RESEARCH ARTICLE 53

Development 137, 53-61 (2010) doi:10.1242/dev.041749 Role of Notch signaling in establishing the hemilineages of secondary in Drosophila melanogaster James W. Truman*,§, Wanda Moats, Janet Altman*, Elizabeth C. Marin† and Darren W. Williams‡

SUMMARY The secondary neurons generated in the thoracic central nervous system of Drosophila arise from a hemisegmental set of 25 neuronal stem cells, the (NBs). Each NB undergoes repeated asymmetric divisions to produce a series of smaller ganglion mother cells (GMCs), which typically divide once to form two daughter neurons. We find that the two daughters of the GMC consistently have distinct fates. Using both loss-of-function and gain-of-function approaches, we examined the role of Notch signaling in establishing neuronal fates within all of the thoracic secondary lineages. In all cases, the ‘A’ (NotchON) sibling assumes one fate and the ‘B’ (NotchOFF) sibling assumes another, and this relationship holds throughout the neurogenic period, resulting in two major neuronal classes: the A and B hemilineages. Apparent monotypic lineages typically result from the death of one sibling throughout the lineage, resulting in a single, surviving hemilineage. Projection neurons are predominantly from the B hemilineages, whereas local are typically from A hemilineages. Although sibling fate is dependent on Notch signaling, it is not necessarily dependent on numb, a gene classically involved in biasing Notch activation. When Numb was removed at the start of larval , both A and B hemilineages were still generated, but by the start of the third larval instar, the removal of Numb resulted in all neurons assuming the A fate. The need for Numb to direct Notch signaling correlated with a decrease in NB cell cycle time and may be a means for coping with multiple sibling pairs simultaneously undergoing fate decisions.

KEY WORDS: Neurogenesis, Hemilineage, Notch, Numb, Neuroblasts, Drosophila

INTRODUCTION is determined by a sequence of transcription factors that are passed How such a great diversity of cell types is generated within the on to successive GMCs through time and establish neuronal fates nervous system during development remains a major unresolved within a given lineage (Kambadur et al., 1998; Isshiki et al., 2001; question in neurobiology. In vertebrates and invertebrates, both Grosskortenhaus et al., 2005). In the embryo, the daughter neurons inductive (Briscoe, 2009; Edlund and Jessell, 1999) and lineage- produced by the GMC division typically have distinct identities, and based mechanisms are involved in producing this diversity (Desai this difference is controlled by Notch signaling (Spana and Doe, and McConnell, 2000; Cayoutte et al., 2006), but different regions 1996; Skeath and Doe, 1998). of the nervous system may be biased towards one end of this The bulk of the activity of most NBs is devoted to making spectrum or the other. Within insects, lineage-based mechanisms are secondary neurons. The secondary neurons constitute a more responsible for the vast majority of neuronal diversity, with the homogeneous population than the initial, primary set. In insects with possible exception of the optic lobes. In the central brain and ventral complete metamorphosis, like Drosophila, most of the secondary ganglia, the neuronal stem cells (the neuroblasts, NBs) are neurons are born during a larval phase of neurogenesis. Studies on identifiable as individuals and each makes a characteristic set of the generation of secondary neurons in the caterpillar of the tobacco progeny (e.g. Bossing et al., 1996; Schmidt et al., 1997; Schmid et hornworm, Manduca sexta (Witten and Truman, 1991), indicated al., 1999). NBs go through asymmetric, self-renewing divisions, that the GMC divides to make daughters of divergent phenotypes each resulting in a neuronal precursor cell, the ganglion mother cell and that this process is then reiterated scores of times to generate two (GMC). Although there are now known to be exceptions (Bello et major classes of interneurons. Similarly, in grasshoppers, Jia and al., 2008; Bowman et al., 2008; Boone and Doe, 2008), the GMC Siegler (Jia and Siegler, 2002) showed that the GMCs from the usually undergoes a terminal division, producing two daughter median in the thorax consistently produce an engrailed neurons. The initial progeny made by a NB are often highly diverse positive and engrailed negative daughter, which become a local and are termed the primary neurons (Hartenstein et al., 2008). Their and a projection cell, respectively. Region-specific cell identities are based on the birth order of the GMCs, and this ordering death of one sibling then sculpts the final lineage composition in a given segment. These examples of diverse classes of cells being generated throughout a lineage is in contrast to neurogenesis in the Department of Biology, Box 351800, University of Washington, Seattle, WA 98195, , where sibling neurons generated at any given USA. time are morphologically indistinguishable (Lee et al., 1999). *Present address: Janelia Farm Research Campus, Howard Hughes Medical Institute, In this paper, we present a comprehensive analysis of the role of 19700 Helix Drive, Ashburn, VA 20147, USA Notch signaling in generating neuronal phenotypes within the †Present address: Department of Biology, Bucknell University, Lewisburg, PA 17837, USA secondary lineages of the segmental central nervous system (CNS). ‡Present address: MRC Centre for Developmental Neurobiology, King’s College The universal pattern is for a GMC to produce two neurons of London, Guy’s Hospital Campus, London SE1 1UL, UK different phenotypes, ‘A’ and ‘B’, with cell death involved in making §Author for correspondence ([email protected]) some lineages monotypic. A clear division of phenotype between

