Genetic Specification of Axonal Arbors: atonal ... - Semantic Scholar

Neuron, Vol. 37, 41–51, January 9, 2003, Copyright 2003 by Cell Press

Genetic Specification of Axonal Arbors: atonal Regulates robo3 to Position Terminal Branches in the Drosophila Nervous System Marta Zlatic, Matthias Landgraf, and Michael Bate* Department of Zoology University of Cambridge Downing Street Cambridge CB2 3EJ United Kingdom

Summary Drosophila sensory neurons form distinctive terminal branch patterns in the developing neuropile of the embryonic central nervous system. In this paper we make a genetic analysis of factors regulating arbor position. We show that mediolateral position is determined in a binary fashion by expression (chordotonal neurons) or nonexpression (multidendritic neurons) of the Robo3 receptor for the midline repellent Slit. Robo3 expression is one of a suite of chordotonal neuron properties that depend on expression of the proneural gene atonal. Different features of terminal branches are separately regulated: an arbor can be shifted mediolaterally without affecting its dorsoventral location, and the distinctive remodeling of one arbor continues as normal despite this arbor shifting to an abnormal position in the neuropile. Introduction During the development of neural circuits, neurons of different kinds form distinctive terminal arbors within particular domains of the central nervous system. These terminal branch patterns reveal a transition from axon growth and guidance to synaptogenesis, and the positioning of such arbors is an integral part of determining the emerging pattern of connectivity. In general, we know that the growth of axons in the CNS and in the periphery is governed by the responses of growth cones to external guidance cues. In Drosophila, for instance, the midline repellent Slit regulates the positioning of axon fascicles at particular distances from the midline, and individual growth cone responses to Slit are mediated by cell-specific expression patterns of different combinations of Robo receptors (Rajagopalan et al., 2000; Simpson et al., 2000a). Similarly, vertebrate homologs of Slit are expressed by midline structures and are thought to regulate the formation of axonal trajectories relative to these sites. In addition to their repulsive function, vertebrate Slits promote the branching of sensory axons and cortical dendrites (for review of Slit proteins and their functions, see Wong et al., 2002). However, we still know relatively little about the intrinsic properties of neurons that lead to individual patterns of branching in the CNS or of the signals to which such terminating axons respond. To show how axon growth is regulated to produce an organized pattern of terminal arbors, we need to be able to analyze the link between *Correspondence: [email protected]

the genetic specification of neurons of different classes and their distinctive patterns of growth within the developing CNS. The sensory system of the Drosophila embryo provides us with a model system in which these issues can be studied. The pattern of sense organs has been characterized, with some 42 neurons in each abdominal hemisegment, consisting of three functional types: chordotonal (ch), multidendritic (md), and external sensory neurons (es) (Hartenstein, 1988). The distinctive terminal branches formed by individual sensory axons in each of these classes have been described and the genetic specification of the different classes of sense organs is increasingly well understood (Hassan and Bellen, 2000; Merritt and Whitington, 1995; Schrader and Merritt, 2000). Thus, we have the opportunity to dissect out the way in which such genetic information can be used to specify the characteristics of a terminal arbor within the three-dimensional structure of the CNS. We can distinguish two characteristics of sensory neurons that contribute to the final specification of their terminal arborization: position and modality. Here, we deal specifically with the question of modality-dependent arborization and, as a model for understanding the mechanisms involved, we use the distinctively different projections formed by the ch and md neurons, respectively. We can reduce the complexity of individual arbors by focusing separately on the essential features that give each arbor its characteristic identity and we begin by concentrating on arbor positioning within the mediolateral axis of the CNS. We work within a framework provided by a prominent set of axon fascicles that are labeled by antibodies to the cell adhesion molecule Fasciclin II (FasII) (Lin et al., 1994), and this enables us to detect small changes in the position and/or structure of terminal arbors. The axons of the ch neurons branch and terminate on an intermediate axon fascicle, while those of the md neurons terminate on a medial fascicle. There are additional distinguishing features in the two projections, such as their positions in the dorsoventral axis and the late embryonic remodeling of the arbor formed by the dorsal bipolar dendrite (dbd) neuron (an identified member of the md class). Using this system, we can ask the following questions about the way in which cell type-specific arborization is patterned in the developing CNS. First, we know that the Slit/Robo system positions axon fascicles in the mediolateral axis of the CNS. We can ask whether the same system directly or indirectly controls the positions at which terminal arbors are formed in this axis. Next, we can find out whether different aspects of the same arbor, such as dorsoventral as opposed to mediolateral positioning, are coordinately controlled or independently specified. We can also show whether the likely targets of terminating neurons influence the position at which termination actually occurs. Finally, we can ask what are the genetic controls that specify the different characteristics of a terminal arbor. One of our findings is that arbor position in the mediolateral axis is determined in a binary fashion by the expression (ch neurons) or nonexpression (md neurons) of the Robo3 receptor

