Targeted Neuronal Cell Ablation in the Drosophila Embryo: Pathfinding by Follower Growth Cones in the Absence of Pioneers

David M. Lin,* Vanessa J. Auld,*t reach their targets in the CNS (Wigglesworth, 1953). The and Corey S. Goodman ability of the initial axons to guide later growth cones has Howard Hughes Medical Institute led to the suggestion that pioneering growth cones might Division of Neurobiology be endowed with special pathfinding abilities. Do subse- Department of Molecular and Cell Biology quent growth cones possess the same pathfinding abili- Life Science Addition, Room 519 ties, or are they different from the pioneers? University of California, Berkeley Ablation experiments in a variety of organisms thus far Berkeley, California 94720 have provided a range of conflicting answers to this ques- tion. In the grasshopper embryo CNS, the G growth cone turns anteriorly along the P axons in the NP fascicle, a Summary pathway formed by the three descending P and the two ascending A axons. Although the A and P axons normally We developed a rapid method that uses diphtheria fasciculate and follow one another, either can pioneer the toxin, the flp recognition target sequences, and the complete pathway on its own. Furthermore, any one of GAL4-UAS activation system, to ablate specific neu- the P axons will suffice, since when any two of them are rons in the Drosophila embryo and to examine the con- ablated the remaining P pioneers the pathway. When the sequences in large numbers of embryos at many time three P axons are ablated, the G growth cone stalls, and points. We used this method to show that, in the ab- neither follows the A axons nor any other longitudinal ax- sence of the aCC axon, which pioneers the interseg- ons; however, if only one P remains, G will follow it (Raper mental nerve in the PNS, the three U follower axons et al., 1983a, i983b, 1984; Bastiani et al., 1984). if one are delayed and make frequent errors. However, the considers all three P axons as the pioneers, then the pio- pathway ultimately forms in most segments. We also neers are absolutely required for guidance of the G growth ablated the axons that pioneer the first longitudinal cone. However, if one considers P1 as the pioneer, then pathways within the CNS and observed similar results; P2 and P3 clearly have the same ability and can take its the formation of longitudinal pathways is delayed and place. disorganized in 70% of segments, but these tracts ulti- Similar results were obtained in the zebrafish, in which mately form in 80% of segments. Thus, pioneers facili- elimination of the first of the three primary motoneurons tate the development of PNS and CNS axon pathways; (CAP) or elimination of any two of the three does not pre- in their absence, followers are delayed and make nu- vent the remaining primary motoneuron(s) from extending merous errors. However, pioneers are not absolutely along the appropriate pathway (Eisen et al., 1989; Pike required, as these embryos display a remarkable abil- and Eisen, 1990). Thus, among certain groups of neurons ity to correct for the loss of the pioneering neurons. (e.g., the three P neurons in the grasshopper and the three primary motoneurons in the zebrafish), there does not ap- Introduction pear to be a special distinction between the first versus the second or third growth cone's ability to pathfind, whereas a In 1910, Ross Harrison coined the term "pioneer" based different type of follower axon (e.g., the G growth cone in on his examination of the formation of axon pathways in grasshopper) may in fact have a very different pathfinding the frog. "The fibers which develop later," he wrote, "follow, ability as compared with the pioneer(s). in the main, the paths laid down by the pioneers." Since These same issues have been examined in a variety of then, studies in a wide variety of organisms from insects other systems, with a variety of results. In the developing to mammals have confirmed Harrison's initial observation limbs in the grasshopper embryo, a pair of pioneer neurons that axon pathways are established early in development, arise at the tips of limb buds and extend axons toward the when distances are short and the terrain relatively simple CNS (Bate, 1976; Bentley and Keshishian, 1982). When (e.g., Bate, 1976; Bate and Grunewald, 1981; Edwards et these neurons are eliminated, follower growth cones are al., 1981; Goodman et al., 1982; Bentley and Keshishian, either able (Keshishian and Bentley, 1983) or unable 1982; Ho and Goodman, 1982; Raper et al., 1983a; Ku- (Klose and Bentley, 1989) to reach the CNS, depending wada, 1986; McConnell et al, 1989; Chitnis and Kuwada, upon the time of axon outgrowth. 1990; Wilson et al., 1990). Axons that arrive much later In the fish CNS, a particular dorsal spinal cord pathway in development often need these guide fibers to find their is pioneered by the axons from a population of transient way through a more complex and changing environment. Rohon-Beard neurons. When these neurons are ablated, For example, transection of a peripheral nerve in the insect the follower growth cones do not extend properly (Kuwada, Rhodnius prolixis causes subsequent sensory axons that 1986). In related experiments, elimination of a brain tract normally follow this nerve to form large whirls and never in the zebrafish embryo increases pathfinding errors by *These authors contributed equallyto this work. follower growth cones, but does not absolutely prevent tPresent address: Departmentof Zoology,University of British Colum- them from finding their correct pathway (Chitnis and Ku- bia, Vancouver,B. C. V6T1Z4, Canada. wada, 1991). Neuron 708