Accepted 24 October 2009 these A and B cell types suggest that the circuitry of the thoracic DEVELOPMENT 54 RESEARCH ARTICLE Development 137 (1) nervous system is generated in developmental units we term secondary neurons produced during the postembryonic neurogenic ‘hemilineages’. The accompanying paper by Lin et al. (Lin et al., phase are much more homogeneous (Truman et al., 2004; Pereanu 2010) shows that this pattern also holds the antennal lobes in the brain. and Hartenstein, 2006; Brown and Truman, 2009; Zhou et al., 2009). As seen in Fig. 1A, a NB typically generates either one or two classes MATERIALS AND METHODS of secondary neurons, as defined by their pattern of neurite Fly stocks projection. In the latter case, the two classes are based the division of Flies were reared on the standard yeast-cornmeal-molasses diet. Mitotic the GMC, with the two daughters showing distinct growth decisions clones were generated using the mosaic analysis using a repressible cell (Fig. 1B). This difference is then repeated for all of the GMCs marker (MARCM) technique (Lee and Luo, 1999). We used the pan- neuronal driver, elavC155 GAL4 (Lin and Goodman, 1994) to obtain a range generated during the second neurogenic phase. Skeath and Doe of clones that covered all of the thoracic lineages. Wild-type clones were showed that Notch signaling is responsible for the differences in generated in flies of the genotype: GAL4C155, hsFLP, UAS-mCD8::GFP; sibling fates during GMC divisions in the embryo (Skeath and Doe, FRT2A, tubP-GAL80/FRT2A. Notch null clones were produced using the 1998), and we hypothesized that this mechanism probably also null allele N55e11 (Heitzler and Simpson, 1991) in the genotype: extends into the secondary phase of neurogenesis. Immunostaining elavC155,N55e11,FRT 19A/tub-GAL80, hs-flp, FRT 19A; UAS- of neuroblast clones for Notch showed the prominent membrane mCD8::GFP/UAS-mCD8::GFP. Clones that showed constitutive Notch localization of Notch in the NB, GMCs and an adjacent cluster of signaling were produced by expressing the intracellular domain of Notch young neurons (Fehon et al., 1991), but also, typically, two of the CA C155 [UAS-Notch (Larkin et al., 1996)] using the genotype: elav , FRT young neurons had nuclear Notch (Fig. 1C,D) suggesting that these 19A/tub-GAL80, hs-flp, FRT 19A; UAS-mCD8::GFP/UAS-mCD8::GFP; were in the process of establishing their sibling fates. Therefore, we UAS-NotchCA/+. Numb activity was removed using the numb2 null allele (Frise et al., 1996) in the combination: elavC155, UAS-mCD8::GFP, hs- examined Notch signaling in the secondary lineages using both loss- flp/elavC155, UAS-mCD8::GFP, hs-flp; tub-GAL80, FRT 40A/y+,numb2, ck, of-function and gain-of-function approaches. FRT40A. Cell death was inhibited in homozygous clones that were mutant MARCM clones were induced postembryonically to include only for the initiator caspase dronc (Kondo et al., 2006; Williams et al., 2006) the secondary neurons born during the larval neurogenic period. using flies of the following genotype: hs-flp, elavC155GAL4, UAS- Notch loss-of-function clones were homozygous mutant for the null mCD8::GFP/+; tubP-GAL80, FRT 2A/droncDA8, FRT2A. allele N55e11 (Heitzler and Simpson, 1991). During Notch signaling For inducing MARCM clones in recently hatched larvae, eggs were the receptor is cleaved and the intracellular domain translocated to collected over a 1- to 2-hour period, maintained at 25°C for 24 hours, and the nucleus (Struhl et al., 1993). We generated Notch gain-of- the larvae then heat-shocked at 37°C for 45 minutes to 1 hour. Brief egg function clones either by expressing the intracellular domain of collections were also maintained for 72 hours before heat shock to induce Notch, which serves as a constitutive activator [NotchCA (Larkin et clones around the start of the third larval instar. al., 1996)], or by making MARCM clones that were null for numb, Immunocytochemistry a negative regulator of Notch (Knoblich et al., 1995; Spana and Doe, Dissected nervous systems were fixed in buffered 3.7% formaldehyde for 1996). Fig. 2 summarizes the effects of manipulating Notch about an hour at room temperature and then washed three times in PBS-TX signaling in the 25 secondary lineages in the ventral CNS. Nervous (phosphate buffered saline [pH 7.2] with 1% Triton-X100). Fixed samples systems were counterstained for neurotactin and the clones were blocked in 2% normal donkey serum (Jackson ImmunoResearch Labs, identified by the neurotactin-positive bundle in which their neurites West Grove, PA, USA) in PBS-TX for 30 minutes and then incubated in project (Truman at al., 2004). As a control for the Notch loss-of- primary antibodies for 1 to 2 days at 4°C. Primary antibodies were: 1:50 mouse anti-Notch MAb [MAb C17.9C6 (Fehon et al., 1990)]; 1:1000 rat anti-mCD8 function analysis, we looked at CNSs that were counterstained for (Caltag Laboratories, Burlingame, CA, USA); 1:20 mouse anti-neurotactin Notch and confirmed the loss of Notch protein in the clones (data MAb [F4A (de la Escalera et al., 1990)]. After three to four rinses to remove not shown). the primary antisera, tissues were incubated overnight in combinations of The cell clusters from NBs 1, 3, 6, 8, 11, 12, 13 and 19 have two FITC-conjugated and Texas Red-conjugated secondary antibodies at 1:500 primary neurite bundles of roughly equivalent size (e.g. Fig. 3A-D). dilution (Jackson ImmunoResearch Labs). Nervous systems were then rinsed, This dichotomy was also seen in their respective GMC clones (Fig. mounted on polylysine-coated coverslips, dehydrated through an ethanol 1B and data not shown), showing that the two neurite trajectories series, cleared in xylene and mounted in DPX (Fluka, Bachs, Switzerland). reflect the different fates of the two siblings. When Notch was either Image analysis removed or constitutively activated in these lineages, we saw only Confocal image stacks were typically collected at 63ϫ on either a BioRad one neurite bundle, characteristic of a single sibling type, but the MRC 600 or a Zeiss 510 confocal microscope. Image stacks were processed bundle differed under the two conditions (Fig. 2). For example, as using Image J (http://rsb.info.nih.gov/ij/). The z-projections for a given clone seen in Fig. 3, for lineages 1, 3, 8 and 12 the Notch loss-of-function (green) included all of the sections from the cell body cluster to the end of condition resulted in the presence of the 1i, 3id, 8c and 12c neurite the neurite bundle. The z-projection for the reference channel (magenta) bundles, respectively, whereas the 1c, 3il, 8i and 12i bundles were typically included only the sections in the neuropil region that showed the present in the NotchCA clones. Relative to Notch signaling, the latter neurotactin-positive bundles needed for lineage identification. Images were sibling is considered the ‘A’ fate (NotchON), whereas the former only globally adjusted for intensity and background. shows the ‘B’ fate (NotchOFF) (Skeath and Doe, 1998). Notch null We collected cell number data in NB clones by placing a mark on z-stack CA at the center of each cell. Each cell was marked only once and a count of the clones always produced a single sibling type. However, the Notch total marks yielded the total number of cells. clones sometimes contained one or two neurons of the ‘B’ type and the remainder of the ‘A’ type (Fig. 3M,N). We assume the presence of a few ‘B’ types in the NotchCA clones is because after clone RESULTS induction one or two neurons may still commit to a NotchOFF fate Effects of Notch on neuronal fates of the before sufficient Notchintra protein is made to suppress the ‘B’ secondary neurons of the thorax phenotype. In a given lineage, the primary neurons generated during the The shift in cellular composition of these clones was the result of embryonic phase of neurogenesis are typically diverse (Bossing et a change in cell fate and not selective cell death. This switch in fate