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Figure 1. Projection Patterns of dbd- and chTerminal Branches in the Neuropile (A and B) Diagrams of wild-type projection patterns of dbd- (red) and ch- (green) terminal arbors with respect to the FasII CNS axon fascicles. Part of the neuropile of one half segment is depicted as a 3D image in (A) and as a transverse section in (B). dbd branches adjacent to the medial dorsal M1 fascicle, and ch-terminal arbors form on the ventral intermediate C3 fascicle. Dotted lines demarcate medial, intermediate, and lateral regions of the neuropile. (C–G) Representative images of terminal arbors (green) of ch (C and F) and dbd (D, E, and G) neurons with respect to FasII fascicles (red) at different stages of development. Images show projections of confocal z series of single sensory neurons filled intracellularly with Lucifer yellow. Anterior is to the left. Arrowheads show the midline. Abbreviations: l, lateral; i, intermediate; m, medial FasII fascicles. Scale bar equals 10 ␮m, except in (D), where it equals 15 ␮m. (C and F) Terminal ch projections: lch5-3 at 15 hr (C) and chv’ in a first instar larva (24 hr) (F). ch axons branch on the ventral intermediate C3 fascicle. (D) md neurons in the embryo branch just lateral to the medial FasII fascicles. Representative projection of the dbd at 15 hr; branches extend parallel to the dorsomedial M1 fascicle. (E) dbd terminal arbors remodel in the late embryo. At 19 hr, the dbd projection is intermediate between the larval and the early embryonic projections. The anteroposterior branches (asterisks) retract while new arbors (arrows) form at the original branch point and extend ventrally. (G) In L1 (24 hr), the embryonic anteroposterior branches of the dbd terminal arbor have completely retracted. New branches (arrows) extend ventrally from the branch point, which is adjacent to the medial FasII fascicle.

for the midline repellent Slit. We find that Robo3 expression is one of a suite of properties characteristic of ch neurons that depend on the expression of the proneural gene atonal (ato) in the precursors of these cells. Thus, while ectopic expression of Robo3 in md neurons switches their terminal arbor from a medial to an intermediate fascicle in the mediolateral axis, other properties of the arbor (such as late remodeling of the dbd arbor) are unaffected. In contrast, ectopic expression of the proneural gene ato in the precursors of md neurons transforms all aspects of their central projection to form a ch-like arborization. Results Sensory Terminal Arbors Form at Cell Type-Specific Locations in the CNS A previous study documented the patterning of sensory projections by measuring their relative mediolateral position within the connectives (Merritt and Whitington, 1995). We have refined this method by staining individual sensory neurons with Lucifer yellow against a background of CNS fascicles labeled with a FasII antibody. FasII labels a subset of CNS axon fascicles (Figures 1A and 1B): two fascicles in the medial region of the neuropile (dorsal M1 and ventral M2), four fascicles in the intermediate region (dorsal D1 and central C1–C3,

of which C3 is the most ventral), and three fascicles laterally (dorsal D2, central C4, and ventral V) (Lin et al., 1994; Prokop et al., 1998). We labeled representatives of two classes of sensory neurons in 15-hour-old embryos and first instar larvae (L1) and mapped their CNS projections with reference to the FasII fascicles. At 15 hr, ch axons (n ⫽ 20) bifurcate on the most ventral intermediate FasII-fascicle C3 (Figure 1). This pattern of projection is maintained in L1. In contrast, md neurons at 15 hr bifurcate adjacent to the medial FasII fascicles (n ⫽ 20), with three exceptions (vbd, v’td2, and vpda) (Merritt and Whitington, 1995), which we do not consider further. Dorsoventrally, the md projections are found on the ventral medial fascicle M2, with the exception of the dbd neuron (Merritt and Whitington, 1995), which bifurcates adjacent to the most dorsomedial FasII-fascicle, M1 (n ⫽ 20) (Figure 1). We chose to focus on the dbd because the dorsal location of its projection provides a further distinction from the ventrally projecting ch neurons. Alone among sensory neurons, the dbd projection undergoes a drastic reorganization, late in embryogenesis (Schrader and Merritt, 2000). As a result, in L1 the branches no longer run parallel to the FasII fascicles. Instead, they grow ventrally, forming a V shape, although they still remain in the medial region of the neuropile (Figures 1E and 1G). This late remodeling is an active consequence of the dbd neuron identity: dbd neurons

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Figure 2. Robo3 Is Differentially Expressed in ch but not md Neurons Immunofluorescent visualization of Robo (A) and Robo3 (D) expression in the PNS (visualized with anti-HRP, red) at 15 hr. (A–C) Robo (green) can be detected in all sensory neurons (red) at this stage as punctate cytoplasmic staining. Close-up of the dorsal cluster containing dbd and several md and es neurons. (D–F) Robo3 (green) is expressed in ch neurons but not in md neurons. Close-up of the lateral ch organs and the dorsal cluster of es and md neurons including the dbd. Abbreviations: esl, es neurons of the lateral cluster; esd, es neurons of the dorsal cluster; dmds, md neurons of the dorsal cluster; dbd, dorsal bipolar dendrite neuron; chl, ch neurons of the lateral cluster. Anterior is to the left. Scale bar equals 10 ␮m.