Pioneers can also be required for target selection. The short term, the three U growth cones that normally follow transient population of subplate neurons help to pioneer this axon are delayed and make frequent pathfinding er- the pathway between the thalamus and cortex (McConnell rors. In the long term, the ISN pathway eventually does et al., 1989). When the subplate neurons are ablated, the form in 88% of segments. Thus, in this case, the followers, axons from a particular thalamic nucleus extend in the although not as facile in navigating on their own as is the white matter under the cortex, but fail to enter their correct pioneer, are nevertheless ultimately able to form the path- target region of cortex (Ghosh et al., 1990). way. We also ablated the axons that pioneer the first longi- In the present paper, we re-explore the role of pioneers tudinal pathways within the CNS and observed similar re- in the Drosophila embryo using a novel and rapid ablation sults. In the short term, the formation of longitudinal method that allows us to ablate specific neurons and ex- pathways is delayed and disorganized in 70% of seg- amine the consequences in large numbers of embryos at ments. However, in the long term, the longitudinal tracts many different time points after ablation. In this way, we ultimately form in 80% of segments. have been able to examine both the short- and long-term consequences of ablating these early differentiating Results neurons. Toxins such as diphtheria or ricin act by inhibiting protein Creation of Blue Death Construct translation; given sufficient time, a single molecule of diph- and Transformants theria toxin can completely disrupt cellular function (Ya- To prevent random expression of diphtheria toxin, the cod- maizumi et al., 1978). Diphtheria toxin is composed of ing region was disrupted by insertion of a heterologous two distinct subunits (for review, see Collier, 1975). By gene (Figure 1). This insert acts as a "mask" that prevents expressing only the A chain subunit (DT-A) within a cell, translation of the toxin so that germline transformants can the ablation can be made cell autonomous. DT-A has been be maintained. The mask is bounded by tip recogni- used successfully to ablate various cell types in mice (e.g., tion target (FRT) motifs and can be excised by the yeast Palmiter et al., 1987; Usukura et al., 1994; Lowell et al., site-specific recombinase flp (Golic and Lindquist, 1989). 1993). Stable transformants in flies, however, cannot be This is functionally identical to the "tip-out" cassette de- easily obtained or maintained without modifying the func- scribed by Struhl and Basler (1993) and by D. Buenzlow tion of DT-A. Though examples of transformants with wild- and R. Holmgren (personal communication). Removal type DT-A gene expression do exist (Kalb et al., 1993; of the mask via flp regenerates the coding sequence, Bellen et al., 1992), this seems to be feasible only if the allowing functional toxin to be produced. After excision of toxin is expressed in tissues that are not required for viabil- the insert, a single FRT remains in the genome whose ity or fertility. Because of the difficulties associated with the wild-type "Masked" Toxin -- Blue Cells DT-A gene, several methods have been developed to regulate toxin expression. Toxin expression can be controlled FRT FRT by inserting a suppressible amber codon in the coding region (Kunes and Steller, 1991) or by generating tempera- ~lllllllllUAS ture- and cold-sensitive mutations in the gene (Bellen et A/Ik~J~~ al., 1992; Moffat et al., 1992). These modified toxins can all be used to ablate cells, but 4 or more hours may be I + flp RNA required before visible necrotic phenotypes are observed ~, ...... r~ (Moffat et al., 1992). UAS These modified toxins have proven useful for studying pupal or adult development, where several days are required for maturation. The embryo, however, develops at a much faster pace. Many of the critical early pathfinding events occur at stage 13, which lasts approximately 1 hr (Campos-Ortega and Hartenstein, 1985). Any toxin that is used to ablate neurons in the embryo must act within Functional Toxin this time frame. Diphtheria toxin can stop protein synthesis