al., 1996; Schmidt et al., 1997; Schimid et al., 1999). By contrast, the was clearly shown in GMC clones that lacked Notch activity. For the DEVELOPMENT Notch control of hemilineage identity RESEARCH ARTICLE 55

Fig. 1. Relationship of sibling differences to overall lineage phenotypes. (A,B)z-projection of a lineage 6 neuroblast (A) and GMC (B) clone. The primary neurites from the two siblings form the six contralateral intermediate (6ci) and six contralateral dorsal (6cd) bundles. The schematics show how the primary neurites relate to the segmental commissures and leg neuropils (LNp). (C,D)The appearance of Notch immunostaining in the nuclei of two young neurons in a lineage 1 MARCM clone. (C)A z-projection of the cell cluster. (D)An optical section showing two neurons with nuclear Notch immunostaining; insets are magnified views of the neurons in this and the adjacent section. Yellow arrows: nuclear Notch staining. Commissures: ad, anterior dorsal; pd, posterior dorsal; ai, anterior intermediate; pi, posterior intermediate; v, ventral. above lineages, wild-type GMC clones always show the two Lineages 20, 21 and 22 were difficult to resolve. Each NB daughters that make different outgrowth decisions (e.g. Fig. 1B). In produces one to two motoneurons as well as a large number of Notch null GMC clones, by contrast, the two daughters were always interneurons that project to the leg neuropil (Truman et al., 2004). the same (e.g. the lineage 8 and 12 GMC clones in Fig. 3I,J). GMC For the Notch null clones, we saw many examples of two to four clones that expressed NotchCA typically showed both sibling types, motoneurons that were associated with a detached cell cluster but for reasons stated above, we think that this is because of the comprised of the NB, GMCs and very young neurons that lacked delay involved in the GAL4 system so that the daughter cells make neurites. We recovered no Notch null clones that contained the their fate decisions before sufficient Notchintra protein has been interneurons for these three lineages. By contrast, we recovered made. Cell counts support the conclusion that the Notch many NotchCA clones that contained the lineage 21 or 20/22 manipulation results in fate changes throughout a given lineage. In interneurons. These sometimes had an associated motoneuron, but the case of lineage 1, for example, control (125±10 s.e.m.; n5), its presence is probably due to this cell being generated before the Notch null (106±4 s.e.m.; n5) and NotchCA clones (118±6 s.e.m.; production of sufficient Notchintra, as discussed above. n5) had roughly the same number of neurons, despite the NB 0 makes a midline cluster of local interneurons but wild-type differences in cell composition under the three conditions. clones occasionally include a projection with a bifurcating Neuroblasts 0, 4, 9, 14, 20, 21 and 22 also generate two classes axon, the phenotype of the VUM cells produced by this neuroblast of neurons, but only a few of one type and many of the other. For example, lineages 20, 21 and 22 include one or two motoneurons as well as a large number of local interneurons that supply the leg neuropil (Truman et al., 2004). We had previously thought that NBs 4 and 14 produced only one type of neuron (Truman et al., 2004), but we have since found that they produce one or two interneurons that have trajectories that markedly differ from the remainder of neurons in the lineage. These rare neurons are among the first postembryonic neurons to be born and are often missing from neuroblast clones. The effect of Notch manipulation on these unequal lineages was also straightforward. Under one condition, a large cell cluster containing only the major neuron type was evident, whereas under the other condition, the minor cell type, with numbers typically doubled, was seen along with a slightly separated NB with its cluster of GMCs and young Fig. 2. Summary of the results of removing Notch or having neurons, but the latter apparently dying soon after their birth. For constitutively active Notch on the phenotypes of NB MARCM example, NB 14 generates a large number of interneurons that clones. (A)The frequency of particular lineages (for monotypic project through the ventral commissure to the contralateral leg lineages) or hemilineage bundles under the two conditions. (B)The neuropil and one or two neurons projecting to the dorsal neuropil frequency of ‘disembodied’ lineages that included the NBs, GMCs and (Fig. 4D). In Notch null clones, we saw only the dorsal-projecting a few young neurons, but few or no neurites emerging from the neurons accompanied by a detached cell cluster with the NB (Fig. cluster. The NB was identified by the location of the cluster and the lack of its characteristic neurotactin-positive, neurite bundle. Notch null, 4H). In such cases, neurotactin staining showed that the lineage white bars; constitutively active Notch (NotchCA), black bars. Numbers 14 ventral bundle was missing from that side (data not shown). In refer to the NBs; 20/22, combined data for lineages 20 to 22; in, local CA MARCM clones expressing Notch , by contrast, lineage 14 leg interneurons, mn, motoneurons. Other designations are neurite clones contained only the ventral projecting neurons. The size of bundles: c, contralateral; ci, contralateral intermediate; cd, contralateral the cell cluster was roughly twice that seen under wild-type dorsal; d, dorsal; i, ipsilateral; id, ipsilateral dorsal; ii, ipsilateral