reinitiate growth at about 18 hr by sending out two side branches from the previously formed longitudinal branches. These side branches form close to and on either side of the longitudinal branch point. They grow ventrally away from the longitudinal embryonic branches and the M1 fascicle. The newly formed side branches and the initial branch point form the base of the larval V-shaped projection. At the same time as the new side branches are extending, the old longitudinal branches are retracting. Thus, at about 19 hr the projection of the dbd neuron has an intermediate form consisting of four branches: the initial, longitudinal ones are slightly retracted and the later, side ones are half extended (Figure 1E). Clearly, the late remodeling of the dbd neuron is an active process, characteristic of this neuron, that involves the growth of new branches and the retraction of existing ones. Thus, the ch and dbd neurons form terminal arbors that are distinguished by three features: (1) mediolateral position, (2) dorsoventral position, and (3) late stage remodeling. We can now ask how these distinctive features are controlled. Slit Receptors Are Differentially Expressed in ch and md Neurons Our first goal was to show whether any of the Robo receptors for Slit are expressed by sensory neurons when their axons are branching in the CNS (at 14–15 hr). We labeled sensory neurons with anti-HRP and with antibodies to either Robo, Robo2, or Robo3. At 15 hr we detected Robo protein in the cell bodies of both md and ch neurons (Figures 2A–2C), while Robo2 was undetectable in any sense organ at this stage (data not shown). Interestingly, Robo3 showed a differential expression pattern dependent on cell type, being present in ch but not in md neurons (Figures 2D–2F). These data suggest that (1) both md and ch neurons are likely to

be sensitive to Slit, thus confining their projections in general to the ipsilateral half of the developing CNS; and (2) expression of Robo3 could be decisive in determining the cell type-specific ordering of sensory projections within the ipsilateral neuropile. To test these predictions, we first analyzed the projection patterns of sensory neurons in 15 hr embryos mutant for robo (Kidd et al., 1998a). robo Regulates Midline Crossing but not Cell Type-Specific Positioning of Sensory Projections In robo mutants, the terminal branches of dbd neurons cross the midline (9/10) and often form bilateral projections that are mirror images of each other (Figures 3B and 3D). The terminal branches of ch neurons are unaffected in 6/10 embryos. However, in 4/10 embryos, a branch crosses the midline (Figures 3A and 3C). The ch axons in these embryos, like those of the dbd neurons, form a mirror image contralateral projection, which finds the ch-specific intermediate FasII fascicle on the opposite side of the nervous system. Thus, for both dbd and ch neurons, where bilateral projections develop, the mirror image projection forms at a location in the mediolateral axis that is appropriate to the cell type concerned. The conservative nature of these abnormal, contralateral arbors indicates that, while Robo directly or indirectly ensures that the longitudinal branches of the sensory neurons are confined to one side of the midline, additional factors must operate to determine the cell type-specific location of terminal branches within the developing neuropiles. Robo3 Is Required for the Correct Positioning of ch Terminal Arbors Next, we tested the function of robo2 and robo3 in patterning the projections of ch neurons. In general, in robo2 mutant embryos, ch projections are unaffected,

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Figure 3. Robo Regulates Midline Crossing of Sensory Projections (A and B) Central arbors (green) of a ch (A) and a dbd (B) neuron with respect to FasII fascicles (red) in 15 hr robo/CyO control embryos. ch-terminal arbors extend on an intermediate FasII fascicle (A), while the dbd branches alongside a medial FasII fascicle (B). (C) ch projection in a 15 hr robo mutant embryo. One branch formed correctly on the remnants of the intermediate FasII fascicle (asterisk). The axon continues across the midline and forms a second contralateral branch (arrow), which forms on the intermediate FasII fascicle, appropriate for ch projections. (D) dbd projection in a 15 hr robo mutant embryo. The terminal branches of the dbd neuron aberrantly cross the midline, but remain in apposition to the medial FasII fascicle, which is appropriate for the dbd terminal projection. Anterior is to the left. Arrowheads point to the midline. Abbreviations: l, lateral; i, intermediate; and m, medial FasII fascicles. Scale bar equals 10 ␮m.

although in 2/10 embryos we did observe a slight medial shift. This, like the antibody stainings (see above), suggests that Robo2 does not play a major part in patterning the ch projections. In 9/10 robo3 mutant embryos, ch projections are shifted to the medial region of the neuropile like the CNS axon fascicles (Figure 4B). These results reveal a requirement, direct or indirect, for Robo3 in the normal

positioning of ch projections. Additionally, they suggest that the bilateral symmetry of ch projections in robo mutants depends on the repellent action of Slit acting through the Robo3 receptor. The dbd neuron projection is unchanged in both robo2 and robo3 mutants (data not shown). To address the question of whether the effect of the robo3 mutation on ch neurons is primary or a secondary Figure 4. Robo3 Is Necessary to Position ch Projections in the Intermediate Neuropile

(A) ch projections (green) in a 15 hr robo3/ CyO control embryo form on an intermediate FasII fascicle (red). (B) In a 15 hr robo3 mutant embryo, ch-terminal projections and most of the intermediate FasII fascicles have shifted medially. ch arbors shift medially even when some intermediate FasII fascicles retain an intermediate position (arrows). Unlike in robo mutants, ch projections do not cross the midline in robo3 mutants. (C) Restoration of robo3 in the PNS alone is sufficient to rescue positional defects of ch projections in robo3 mutant embryos. Projection of a lateral ch neuron in a 15 hr robo3 mutant embryo in which robo3 was restored only in the PNS. In such embryos, ch-terminal arbors form in the intermediate region of the neuropile (arrow), even though their normal substrates for arborization (intermediate FasII fascicles) are shifted medially. Inset: Immunofluorescent visualization of Robo3 expression (green) in the CNS (antiHRP, pink) of a 15 hr robo3;PO163-GAL4/UAS-robo3 embryo. Robo3 is seen in the axons and terminal projection of PNS neurons in the CNS that are otherwise devoid of Robo3. (D) Downregulation of Robo receptors by ectopic Comm expression in the PNS shifts ch projections medially: central projections of two lateral ch neurons in a 15 hr PO163-GAL4;UAS-comm embryo. The bifurcation point and the branches of ch axons are shifted medially, resembling the projection pattern of md neurons. The ch branches shift independently of the FasII fascicles, which are unaffected in this experiment. In some cases, as shown here, an additional contralateral branch forms that bifurcates on the contralateral side (arrow). Anterior is to the left. Arrowheads indicate the midline. Abbreviations: l, lateral; i, intermediate; and m, medial FasII fascicles. Scale bar equals 10 ␮m.