in less than 1 hr, depending upon the cell type (Collier, ...... uAs 1975). We have developed a novel method called "Blue Death" Figure 1. Structure of the UAS-Blue Death Reporter using diphtheria toxin to study growth cone guidance in The "flp-out" method was used to regulate expression of diphtheria the embryo. This has been achieved by maintaining toxin toxin. The lacZ gene {which encedes 13-galactosidase) is fused to an function as close to that of wild type as possible (see Re- hspTO pely(A)+ sequence, inserted downstream of the ATG of the toxin, sults for design of constructs). We use this new cell abla- and bounded by FRTs (circled arrows). This Blue Death cassette is tion method in conjunction with the fushi tarazu (ftz) neuro- inserted into the UNS vector downstream of a minimal hspTO premeter (hooked arrow) and UAS sequences (boxes). Upon injection of tip genic (ftz,g) control element (Hiromi et al., 1985) to study sense RNA, site-specific recombination removes the lacZ "mask" to the role of the aCC axon, which pioneers the intersegmen- generate the toxin coding sequence. A single 22 amino acid FRT re- tal nerve (ISN) pathway. In the absence of the aCC, in the mains in the coding region. Targeted Neuronal Cell Ablation in Drosophila 709

sequence is in frame with that of the toxin. Because the to the ftzng control element, expression of 13-galactosidase action of flp can be mosaic (Golic and Lindquist, 1989), the is detected in the first ftzng-expressing neural precursor, lacZgene was chosen to act as the mask. In the absence of MP2, at stage 10. If instead a ftzn~GAL4 effector strain tip, cells containing the masked toxin will express ~-galac- is crossed to a lacZ reporter, ~-galactosidase activity is tosidase. The use of lacZ as the mask for toxin function detected in MP2 at late stage 11. This 1-1.5 hr delay in led us to call this method Blue Death (Figure 1). activation is typical of the GAL4 system (Brand and Perri- To maximize the usefulness of this construct, the Blue mon, 1993). Expression is first detected in MP2 (and its Death cassette was placed in a UAS~ reporter (R. Kostri- two siblings, dMP2 and vMP2), and is followed soon there- ken, unpublished data) to take advantage of a large bank after by the aCC, its sibling pCC, and the MP1 neurons. of available GAL4 enhancer trap lines (e.g., Lin and Good- The aCC growth cone normally pioneers the ISN at stage man, 1994). The GAL4 system uses a binary activation 12/1 to 12/0; the three U growth cones follow aCC out the method to induce expression of a given protein (Brand ISN at stage 12/0-early 13. At stage 12/0 to stage early and Perrimon, 1993; Fischer et al., 1988). One advantage 13, only a small group of neurons strongly express I~-ga- to using this system is that the expression induced by lactosidase as driven by GAL4 in this system, including GAL4 is usually greater than that from the native promoter aCC, pCC, MP1, dMP2, and vMP2; a few other neurons (Lin and Goodman, 1994). By placing the Blue Death con- are beginning to express ~-galactosidase at a low level. struct in a reporter (UAS~-Blue Death), its activation by As development proceeds, ftz is expressed at higher levels GAL4 will produce high levels of toxin. by other neurons as well, ultimately reaching 30 of the One viable transformant was obtained, and this insert 200 neurons per hemisegment by stage late 13. Many of was re-excised using a stable source of transposase (Rob- the ftzng-expressing neurons are motoneurons, while the ertson et al., 1988) to obtain a second, independent inser- rest are interneu rons whose axons fasciculate in the CNS tion. Both inserts were tested by crossing to a GAL4 in the vMP2, MP1, and FN3 longitudinal axon pathways effector. As expected, progeny embryos displayed 13-galac- (Lin et al., 1994). tosidase activity in the pattern induced by the effector, One feature of the Blue Death system is the ability to proving that both the UASGand the lacZ sequences in the identify target cells by assaying for ~-galactosidase ex- UASo-Blue Death reporters were intact (data not shown). pression. As expected, when Blue Death is crossed to To show that the FRTand toxin sequences were also func- ftzn~-GAL4 in the absence of tip, the aCC, pCC, dMP2, tional, a source of flp was introduced by crossing the vMP2, and MP 1 neurons strongly express 13-galactosidase UASG-Blue Death reporters to a strain containing a heat (data not shown). The identity of these cells was confirmed shock-tip (hs-flp) construct (Golic and Lindquist, 1989). by using the monoclonal antibody (MAb) 1 D4, which recog- The hs-flp;UASG-Blue Death recombinants were then nizes the fasciclin II protein (G. Helt and C. S. Goodman, tested by crossing to a GAL4 effector. In the absence of unpublished data). At stage 13, MAb 1D4 recognizes the heat treatment, embryos from this cross produced identified ftz-positive neurons MP1, vMP2, dMP2, pCC, [3-galactosidase activity in the expected pattern. When and aCC. It also recognizes the U motoneurons, which sit these embryos were heat shocked, no 13-galactosidase ac- just ventral and medial to aCC, and which do not express tivity could be detected, suggesting that the FRTs were ftz. Although we and others have observed a lack of seg- intact and could be excised by tip. A minimum time of 45 ment-to-segment consistency in the induction associated min at 36°C was required to produce significant flipping with using GAL4 (Brand and Perrimon, 1993), approxi- activity. When these heat-shocked embryos were stained mately 95% of the expected cells stain with the ~-galac- with various antibodies, severe defects in axon outgrowth tosidase antibody, suggesting a high degree of effec- were seen. Unfortunately, similar defects were observed tiveness. with heat-shocked Canton S embryos, indicating that these conditions led to nonspecific axonal defects. Time Course of Toxin Action To provide an alternative to heat shock, we decided to Embryos from a cross between the ftzng-GAL4 effector inject sense-strand tip RNA into embryos. Embryos were and UAS~-Blue Death reporter were either not injected, collected from en masse matings between the UASo-Blue injected with flp antisense RNA, or injected with flp sense- Death reporter strain and a GAL4 effector strain, and in- strand RNA. As expected, uninjected and antisense- jected with flp RNA. As a control, embryos were injected injected embryos appeared normal when stained with MAb with flp antisense RNA. 1D4. When sense-strand flp RNA is injected, however, reduced or no staining of either aCC's or pCC's cell body Combining Blue Death with the ftz.g Control Element or axon is observed with MAb 1D4 (Table 1). As shown As our initial test of this cell ablation method, we used an in Table 1, pCC does not stain (and is presumed dead) effector in which GAL4 is fused to the ftzng control element at stage 12/3 in 54%, at stage 12/2 in 68%, at stage 12/ (Hiromi et al., 1985; ftz,g-GAL4; D. Van Vactor and C. S. 1 in 85%, and at stage 12/0 to early 13 in 84% of the Goodman, un published data; also used by Lin et al., 1994). segments examined. Thus, inhibition of protein synthesis The ftz gene is expressed during neurogenesis in approxi- (and presumably death) is achieved within -60 min of mately 30 of the 200 neurons in each hemisegment (Doe et toxin expression and is close to the maximum effective- al., 1988). However, this pattern is temporally and spatially ness of the GAL4 method (85% versus -95%). Further- dynamic, and at early stages, the ftz gene is expressed more, expression of the toxin in aCC or pCC, which are in only a few neurons. If the/acZ gene is fused directly adjacent to the U motoneurons, does not affect expression Neuron 710