conditions. intermediate; il, ipsilateral lateral; v, ventral. DEVELOPMENT 56 RESEARCH ARTICLE Development 137 (1)

Fig. 3. MARCM clones showing the effects of manipulating Notch signaling on lineages that produce two major classes of interneurons. Green, anti-CD8; magenta, anti-neurotactin showing the arrangement of lineage bundles. Insets are a reduced grayscale image of each clone. (A-D)Wild-type NB clones. (E-H)NB clones that are homozygous Notch null show a single neurite bundle. (I,J)Examples of Notch null, GMC clones for lineages 12 and 8, showing that both siblings have the same neurite projection. (K-N)NB clones that express NotchCA. M and N contain lineages 8 and 12 clones and the grayscale image is shown at full magnification. Both show one axon (8c, 12c) of the other sibling phenotype. Neurite bundle names are as in Fig. 2. during embryogenesis. NotchCA clones showed an expanded set of neuron, whereas the other sibling probably dies. local interneurons (Fig. 4I). Loss of Notch resulted in a compact cell Lineage 15 makes exclusively motoneurons that project to the leg cluster around the NB, with a neurite bundle that entered the imaginal disc (Truman et al., 2004; Baek and Mann, 2009; Brown neuropil but then dwindled away (Fig. 4E). As the neurons with the and Truman, 2009; Brierley et al., 2009). Removal of Notch results abortive neurites are located close to the NB, we conclude that they in a doubling in the size of this neuron cluster from 28±2 (n10) are recently born projection neurons that die soon after their neurite cells in control clones to 57±2 (n7) neurons in Notch null clones. enters the neuropil. Under the NotchCA condition we occasionally saw a clone with a The remaining lineages, from NBs 2, 5, 7, 10, 15, 16, 17, 18, 23 moderate number of motoneurons, but we were unable to and 24, have only a single neuron type. Clones for some of these unequivocally ascribe these to lineage 15 (these neurons show very lineages are recovered only rarely, even under wild-type conditions, weak expression of neurotactin). Also, under wild-type conditions, so we can make only tentative conclusions about them. However, we occasionally saw GMC clones with two lineage 15 motoneurons. others, such as NBs 2, 7, 15 and 16, appear at a high frequency. For We have concluded that the majority of lineage 15 motoneurons lineage 2 clones that lacked Notch, we saw only a cell cluster arise as the ‘B’ sibling, but a few may arise as part of the ‘A’ portion including an NB, GMCs and young neurons without neurites at the of the lineage. A second, smaller motor lineage that was missed in appropriate location, but no neurotactin-positive, neurite bundle Truman et al. (Truman et al., 2004) is lineage 24 (Brown and (Fig. 4F). The lineage 2 cluster and bundle were present in the Truman, 2009), which makes the motoneurons for the proximal leg NotchCA clones (Fig. 4J). The opposite relationship to Notch was muscles (Baek and Mann, 2009) (D. J. Brierley and D.W.W., seen for lineages 7 and 16, with an enlarged cluster of neurons unpublished). These motoneurons arise as the ‘B’ fate in lineage 24 having the expected projection pattern under the Notch-null (Fig. 2). condition (Fig. 2B). For lineage 7, the expression of NotchCA resulted in the loss of the 7c neurotactin bundle and a disembodied The role of cell death in generating monotypic NB and associated cell cluster in the normal NB 7 position. NotchCA lineages expression in lineage 16 was complicated because this treatment The majority of the segmental lineages are either monotypic, like often resulted in an enlarged cell cluster because of the production lineages 2 and 10, or make a few of one neuron type and an of additional NBs (see below). Neurites emerged from this cluster abundance of the other, such as lineages 4 and 14. The Notch data and projected along the expected path but then abruptly terminated, suggest that in these cases, one hemilineage survives while most or similar to the pattern seen in lineage 0 under the Notch null all of the cells of the other hemilineage either die or become another condition. None of the neurons in the small disembodied cell cell type, such as , which are not revealed by the elav driver. We clusters expressed Broad-Z3 (Broad – FlyBase), which is a marker assessed the role of cell death in sculpting the composition of these of intermediate neuronal development (Zhou et al., 2009; data not lineages by generating MARCM clones that lacked the initiator