consequence of the shifted fascicles, we analyzed ch projections in robo3 mutants in which Robo3 expression was restored in the PNS only (robo3; PO163-GAL4/UASrobo3) (Brand and Perrimon, 1993; Hummel et al., 2000; Simpson et al., 2000a). Antibody staining revealed high levels of Robo3 receptor in the axon terminal of PNS neurons (Figure 4C). In these embryos, the FasII fascicles still show a mutant phenotype: the intermediate fascicles are shifted from the intermediate to the medial region, resulting in thick medial FasII-fascicles, a gap in the intermediate region, and thin lateral FasII-fascicles. However, the ch projection was rescued in 7/10 embryos (Figure 4C). In all of these cases, the projection was more lateral than the thick medial FasII fascicle with a clear gap between them. Thus, the ch axons can be shifted independently of the FasII fascicles, and expression of Robo3 in the PNS alone is sufficient to reposition them. Targeted Loss-of-Function: Ectopic Expression of commissureless To confirm that there is an autonomous requirement for Robos in sensory neurons, we targeted reductions in the level of Robo expression to sensory neurons through expression of the Robo antagonist commissureless (comm) (Kidd et al., 1998b). In 15-hour-old embryos of this kind (Figure 4D), the ch projections are shifted adjacent to the ventral medial fascicle M2 (6/10 embryos). Thus, when Comm is overexpressed in sensory neurons, ch projections are transformed to resemble an md-like projection. In 4 out of 10 cases, a side branch crosses the midline. In two of these cases, the side branch forms a contralateral projection, which runs in the equivalent medial longitudinal tract to that of its partner in the opposite hemisegment. Thus, in this instance, where the levels of both Robo3 and Robo are likely to be reduced, the ch neuron behaves like a dbd/md neuron in a robo mutant and not like a ch neuron (Figure 4D). It appears, therefore, that the differentiating feature, the crucial switch in behavior between these two neurons with respect to mediolateral position, is the presence or absence of the Robo3 receptor. In its presence, axons in the mediolateral axis are ch-like; in its absence, axons are md-like. robo3 Is Sufficient for the Modality-Specific Mediolateral Positioning of Sensory Projections If the decisive difference between these neurons is the expression of Robo3, then it should be possible to switch the dbd projection to a ch-like projection simply by misexpressing robo3 in these neurons. In 15-hourold embryos in which two copies of robo3 are ectopically expressed in the PNS, the dbd projection, which is normally positioned just before the medial fascicle, is indeed shifted laterally to the intermediate fascicle (7/10 cases) (Figures 5A and 5B). In 2 out of these 7 cases, two branches are formed, one in the correct position and the other behaving like a ch axon, aligning itself with the intermediate fascicle. When the distinction between the ch and the dbd projections has been lost in the mediolateral axis, it is still maintained in the dorsoventral axis, with the dbd projecting to the dorsal intermediate fascicle C1 and the ch projecting to the ventral interme-

diate fascicle C2. Furthermore, in embryos that ectopically express Robo3 in the PNS, dbd projections still undergo late stage remodeling (both branch retraction and new branch outgrowth), despite being located in the incorrect region of the neuropile (Figures 5C and 5D). slit Is the Functional Ligand Controlling ModalitySpecific Branching of Sensory Projections To exclude the possibility that the functional ligand for Robo3 or Robo receptors in this system is not Slit, but some target-derived molecule, we looked for a genetic interaction between the amorph slit2 allele and robo3 based on the ch-projection phenotype. We found that in both slit2/⫹ and robo3/⫹ embryos, 10/10 ch-projections look wild-type. However, an interaction between these genes is clearly evident when ch projections are examined in the double heterozygotes. Forty percent of ch projections in slit2/⫹;robo3/⫹ embryos are abnormal, with branches on both the ventral intermediate C2 fascicle and the ventral medial M2 fascicle (Figure 6D). The FasII fascicles appeared normal in these embryos, with the intermediate fascicles shifting medially in only 10% of segments observed. We also analyzed ch projections in embryos homozygous for the amorph slit2 allele at 15 hr. Four out of ten ch neurons in these embryos failed to branch, continued growing across the midline, and exited the CNS contralaterally (Figure 6B). Six out of ten ch neurons did not exit the CNS, but instead the axons turned on the midline and grew anteriorly (posterior segments) or posteriorly (anterior segments) along the midline. Only one of these ch neurons formed a proper T-shaped arbor with branches extending both anteriorly and posteriorly along the midline. Similar results were obtained at 18 hr (5/10 unbranched ch axons), suggesting that branching is not simply delayed. Thus, Slit may be a required component for branching of sensory neurons as well as giving them positional cues as to where to terminate. ato Can Activate robo3 Expression in md Neurons Our previous experiments show that robo3 expression is sufficient to mediate a switch between two cell typespecific patterns of arborization in the mediolateral axis of the CNS. Since the pattern of termination is tightly linked to the type of sense organ involved, it seemed likely that robo3 expression might be one of a set of characteristics associated with a specific class of sensory neurons and dictated by genes operating to determine neuron identity. The development of sense organs is prefigured by the expression of proneural genes that initiate the formation of different types of precursors (Hassan and Bellen, 2000). In the case of ch organs, atonal (ato) expression prefigures and is required for the formation of ch organ precursors. In ato mutants, ch organs generally fail to form, and under some conditions ectopic expression of ato drives the formation of supernumerary ch organs in adult flies (Jarman et al., 1993, 1995). Thus, ato is a candidate gene to regulate the characteristic location of ch arbors by activating the expression of robo3. Since ch organs fail to form in the absence of ato, we asked whether ectopic expression of ato would be sufficient to activate robo3 and

