) for R9D11 and R19H09 flies, the Bloomington stock center for fly stocks, Jim Skeath and DHSB for antibodies, and Sen-Lin Lai for comments on the manuscript. OAB was supported by an NIH training grant, JQB was supported by an HHMI postdoctoral fellowship, and CQD was supported by the HHMI.
- Doe CQ: Neural stem cells: Balancing self-renewal with differentiation. Development. 2008, 135: 1575-1587. 10.1242/dev.014977.View ArticlePubMedGoogle Scholar
- Knoblich JA: Mechanisms of asymmetric stem cell division. Cell. 2008, 132: 583-597. 10.1016/j.cell.2008.02.007.View ArticlePubMedGoogle Scholar
- Bello BC, Izergina N, Caussinus E, Reichert H: Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development. Neural Develop. 2008, 3: 5-10.1186/1749-8104-3-5.PubMed CentralView ArticleGoogle Scholar
- Boone JQ, Doe CQ: Identification of Drosophila type II neuroblast lineages containing transit amplifying ganglion mother cells. Dev Neurobiol. 2008, 68: 1185-1195. 10.1002/dneu.20648.PubMed CentralView ArticlePubMedGoogle Scholar
- Bowman SK, Rolland V, Betschinger J, Kinsey KA, Emery G, Knoblich JA: The Tumor Suppressors Brat and Numb Regulate Transit-Amplifying Neuroblast Lineages in Drosophila. Dev Cell. 2008, 14: 535-546. 10.1016/j.devcel.2008.03.004.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee CY, Robinson KJ, Doe CQ: Lgl, Pins and aPKC regulate neuroblast self-renewal versus differentiation. Nature. 2006, 439: 594-598. 10.1038/nature04299.View ArticlePubMedGoogle Scholar
- Wang H, Ouyang Y, Somers WG, Chia W, Lu B: Polo inhibits progenitor self-renewal and regulates Numb asymmetry by phosphorylating Pon. Nature. 2007, 449: 96-100. 10.1038/nature06056.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang H, Somers GW, Bashirullah A, Heberlein U, Yu F, Chia W: Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts. Genes Dev. 2006, 20: 3453-3463. 10.1101/gad.1487506.PubMed CentralView ArticlePubMedGoogle Scholar
- Bello B, Reichert H, Hirth F: The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila. Development. 2006, 133: 2639-2648. 10.1242/dev.02429.View ArticlePubMedGoogle Scholar
- Betschinger J, Mechtler K, Knoblich JA: Asymmetric segregation of the tumor suppressor brat regulates self-renewal in Drosophila neural stem cells. Cell. 2006, 124: 1241-1253. 10.1016/j.cell.2006.01.038.View ArticlePubMedGoogle Scholar
- Lee CY, Wilkinson BD, Siegrist SE, Wharton RP, Doe CQ: Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal. Dev Cell. 2006, 10: 441-449. 10.1016/j.devcel.2006.01.017.View ArticlePubMedGoogle Scholar
- Pereanu W, Hartenstein V: Neural lineages of the Drosophila brain: a three-dimensional digital atlas of the pattern of lineage location and projection at the late larval stage. J Neurosci. 2006, 26: 5534-5553. 10.1523/JNEUROSCI.4708-05.2006.View ArticlePubMedGoogle Scholar
- Truman JW, Moats W, Altman J, Marin EC, Williams DW: Role of Notch signaling in establishing the hemilineages of secondary neurons in Drosophila melanogaster. Development. 2010, 137: 53-61. 10.1242/dev.041749.PubMed CentralView ArticlePubMedGoogle Scholar
- Weng M, Golden KL, Lee CY: dFezf/Earmuff maintains the restricted developmental potential of intermediate neural progenitors in Drosophila. Dev Cell. 2010, 18: 126-135. 10.1016/j.devcel.2009.12.007.View ArticlePubMedGoogle Scholar
- Li L, Vaessin H: Pan-neural Prospero terminates cell proliferation during Drosophila neurogenesis. Genes Dev. 2000, 14: 147-151.PubMed CentralPubMedGoogle Scholar
- Choksi SP, Southall TD, Bossing T, Edoff K, de Wit E, Fischer BE, van Steensel B, Micklem G, Brand AH: Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells. Dev Cell. 2006, 11: 775-789. 10.1016/j.devcel.2006.09.015.View ArticlePubMedGoogle Scholar
- Torii M, Matsuzaki F, Osumi N, Kaibuchi K, Nakamura S, Casarosa S, Guillemot F, Nakafuku M: Transcription factors Mash-1 and Prox-1 delineate early steps in differentiation of neural stem cells in the developing central nervous system. Development. 1999, 126: 443-456.PubMedGoogle Scholar
- Dyer MA: Regulation of proliferation, cell fate specification and differentiation by the homeodomain proteins Prox1, Six3, and Chx10 in the developing retina. Cell Cycle. 2003, 2: 350-357.View ArticlePubMedGoogle Scholar
- Laerm A, Helmbold P, Goldberg M, Dammann R, Holzhausen HJ, Ballhausen WG: Prospero-related homeobox 1 (PROX1) is frequently inactivated by genomic deletions and epigenetic silencing in carcinomas of the bilary system. J Hepatol. 2007, 46: 89-97. 10.1016/j.jhep.2006.07.033.View ArticlePubMedGoogle Scholar