shown). This pattern suggests that one sibling survives to become a caspase, dronc (Nedd2-like caspase – FlyBase) (Kondo et al., 2006). DEVELOPMENT Notch control of hemilineage identity RESEARCH ARTICLE 57

Fig. 4. Effects of Notch manipulation on monotypic lineages. Examples of MARCM clones for lineages that are monotypic (A,B) or produce a major and minor class of neuron (C,D). Green, anti-CD8; magenta, anti-neurotactin. Insets are a reduced grayscale image of each clone, except for F, which shows the neurotactin channel. (A-D)Wild-type NB clones. (E-H)NB clones that are homozygous Notch null. (E)A lineage 0 clone with the neuroblast (N) associated with a compact cell cluster containing a neurite bundle that dwindles as it enters the neuropil. (F)A lineage 2 clone consisting of the NB and a few associated cells but no emerging neurites. The neurotactin-positive bundle of its wild-type, contralateral homolog is indicated by the yellow arrowhead; this bundle is missing on the side with the clone. (G,H)NB clones showing just the rare cell-type and a slightly separated NB with a small compact ball of associated cells. (I-L)NB clones that express NotchCA. Neurite bundle names are as in Fig. 2.

As summarized in Fig. 5A-E, blocking cell death resulted in a and 18, but for the remainder (lineages 10, 17 and 23) a new class of striking increase in the numbers of the rare neuron types in lineages interneurons appeared. Taking these results together with the Notch 0, 4, 14, 20/22 and 21. In addition, in most of the purely monotypic data, we conclude that for the monotypic lineages one sibling lineages, we saw the appearance of neurons of novel morphology consistently lives, resulting in a single hemilineage. (Fig. 5G-L). In lineage 2, the new cells also projected to the dorsal ipsilateral neuropil, but to a more lateral extent than the normal Numb loss-of-function and neuronal fates sibling (Fig. 5H). In the other lineages, the new cells differed A way to bias the cells to activate Notch signaling is through the loss markedly from those seen in the wild-type lineage. For the two of numb function (Knoblich et al., 1995; Karacavich and Doe, 2005; motor lineages, the new cells were interneurons (Fig. 5G,K). By Spana and Doe, 1996; Frise et al., 1996; Guo et al., 1996). However, contrast, with the blockade of cell death, lineage 5 now contained unlike the situation reported for the embryo (Skeath and Doe, 1998), motoneurons as well as its normal type of interneuron (Fig. 5F). We we found that the NBs were refractory to the loss of Numb during could not resolve the effects of cell death blockade in lineages 7, 16 early larval growth. For clones induced soon after hatching and then

Fig. 5. Examples of MARCM NB clones that are null for the caspase dronc. (A-E)Lineages in which one cell type is typically represented by only one or two individuals. Blocking cell death results in the addition of more neurites from the cells of the rare phenotype (red arrow). (A)Lineage 0, (B) lineage 4, (C) lineage 14, (D) two examples of lineage 21, (E) lineage 20/22. (F-L)Monotypic lineages having one neurite bundle (yellow arrow) acquire a new neuronal class (red arrow) when cell death is blocked. (F)Lineage 5, (G) lineage 15, (H) lineage 2, (I) lineage 10, (J) lineage 17, (K) lineage 24, (L) lineage 23. Wild-type examples are included in Fig. 9. DEVELOPMENT 58 RESEARCH ARTICLE Development 137 (1)