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Figure 5. Robo3 Is Sufficient to Position Sensory Neuron Projections in the Intermediate Neuropile (A and C) Dbd projections in 15 hr PO163GAL4 control embryos (A) and L1 (C) form adjacent to a medial FasII fascicle. (B and D) Ectopic expression of Robo3 shifts dbd projections from the medial to an intermediate region of the neuropile, transforming them into ch-like projections with respect to the mediolateral axis. Ectopic Robo3 does not affect the dbd-specific dorsal positioning (B) or late stage remodeling, though the V-shaped side branches of the dbd (arrows) are slightly shorter than in wild-type (D). Note that the images are maximum projections of confocal z series; this obscures the extent of the late stage dbd projections, which are almost perpendicular to the FasII fascicles. Anterior is to the left. Arrowheads indicate the midline. Abbreviations: l, lateral; i, intermediate; and m, medial FasII fascicles. Scale bar equals 10 ␮m.

transform the central projections of the dbd neurons. We used scabrous-GAL4 to drive ato expression throughout the nervous system (Mlodzik et al., 1990) because the PNS-specific PO163-GAL4 driver first becomes active after the time at which ch neurons are specified and ato is expressed in wild-type embryos (Hassan et al., 2000). Ectopic expression of ato with scabrous-GAL4 lead to the widespread activation of robo3 expression in sense organs. While in the wild-type, Robo3 is only detectable in ch neurons, in embryos ectopically expressing ato, it is clearly detectable in all md neurons, including dbd, as well as in ch neurons (Figures 7A–7F). Ato is therefore sufficient to activate robo3 expression in md neurons.

Ectopic ato Is Sufficient to Transform dbd into ch-Type Projections We analyzed projection patterns of dbd neurons in scabrous-GAL4;UAS-ato first instar larvae (Figures 7G–7I). We observed transformation of the projection in 5/10 cases. In three instances, the transformation was complete, with the dbd projection now being on an intermediate fascicle. The projection was shifted both mediolaterally and dorsoventrally and was now positioned on the more ventral fascicle, C3, characteristic of ch terminals. In addition, there was no late remodeling of the projection expected for dbd (Figure 7I). In two further instances, the transformation was incomplete in that the

Figure 6. slit and robo3 Interact in Positioning Sensory Terminals (A and C) Ch projections (green) project to their normal position on an intermediate FasII fascicle (red) in 15-hour-old embryos with a single copy of either slit (A) or robo3 (C). (B) Projections of two ch neurons (green) in a 15-hour-old slit2 mutant embryo. All FasII fascicles (red) have collapsed into a single tract on the midline. One ch neuron has crossed the midline and stalled on the other side (arrow) in the CNS, while the other has exited the CNS and stalled unbranched in the periphery (asterisk). (D) Single ch projection (green) in a 15-hourold slit2/robo3 double heterozygote. FasII fascicles appear normal (red). The ch projection is abnormal, indicating a genetic interaction between robo3 and slit2. The ch axon terminal has two intermediate branches (arrows) and one md-like medial branch (asterisk) adjacent to the medial FasII fascicle. Anterior is to the left. Arrowheads indicate the midline. Abbreviations: l, lateral; i, intermediate; and m, medial FasII fascicles. Scale bar equals 20 ␮m (A and B) and 10 ␮m (C and D).


Figure 7. ato Controls Cell Type-Specific Mediolateral Positioning of Sensory Arbors through the Robo3 Receptor Robo3 expression (green) in the PNS (visualized with anti-HRP, red) at 15 hr. (A–C) In scabrous-GAL4 controls, Robo3 is expressed in ch but not in md neurons. Closeup of lateral ch organs and dorsal cluster of md neurons including dbd. (D–F) Ectopic expression of ato in the PNS in all SOPs results in the expression of Robo3 (green) throughout the PNS (red) including md neurons, which do not express Robo3 in the wild-type. The same region is shown as in (A)–(C). (G) Dbd projection in a scabrous-GAL4 control L1. The dbd axon branches adjacent to a medial FasII fascicle (red) and arbors (green) extend ventrally. (H and I) Projections of dbd sensory neurons of a L1 ectopically expressing ato in the nervous system (scabrous-GAL4; UAS-ato). Ectopic ato upregulates Robo3 expression in dbd and transforms dbd into ch-like projections. The extent of this dbd to ch transformation varies and may depend on both level and time of onset of ectopic ato expression. (H) Terminal arbor of a dbd neuron that is intermediate in morphology and neuropile position between a dbd and ch projection. Branches are positioned halfway between the intermediate and the medial FasII fascicles. The arbor has undergone partial late phase remodeling, but one side has maintained the early embryonic anteroposterior projection (asterisk). The new branches that form the V-shape are abnormally short (arrows). (I) Central projection of a dbd neuron that has been completely transformed to a chlike terminal arbor. The branches are on the ventral-most intermediate FasII fascicle characteristic of ch neurons. No aspects of the dbd-specific late stage remodeling are observed. Note that the images are maximum projections of confocal z series; this obscures the extent of the late stage dbd projections perpendicular to the FasII fascicles. Abbreviations: esl, es neurons of the lateral cluster; dmds, md neurons of the dorsal cluster; dbd, dorsal bipolar dendrite neuron; chl, ch neurons of the lateral cluster; l, lateral; i, intermediate; and m, medial FasII fascicles. Anterior is to the left. Arrowheads indicate the midline. Scale bar equals 10 ␮m.