Table 1. Time Course and Effectiveness of Ablating pCC Injection of flp Sense RNA into ftz,g-GAL4/UAS-Blue Death Embryos Stage Stage Stage Stage 12/3 12/2 12/1 12/0 to Early 13 pCC present 41 (46%) 37 (32%) 11 (15%) 13 (16%) pCC absent 48 (54%) 80 (68%). 64 (85%) 66 (84%) n (# segments) 89 117 75 79

of MAb 1D4 by the U motoneurons, confirming that the Significantly fewer defects are detected by stage mid 13, toxin acts in a cell-autonomous fashion. Thus, by using and fewer still by stage late 13. Thus, given the rapid rate the ftzng-GAL4 effector, the aCC, pCC, dMP2, vMP2, and of Drosophila embryonic development, this error correc- MP1 neurons can be killed at or about the time they begin tion in the U growth cones occurs over a period of about to extend their growth cones. For the purposes of the ex- 30 min. By stage 16, only 12% of segments have abnor- periments described below, this method has allowed us mal ISN trajectories, most of these either displaying a to ablate aCC (and only a very few other neu rons) between branched peripheral nerve or crossovers from one seg- stage 12/5 and 12/0, and to examine the consequences ment to another in the periphery. on pathfinding by the U growth cones between stage 12/ 0 and stage early 13. To our knowledge, no other cells in A Drosophila E grasshopper the vicinity of the ISN pathway are ablated by this method during this time period.