Fig. 6. Examples of MARCM NB clones that are homozygous for a numb null mutation (numb2). (A,B)Bundles from both sibling types are evident in numb clones induced at 24 hours AEL and examined at wandering. The lineage 1 clone (A) was among clones from other lineages, but the 1i and 1c bundles are readily distinguishable. (C,D)numb clones induced at 72 hours AEL and examined at wandering showed only the A sibling in lineage 8 (D), or only a few axons of the B type (bundle 1i) and the remainder of the A type (bundle 1c) in lineage 1 (C). Neurite bundle names are as in Fig. 2. examined in wandering larvae, we typically saw that both types of produced extra NBs. The pattern of extra NBs seen after loss of siblings were present (Fig. 6). Comparison of bundle size between numb was similar to that seen when the same set of NBs was the two siblings was problematic, because in many cases the loss of examined for their response to expression of NotchCA (Fig. 8B). Numb resulted in a duplication of the NB, and this often resulted in Again, lineages 0, 6 and 12 were unaffected, while 9, 13 and 16 the disproportionate production of ‘A’ siblings (see below). showed multiple NBs. A lineage that responded differently to the We also generated a smaller set of clones at the start of the third two treatments was lineage 19, which often showed supernumerary instar, inducing the clones by heat shock at 72 hours after egg laying NBs after loss of numb, but did not do so in seven NB clones that (AEL) and examining the morphology of the resulting clones at expressed NotchCA. wandering. Lineages such as 1, 8 and 11 now showed clones that responded as expected, with the A phenotype being the sole (Fig. DISCUSSION 6D) or predominant (Fig. 6C) phenotype in the clone. The role of Notch signaling in establishing sibling difference in secondary neurons and the Notch and neuroblast duplication ‘hemilineage’ Studies of the Drosophila brain have shown that establishing The great diversity of cell types within the nervous system has constitutive Notch activity, either through loss of numb or by been appreciated since the studies by Cajal. Understanding the expression of Notchintra, results in the generation of rules that are used for generating such diversity remains one of the supernumerary NBs (Bowman et al., 2008). We found that this phenomenon also occurs in the ventral CNS but on a reduced scale (Fig. 7). The extra NBs in MARCM clones were identified by their large size and their expression of grainy head (Fig. 7A), a marker for NBs (Almeida and Bray, 2005). For NB clones that were induced around hatching and were null for Numb, 35% (n324) showed supernumerary NBs when examined late in the wandering stage. Unlike the massive increase in NBs seen in some brain lineages, the increase in NBs in the ventral CNS was modest after numb removal, with numbers of extra stem cells ranging from one to eight. When multiple NBs were present, each was typically associated with a set of young neurons with neurites that showed the projection and targeting that were typical of neurons generated at that site. The size and geometry of the cell cluster associated with each NB suggested that the generation of a supernumerary NB typically occurred early in the neurogenic period. For example, Fig. 7B,C show clones for lineages 3 and 9, which have two and three NBs, respectively. Each NB is associated with a separate cluster of progeny and the fasciculated bundles from each cluster join at the base of the clone where they enter the neuropil. Such large and relatively equivalent clusters could only arise if the NB replication occurred early in larval life. The conclusion that NB duplication typically occurs early in Fig. 7. Effects of Notch activation on NB duplication. MARCM NB postembryonic neurogenesis is also supported by the results of clones showing that loss of numb (A-D) or constitutive Notch signaling inducing numb clones at 72 hours AEL. We generated only a (E) results in clusters with multiple NBs. (A)Optical section through a limited number of such clones, but these included examples for clone (green) showing the expression of grainy head (magenta) in the lineages, 8, 9, 11, 16, 19 and 20/22. For early-induced clones in supernumerary NBs. (B,C)Examples of lineages 3 and 9, which have these lineages, 82% (n67) showed supernumerary NBs, whereas supernumerary NBs (insets); each NB is at the end of a discrete cell cluster (arrows) and their fasciculated neurites join at the neuropil into a only 9% (n11) of the late-induced clones had extra NBs. Overall, common bundle that projects to their normal targets. (D,E)Examples of only 6% (n34) of the 72-hour clones produced extra NBs. lineage 13 clones that are numb null or express NotchCA, respectively. An intriguing feature of the loss of numb function was that the Both treatments induced supernumerary NBs (insets). With loss of thoracic NBs differed markedly in their response to this loss. For numb (D), the 13c bundle was of normal size, whereas 13i was example, as seen Fig. 8A, lineages 0, 6 and 12 rarely, if ever, showed hypertrophied. With NotchCA expression (E), bundle 13i was

a supernumerary NB, whereas lineages 9, 13 and 16 consistently hypertrophied but 13c was missing. N, neuroblast. DEVELOPMENT Notch control of hemilineage identity RESEARCH ARTICLE 59