projection was intermediate between ch and dbd, both with respect to position and late morphogenesis (Figure 7H). Despite these major changes in sense organ projection patterns, the general effect of ectopic expression of ato throughout the nervous system was subtle. The major CNS axon fascicles were wild-type, and the embryos hatched. Also, ectopic ato failed to transform embryonic dbd neurons into ch neurons at the level of cell body morphology. In 2/10 cases, the dbd neuron had only one of its characteristic dendrites and this was shorter than in wild-type. Even here, however, the neuron was not ch-like at the cell body. There is an obvious distinction between the results of these experiments and those obtained by expressing robo3 in the same neurons. Robo3 shifts the mediolateral position but does not affect the dorsoventral location or remodeling (Figure 5). Ato expression achieves a more complete transformation in that the projection shifts in both axes and remodeling is blocked (Figure 7I). Thus, Robo3 is one of a suite of properties regulating

cell type-specific projections of sensory neurons. Additional factors present in dbd, but missing in ch neurons, must dictate both dorsoventral location and late cell type-specific remodeling of dbd. Discussion Conserved Neuropile Architecture within which Axons Terminate The terminal arbors of sensory neurons in the Drosophila embryo form within a developing neuropile that consists largely of commissural and longitudinal axon fascicles together with the intervening branches and dendrites of other neurons. This mass of axons and dendrites is an organized three-dimensional structure within which there are clear signs of a conserved architecture that recurs throughout the insects and probably more widely in the arthropod phylum (Boyan and Ball, 1993). At its most fundamental level, there is a subdivision in which the principal arborizations of the motoneurons form dorsally, whereas sensory terminals are generally found

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more ventrally. The endings of sensory neurons themselves terminate in distinctive regions of the more ventral neuropile that have an overall similarity across the insects: the mechanosensory endings in the most ventral sector and the proprioceptive endings more dorsally (Schrader and Merritt, 2000). This suggests that connections form in a structured environment that imposes an order on the developmental process. It is the nature of this ordering process with which this paper is concerned. We have focused our analysis on the distinctive terminal arborizations formed by two different types of sensory neurons, the ch neurons and the dbd neurons. The first detailed maps of sensory terminals in the developing CNS of the Drosophila embryo and larva revealed that the arborizations of the sensory axons are as characteristic of particular sense organs as the structures of the sense organs themselves (Merritt and Whitington, 1995; Schrader and Merritt, 2000). For example (and this is confirmed and extended here), ch organs have arbors that form at an intermediate and fairly ventral level in the neuropile. In contrast, the arbors of most md neurons develop at a more medial and slightly more ventral location. Within the md class, however, four neurons have specialized projections. In particular, the dbd neuron has a dorsal projection that is uniquely remodeled toward the end of embryogenesis. Interestingly, dbd and the other neurons with specialized arbors correspond to identifiable subsets of cells within the md class that are distinguished both by their peripheral structures and by patterns of gene expression. Our first finding (Figure 8) is that in wild-type embryos, md and ch arborizations are closely aligned with elements of the FasII-positive axon tracts that form a conspicuous set of longitudinal pathways in the embryonic and larval nervous systems (Lin et al., 1994; Prokop et al., 1998). These consistent patterns of termination allow us to define precisely, in two dimensions (the dorsoventral and mediolateral axes), the different coordinates at which ch and dbd terminals will normally form in developing neuropile. Location in the Mediolateral Axis How are these coordinates specified for each neuron type? Previous work in Drosophila has shown that the Robo receptors for the repellent Slit are responsible for positioning CNS axon fascicles in the medial, intermediate, and lateral tracts on either side of the midline (Rajagopalan et al., 2000; Simpson et al., 2000a). Robo has also been shown to influence dendritic growth and synaptic connectivity in the giant fiber system of the adult fly (Godenschwege et al., 2002). We find that at the time of sensory neuron branch formation, Robo is expressed in dbd/md and ch neurons and acts to confine both kinds of projections to the ipsilateral CNS. In a robo mutant, both sets of neurons form bilateral projections, but the distinctive locations of the terminals persist on both sides of the midline. This distinction depends on the expression of Robo3, which is present in ch neurons but not in dbd/md neurons. When Robo3 is absent or downregulated, ch terminals form at the more medial position characteristic of db/dmd neurons. Similarly, if robo3 is ectopically expressed in dbd/md neurons, their terminals are shifted into the ch region of the neuropile.