- Shimoda M, Takahashi M, Yoshimoto T, Kono T, Ikai I, Kubo H: A homeobox protein, prox1, is involved in the differentiation, proliferation, and prognosis in hepatocellular carcinoma. Clin Cancer Res. 2006, 12: 6005-6011. 10.1158/1078-0432.CCR-06-0712.View ArticlePubMedGoogle Scholar
- Takahashi M, Yoshimoto T, Shimoda M, Kono T, Koizumi M, Yazumi S, Shimada Y, Doi R, Chiba T, Kubo H: Loss of function of the candidate tumor suppressor prox1 by RNA mutation in human cancer cells. Neoplasia. 2006, 8: 1003-1010. 10.1593/neo.06595.PubMed CentralView ArticlePubMedGoogle Scholar
- Pfeiffer BD, Jenett A, Hammonds AS, Ngo TT, Misra S, Murphy C, Scully A, Carlson JW, Wan KH, Laverty TR, Mungall C, Svirskas R, Kadonaga JT, Doe CQ, Eisen MB, Celniker SE, Rubin GM: Tools for neuroanatomy and neurogenetics in Drosophila. Proc Natl Acad Sci USA. 2008, 105: 9715-9720. 10.1073/pnas.0803697105.PubMed CentralView ArticlePubMedGoogle Scholar
- Izergina N, Balmer J, Bello B, Reichert H: Postembryonic development of transit amplifying neuroblast lineages in the Drosophila brain. Neural Dev. 2009, 4: 44-10.1186/1749-8104-4-44.PubMed CentralView ArticlePubMedGoogle Scholar
- Cabernard C, Doe CQ: Apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation in Drosophila. Dev Cell. 2009, 17: 134-141. 10.1016/j.devcel.2009.06.009.View ArticlePubMedGoogle Scholar
- Strauss R: The central complex and the genetic dissection of locomotor behaviour. Curr Opin Neurobiol. 2002, 12: 633-638. 10.1016/S0959-4388(02)00385-9.View ArticlePubMedGoogle Scholar
- Wessnitzer J, Webb B: Multimodal sensory integration in insects--towards insect brain control architectures. Bioinspir Biomim. 2006, 1: 63-75. 10.1088/1748-3182/1/3/001.View ArticlePubMedGoogle Scholar
- Pan Y, Zhou Y, Guo C, Gong H, Gong Z, Liu L: Differential roles of the fan-shaped body and the ellipsoid body in Drosophila visual pattern memory. Learn Mem. 2009, 16: 289-295. 10.1101/lm.1331809.View ArticlePubMedGoogle Scholar
- Otsuna H, Ito K: Systematic analysis of the visual projection neurons of Drosophila melanogaster. I. Lobula-specific pathways. J Comp Neurol. 2006, 497: 928-958. 10.1002/cne.21015.View ArticlePubMedGoogle Scholar
- Hanesch U, Fischbach KF, Heisenberg M: Neuronal architecture of the central complex in Drosophila melanogaster. Cell and Tissue Research. 1989, 257: 343-366. 10.1007/BF00261838.View ArticleGoogle Scholar
- Young JM, Armstrong JD: Structure of the adult central complex in Drosophila: organization of distinct neuronal subsets. J Comp Neurol. 2010, 518: 1500-1524. 10.1002/cne.22284.View ArticlePubMedGoogle Scholar
- Renn SC, Armstrong JD, Yang M, Wang Z, An X, Kaiser K, Taghert PH: Genetic analysis of the Drosophila ellipsoid body neuropil: organization and development of the central complex. J Neurobiol. 1999, 41: 189-207. 10.1002/(SICI)1097-4695(19991105)41:2<189::AID-NEU3>3.0.CO;2-Q.View ArticlePubMedGoogle Scholar
- Miller MR, Robinson KJ, Cleary MD, Doe CQ: TU-tagging: cell type-specific RNA isolation from intact complex tissues. Nat Methods. 2009, 6: 439-441. 10.1038/nmeth.1329.PubMed CentralView ArticlePubMedGoogle Scholar
- Ito K, Suzuki K, Estes P, Ramaswami M, Yamamoto D, Strausfeld NJ: The organization of extrinsic neurons and their implications in the functional roles of the mushroom bodies in Drosophila melanogaster Meigen. Learn Mem. 1998, 5: 52-77.PubMed CentralPubMedGoogle Scholar
- Feinberg EH, Vanhoven MK, Bendesky A, Wang G, Fetter RD, Shen K, Bargmann CI: GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron. 2008, 57: 353-363. 10.1016/j.neuron.2007.11.030.View ArticlePubMedGoogle Scholar
- Miesenbock G: The optogenetic catechism. Science. 2009, 326: 395-399. 10.1126/science.1174520.View ArticlePubMedGoogle Scholar
- Isshiki T, Pearson B, Holbrook S, Doe CQ: Drosophila neuroblasts sequentially express transcription factors which specify the temporal identity of their neuronal progeny. Cell. 2001, 106: 511-521. 10.1016/S0092-8674(01)00465-2.View ArticlePubMedGoogle Scholar
- Yu HH, Chen CH, Shi L, Huang Y, Lee T: Twin-spot MARCM to reveal the developmental origin and identity of neurons. Nat Neurosci. 2009, 12: 947-953. 10.1038/nn.2345.PubMed CentralView ArticlePubMedGoogle Scholar
- Albertson R, Chabu C, Sheehan A, Doe CQ: Scribble protein domain mapping reveals a multistep localization mechanism and domains necessary for establishing cortical polarity. J Cell Sci. 2004, 117: 6061-6070. 10.1242/jcs.01525.View ArticlePubMedGoogle Scholar
- Manning L, Doe CQ: Prospero distinguishes sibling cell fate without asymmetric localization in the Drosophila adult external sense organ lineage. Development. 1999, 126: 2063-2071.PubMedGoogle Scholar
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