lineages that are monotypic, a situation seemingly similar to that seen in the mushroom body, one of the siblings is consistently removed by programmed cell death soon after its birth. Previous studies (Almeida and Bray, 2005) showed that Notch was not needed for the maintenance and division of NBs during the larval neurogenic period. Our data are completely consistent with their findings. Notch also seems dispensable for the early differentiation and survival of the young neurons. With Notch loss- of-function, we sometimes saw an NB with a cluster of immature cells but no neurites from maturing neurons exiting from the cluster, but this was only seen in lineages in which the B (NotchOFF) sibling normally dies. Similarly, expressing NotchCA also resulted in ‘disembodied’ NBs and cell clusters, but only in the lineages in which the A (NotchON) sibling is fated for death. These disembodied Fig. 8. Manipulations that cause NB duplication. Summary of the clusters were especially impressive in lineages, such as lineage 16, analysis of NB MARCM clones that were (A) null for numb function or had (B) constitutive Notch signaling. White bars, clones with a single that also showed supernumerary NBs due to constitutive Notch NB; black bars, clones with supernumerary NBs. activation. Overall, though, these data and those from the loss of the initiator caspase Dronc, show that the abnormal death seen with Notch manipulation is a result of the role of Notch in determining major goals of neurodevelopment, and the insect CNS has cell fate, rather than a requirement for Notch for survival or early contributed significantly to understanding this problem. With the differentiation. possible exception of the optic lobes, the generation of central An interesting question is whether there are global rules for fate neurons is strictly a lineage-related process, with no regulation of determination that apply across the lineages. In monotypic or almost cell fates seen between lineages (Taghert et al., 1984; Witten and monotypic lineages, there is no consistent relationship of the Truman, 1991). In the embryo, the neurons arising from the dominant sibling to the state of Notch signaling. In seven of the division of the GMC typically differ from each other (e.g. Skeath monotypic thoracic lineages the ‘A’ sibling is the dominant surviving and Doe, 1998), but for mushroom body neurons born during cell type, whereas in nine lineages, the ‘B’ sibling is the dominant larval life, the two siblings are indistinguishable (Lee et al., 1999). cell type. Axonal projection, however, does correlate strongly with The mushroom body pattern, however, is quite different from that whether or not the Notch pathway is activated. Only four bundles inferred from studies in the ventral nervous system of Manduca (6ci, 7c, 18c and 19c) project into the longitudinal tracts, and these (Witten and Truman, 1991) and the grasshopper (Jia and Seigler, are the B siblings of their respective lineages. Five lineages produce 2002), which indicate that the two siblings assume different fates. motoneurons (from NBs 15, 20, 21, 22 and 24) and they also The data in this paper and the study on the antennal lineages by Lin represent the ‘B’ fate. In addition, six more lineages [0, 4, 8, 12, 13 et al. (Lin et al., 2010) indicate that the mushroom body pattern is and 19] have one sibling that has a local primary target, whereas the atypical. As seen in Fig. 9, the pattern across the 25 thoracic other sibling projects to the periphery or across a commissure to the lineages is for the GMC to produce two different daughters. In contralateral side of the CNS. Lineage 4 is the only one of this group