Figure 8. Specification of Cell Type-Specific Attributes of Sensory Neuron Terminal Arbors by robo3 and ato Top and bottom show schematic crosssections of the neuropile (left and right hemispheres) with the relative positions of FasII fascicles and dbd- and ch-terminal arbors. In wild-type, dbd and ch arbors form in different mediolateral as well as dorsoventral regions of the neuropile (top left). Ectopic expression of ato is sufficient to transform all aspects of the dbd arbor to those of ch terminal projections (top right). Downregulation of robo3 alters the mediolateral but not the dorsoventral position of ch arbors to that of dbd neurons (bottom left). Ectopic expression of robo3 transforms the mediolateral but not the dorsoventral position of dbd to that of a ch neuron (bottom right).

The action of Robo3 on mediolateral positioning is a direct one. First, either ectopic expression of Robo3 or downregulation of Robo3 function (by expression of comm) in sensory neurons but not their central targets selectively induces lateral shifts of sensory arbors. Second, ch projections can form in an intermediate position independently of their normal substrate for arborization. In robo3 mutants, the intermediate FasII fascicle on which ch arbors normally form shifts medially, as do the ch projections. However, when Robo3 function is selectively restored in the sensory system of robo3 mutants, the ch projections form in the intermediate region of the neuropile, as in wild-type. Independent Specification of Position in Two Axes Our experiments also show (Figure 8) that the locations at which terminals form in the mediolateral and dorsoventral axes are separately specified and we can manipulate one independently of the other. Thus, in all cases where dbd/md or ch terminals are shifted in the mediolateral axis by changing levels of Robo3, the dorsoventral position remains unchanged: a medially shifted ch terminal is ventral; a laterally shifted dbd terminal is


dorsal. This finding reinforces the distinction between two alternative ways of determining the position of sensory axon terminals in the embryonic neuropile. The simplest model would suggest that growing axons are attracted to a specific central target: in this case, the position of the target (however determined) fixes the three-dimensional location of the terminal arbor of the sensory neuron. Our results suggest that this model does not apply: in our experiments, the position of the terminal arbor is regulated by factors that separately specify its position in two dimensions. It is the response of the sensory axon to these different factors rather than to their targets that determines where the terminal arbor will form. ato Regulates ch Arbors We conclude that there are several properties of ch neurons that dictate the position and shape of their terminal arbors, of which Robo3 expression is one. Robo3 expression is specified by the proneural gene ato. Thus, when ato is expressed in other embryonic sensory neurons, these cells too express Robo3. In addition (Figure 8), misexpressing ato can completely transform dbd projections into ch-like projections (although the cell body remains dbd-like). In such cases, both mediolateral and dorsoventral positions are changed and the late stage remodeling does not take place. Thus, ato not only regulates Robo3 expression but also controls the whole suite of properties that dictates the position-specific termination of ch neurons. The mechanism of this regulation, at least for Robo3 expression, is likely to be an indirect one. (1) Transformation (including Robo3 expression) of md into ch-like neurons by ectopic ato requires that ato is expressed early, during the period of sensory organ specification and not as the arbors form. (2) Only primary (not secondary) ch organs express ato, yet all ch neurons express Robo3 and project their terminal arbors into a common region of the neuropile. Previous studies have shown that ectopic ato can transform adult es organs into ch organs and that those transformations are achieved by an active downregulation of cut, which is required for es organ formation but is also expressed in a subset of md neurons (Blochlinger et al., 1988, 1991; Jarman and Ahmed, 1998). If ato upregulated Robo3 by downregulating Cut, then in cut mutant embryos all normally cut-expressing md neurons should be found to express Robo3. We find that this is not the case and that in cut mutants Robo3 is only expressed in the ch neurons, as in wild-type. We also find that md neurons of the dorsal cluster, which normally express Cut, retain their wild-type projections in 15 hr cut mutant embryos (data not shown). A Combinatorial Code Determining Sensory Arbors? While ato regulates ch-like characteristics, what specifies the unique features of the dbd arbor? Alone among md neurons, the dbd neuron projects to the dorsomedial FasII fascicle and undergoes late stage remodeling. The md neurons are a heterogenous population with respect to their expression of proneural transcription factors and identity genes such as cut. Interestingly, the SOPs of the dbd (and ddaE) neurons do not express cut or the

AS-C but a different proneural gene, amos (Brewster et al., 2001; Goulding et al., 2000; Huang et al., 2000). By analogy with the role of ato in determining the ch-like projection pattern, amos may determine aspects of the dbd-specific projection pattern. We also observe that ectopic expression of ato in dbd can generate intermediates, halfway transformations between dbd and ch, both with respect to mediolateral positioning and late stage remodeling. These intermediates may reveal competitive interactions between ch- and dbd-specific transcription factors. Thus, proneural genes could conceivably act in a combinatorial fashion to specify diverse shapes and positions of terminal branches (Merritt and Whitington, 1995). Such a view is reinforced by the fact that it is those sensory neurons, which express a combination of proneural genes in their SOPs, that have distinctive terminal arbors (Merritt and Whitington, 1995). However, there are likely to be additional factors that contribute to specify individual neuronal arbors, in particular in those instances in which terminal projection varies as a function of position. For example, in the cockroach the transcription factor Engrailed controls axon projections and synaptic choice of identified sensory neurons (Marie et al., 2002). Similarly, the prepattern genes araucan and caupolican play a role in establishing the difference in the projection pattern between lateral and medial es neurons in the adult Drosophila notum (Grillenzoni et al., 1998). Termination, Connectivity, and the Assembly of the Neuropile Working from first principles, we might suppose that different kinds of sensory neurons would locate the distinctive positions appropriate for their terminal arbors by seeking out their target interneurons in the developing CNS. However, we find that in its simplest form, this model must be incorrect: in one axis at least, terminating axons detect and are positioned by their response to a cue that is produced not by their targets but by cells on the midline. This might suggest that sensory terminals are located at appropriate positions within the neuropile by factors that are quite independent of the neurons with which they will ultimately form connections. Indeed, an early experiment in the zebrafish embryo showed that if the target Mauthner cell is removed, the conspicuous cap of commissural axon terminals that normally encloses the Mauthner axon hillock continues to form at its proper location in the neuropile (Kimmel et al., 1979). We cannot necessarily conclude from our experiments that sensory terminals in Drosophila are similarly positioned by factors entirely independent of the target. In the periphery, motoneuron axons are guided to their proper muscles by a hierarchy of cues, starting with a transcription factor code that delivers them to particular regions of the muscle field within which they then seek out and synapse with their targets (Thor et al., 1999). In the case of the embryonic sensory neurons, we could also envisage a hierarchy of cues, including target-derived signals, that would contribute to the final projection pattern and certainly to synaptogenesis. When we approach the apparently complex issue of how appropriate connections are formed between neu-

Neuron 50

rons within the meshwork of alternatives presented in the developing neuropiles, there may be two simplifying processes that we should consider. The first, as we have shown here, is that initial patterns of termination may be determined by a combination of cues that separately dictate distinct features of the arbor. The second is that the combination of such cues coupled with specific patterns of receptor expression may lead to the local arborization of pre- and postsynaptic neurons on a common substrate. Such patterns of growth would build a coherent platform on which detailed connectivity could then be established. Experimental Procedures Fly Stocks Wild-type is Oregon-R. For mutant analyses, the loss of function alleles ctdb7 (Blochlinger et al., 1988), roboGA285 (Kidd et al., 1998a), and robo2x135 (Simpson et al., 2000b) and the strong hypomorphic allele robo31 (Rajagopalan et al., 2000) slit2 (Battye et al., 1999) were used. Stocks were made using CyO GFP balancers (Casso et al., 2000). Homozygous mutant embryos were identified by lack of GFP. For ectopic expression, we used the following stocks: PO163-GAL4 (Hummel et al., 2000), scabrous-GAL4 (Budnik et al., 1996), UASrobo3 inserts on the second and the third chromosomes (Simpson et al., 2000a, 2000b), UAS-comm (Kidd et al., 1998b), and UAS-ato (Jarman and Ahmed, 1998). Preparation of Embryos and Larvae for Dye Injection Embryos were staged according to Campos-Ortega and Hartenstein (1985) and referred to in hours after egg laying at 25⬚C. Precise staging was achieved by selecting embryos immediately after formation of the second midgut constriction (13 hr ⫾ 10 min at 25⬚C) and incubating for a further 1 hr 50 min at 25⬚C. Embryos and larvae were dissected as described (Baines and Bate, 1998; Landgraf et al., 1997), fixed with 3.7% formaldehyde in saline for 2.5 min, rinsed with Ca2⫹-free saline (0.075 M phosphate buffer [pH 7.2]), and viewed under a 60⫻ water immersion objective (Olympus) to locate the sensory neuron cell bodies. Lucifer Yellow Injections Intracellular dye injection microelectrodes (60–100 M⍀) were backfilled with 5% Lucifer yellow solution (Molecular Probes). The shaft was backfilled with 0.2 M LiCl. Sensory neuron cell bodies were penetrated using an Olympus BX50-WI microscope and a hydraulic micromanipulator (Narishige). Lucifer yellow was injected by application of a 1.3 nA DC hyperpolarizing current for 2 to 5 min using an Iontophoretic Dye Marker (Digitimer). Preparations were washed with saline, fixed with 3.7% formaldehyde in saline for 30 min, and washed once more. Immunocytochemistry We used the following primary antibodies: anti-FasII (MAb 1D4; 1:10 dilution; Van Vactor et al., 1993), anti-Robo (MAb 13C9; 1:10 dilution; Kidd et al., 1998a), anti-Robo2 (mouse polyclonal; 1:1000 dilution; Simpson et al., 2000a), anti-Robo3 (MAb 14C9; 1:10 dilution; Simpson et al., 2000a), anti-Lucifer yellow (1:1000 dilution, Molecular Probes), and Cy5-conjugated goat anti-Horseradish Peroxidase (1:100 dilution; Jackson ImmunoResearch). Secondary antibodies were used at 1:200 dilutions: Alexa488-conjugated goat anti-mouse or anti-rabbit, Alexa633-conjugated goat anti-mouse (Molecular Probes). Standard immunohistochemical procedures were followed (Patel, 1994), using 0.2% Tween as a detergent for anti-Robo, antiRobo2, and anti-Robo3 and 0.3% Triton-X100 for all other stainings. Immunofluorescence was visualized with a Leica SP confocal microscope. Images are maximum projections of confocal z series processed with Adobe Photoshop software.

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