Fig. 9. Summary of the role of Notch signaling in the 25 lineages of secondary neurons in the ventral CNS. The images show the wild-type morphology of each lineage. The diagrams shows the path of the neurite bundle from each hemilineage (see Fig. 1). Green, Notch-on fate; magenta, Notch-off fate. Open cell bodies show cell types that do not appear in the wild-type clones. Ones that are known to undergo programmed cell death are designated by a cross. When only one cell is filled, the oldest few neurons in the hemilineage survive while the remainder die. DEVELOPMENT 60 RESEARCH ARTICLE Development 137 (1) in which the B sibling stays within its hemineuropil, whereas the A the PAN lineages of the brain in that they respond to constitutive sibling projects across a commissure. The remaining ten lineages are Notch signaling by generating multiple NBs. Their responses are uninformative because they are situations like that in lineage 3, in more muted than the brain NBs, however, with only a few extra NBs which both siblings stay local, or lineage 1, in which both siblings being generated in a given cluster. are projection cells. These last examples notwithstanding, there is a As also seen for an antennal lobe lineage by Lin et al. (Lin et al., strong bias for the A (NotchON) sibling to stay local and for the B 2010), the sensitivity to constitutive Notch signaling is greatest early (NotchOFF) sibling to project to distant targets. Notch signaling in the postembryonic life of the NB. This transient sensitivity to works through the Suppressor of hairless [Su(h)] transcription factor Notch activation suggests that some thoracic NBs may have PAN (Bailey and Posakony, 1995), so one might suspect that targets neuroblast characteristics early after their reactivation but later downstream of Su(h) might promote features of local interneurons establish a traditional mode of behavior for the remainder of their and suppress projection neuron characteristics. lineage. We have not, however, found any GMC clones with more Although the role of Notch in establishing sibling identity is than two siblings, which would be expected if some thoracic consistent across the lineages and through time, that of numb is lineages made a few ‘transiently amplifying’ progeny. We have not not. During embryonic neurogenesis, the loss of numb function systematically induced clones through the early period of larval results in constitutive activation of Notch and the production of neurogenesis, however, so we cannot exclude the possibility that a only A siblings (Skeath and Doe, 1998). Early in the few transiently amplifying GMCs are produced among the earlier postembryonic period, however, we found that the GMCs produce GMCs in these lineages. daughters of both fates despite the loss of numb function (Fig. The production of supernumerary NBs has also been seen in 6A,B), but by the start of the third instar numb becomes essential caterpillars of Manduca sexta, after treatment with hydroxyurea for directing Notch activity (Fig. 6C,D). Therefore, early in the (Truman and Booker, 1986; Witten and Truman, 1991), a drug that secondary phase of neurogenesis, Notch signaling is not dependent blocks nucleotide reductase (Timson, 1975). Although the drug on numb, and other factors must be at play to allow Notch to treatment was devised to kill cycling NBs, we found that there was establish the differences in sibling identity. We do not know the a brief window at the start of postembryonic neurogenesis when nature of these factors. drug treatment caused some NBs to duplicate, rather than die, and It may be important that the lack of a requirement for numb is resulted in twice as many neurons of the appropriate phenotype. It correlated with rate of division of the NB. In the embryo (Campos- may be that some NBs in moths may also have PAN neuroblast Ortega and Hartenstein, 1997) and in the third larval instar (Truman characteristics early in larval life and that hydroxyurea causes a and Bate, 1988) the cell cycle of the NB is less than an hour. In the transiently amplifying precursor to cross a line that changes it into a latter case, we see two or more neurons in a given cluster showing fully fledged NB. nuclear-localized Notch (Fig. 1C,D) suggesting that siblings from It is interesting that the lineages that are the most sensitive (i.e. successive GMCs undergo fate decisions in an overlapping manner. show extra NBs in over 50% of the clones) to either Numb loss or Using Numb protein at this time to bias sibling identity would ensure Notch activation are ones that supply local interneurons to the leg that a given sibling would not be given ambiguous signals from a neuropil (lineages 8, 9, 13, 14, 16, 19, 20, 21 and 22). Providing cousin. When the NBs first reactivate in the second instar, however, PAN neuroblast characteristics to these NBs may be a strategy to they are dividing much more slowly (Truman and Bate, 1988). enhance the cell number or cellular diversity in this highly Assuming that the dynamics of GMC lifespan are similar to those integrative region of the CNS. seen later, we suspect that at these earlier times only one sibling pair in a cluster may be undergoing Notch-dependent decisions at a time. Conclusions This would permit the types of cell-cell interactions, such as seen in In this paper we present a comprehensive analysis of the role of some peripheral sensory precursor cells (Hartenstein and Posakony, Notch signaling in generating neuronal phenotypes within the 1989) or postulated for grasshopper lineages (Doe et al., 1985), to secondary lineages of the thoracic ventral CNS. The universal come into play. The biasing of cells by Numb may be an adaptation pattern is for a GMC to produce two neurons of different for rapid cell cycles when multiple neuron pairs are sensitive at the phenotypes, A and B, with cell death involved in making some same time. lineages monotypic. A clear division of labor between these A and B cell types suggest that the components of circuitry of the thoracic Thoracic NBs show similarities to the PAN nervous system are generated in developmental units we term neuroblasts ‘hemilineages’. We believe that viewing the construction of this part Recently, it has been shown that some NBs, the posterior asense- of the nervous system through the lens of the hemilineage will allow negative (PAN) NBs, differ from the classic scheme in that their us to gain insights into the way by which the genome generates units GMCs undergo additional divisions before making postmitotic of connectivity and how these are constructed into circuits neurons (Bello et al., 2008; Boone and Doe, 2008; Bowman et al., underlying behavior. 2008). PAN neuroblasts respond dramatically to enhanced Notch signaling; clones that either express NotchCA or are numb negative Acknowledgements show a dramatic expansion in the number of NBs in a given cluster, We are grateful to S. Bray, S. Artavanis-Tsakonas, R. Fehon, J. Hirsch, Y. Hiromi, S. Kondo, M. Piovant and H. Ruohola-Baker for fly strains and for antibodies. as some of the GMCs transform into NBs (Bowman et al., 2008). In Work was supported by NIH Grant NS13079 and by the Howard Hughes contrast to the PAN NBs, ‘classic’ brain NBs are unaffected by Medical Institute. Deposited in PMC for release after 6 months. enhanced Notch activity (Bowman et al., 2008). We also find a dichotomy in how the NBs in the ventral CNS References respond to the loss of Numb or expression of NotchCA. Most NBs Almeida, M. S. and Bray, S. J. (2005). Regulation of post-embryonic and GMCs characteristically maintain their normal pattern of neurogenesis by Drosophila Grainyhead. Mech. Dev. 1212, 1282-1293. Baek, M. and Mann, R. S. (2009). Lineage and birth date specify division despite constitutive Notch activation (Fig. 7). A few targeting and dendritic architecture in adult Drosophila. J. Neurosci. 29, 6904-

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