Behavior of the U Growth Cones in the Absence of the aCC Axon The ISN is pioneered by the growth cone of the aCC motoneuron by stage 12/1-12/0. The three U axons follow and fasciculate with aCC's axon by stage 12/0-early 13 (Figure UI-U3 2A). Additional motoneuron axons and growth cones join aCC the nerve as development proceeds. In antisense flp RNA-injected ftzng-GAL41UASG-Blue U1-U3DC o~ Death embryos, the U axons develop normally in 98% of segments (Table 2; n = 245). In sense flp RNA-injected embryos, the aCC neuron is ablated in -85% of segments. In these embryos, the U axons are abnormal in aCC 73% of segments at stage early 13 (n = 77; Figure 3; shown schematically in Figure 2). in 5% of segments, the U axons extend posteriorly and stay within the CNS (Figure 2B; Figures 3A, 3D, and 3F). In 27% of segments, the Figure 2. Behavior of the U Growth Cones in the Absence of aCC in U axons take an abnormal trajectory, extending further Drosophila versus the Behavior of aCC in the Absence of the U Axons posteriorly in the CNS before turning peripherally (Figure in Grasshopper 2C; Figures 3C and 3E). in 40% of segments, the U axons Schematic diagram showing the behavior of the aCC and U growth extend peripherally, but branch into multiple pathways cones in Drosophila and grasshopper, in wild-type embryos and in (Figure 2D; Figures 3A, 3B, and 3E). The time at which embryos in which the pioneer(s) (aCC in Drosophila and the U axons in grasshopper) have been ablated. The Drosophila data are based these phenotypes are most penetrant is stage 12/0-early on the results reported here, whereas the grasshopper data are based 13 (Table 2), precisely the time at which the U growth on previously reported results (du Lac et al., 1986). cones initially contact and follow the aCC axon. (A) In Drosophila, the aCC growth cone is within filopodial grasp of In several embryos, two of which are shown in Figures the segment boundary glial cell, and from the outset, turns laterally 3A and 3D, the mosaic nature of the cell ablation method and pioneers the ISN. The U growth cones arrive at the dorsal surface of the CNS, and then turn and follow aCC's axon. left one aCC (and its sibling pCC) alive in one hemiseg- (B-D) In the absence of aCC, the U growth cones typically display a ment, whereas in all neighboring hemisegments aCC was variety of errors and delays at stage 13, including staying within the ablated. These hemisegments with living aCCs serve as CNS (B), taking an altered trajectory into the periphery (C), and good internal controls, since in each of these hemiseg- branching into multiple pathways (D). See Table 2 for further details. (E) In the grasshopper embryo, the aCC growth cone is further away ments, the U axons fasciculated with aCC's axon and ex- from the segment boundary glial cell that marks the ISN root (Bastiani tended out the ISN with the same timing and trajectory as et al., 1986). The aCC growth cone waits for the U growth cones, which occurs normally, whereas all neighboring hemisegments arrive at the dorsal surface of the CNS, turn posteriorly to pioneer the with ablated aCC axons showed highly abnormal U axons. U fascicle, and then turn laterally to pioneer the ISN. The aCC growth Despite the initial abnormal behavior of the U growth cone follows behind. (F) When the U axons are ablated, the aCC growth cone stalls and cones at stage 12/0-early 13, as development proceeds the is unable to pioneer the pathway on its own (du Lac et al., 1986). U growth cones correct their initial mistakes (Table 2). Bars, 5 p.m (A-D), 10 p.m (E and F). Targeted Neuronal Cell Ablation in Drosophila 711

Table 2. The Effect of Ablating aCC on Pathfinding by the U Growth Cones Control Embryos Injected Experimental Embryos in which flp Sense RNA Is Injected into ftz,g- with flp Antisense RNA GAL4/UAS-Blue Death Embryos Stage Stage Stage Stage Stage 12/0 to Mid 13 12/0 to Early 13 Mid 13 Late 13 16 U axons normal 240 (98°/o) 21 (27%) 114 (69%) 198 (63%) 105 (88%) U axons abnormal (total) 5 (2%) 56 (73o/o) 51 (31%) 117 (37%) 15 (12%) U axons remain in CNS 0 4 (5%) 27 (16%) 0 0 (B in Figure 2) U axons altered trajectory 3 (lO/0) 21 (270/0) 3 (2%) NA NA in CNS (C in Figure 2) U axons branched 2 (10/o) 31 (40%) 21 (13%) 13 (4%) 6(5%) (D in Figure 2) U axons truncated in PNS NA NA NA 104 (33%) 1 (1%) U axons cross-over in PNS NA NA NA 0 8 (6%) n (# segments) 245 77 165 315 120 All percentages are of total number of segments examined. NA, not applicable.

Figure 3. Behavior of the U Growth Cones in the Absence of aCC Photomicrographs of filleted, stage 13, sense flp RNA-injected, ftz, g-GAL41UASa-Blue Death embryos, in all panels, a thin arrow indicates an aCC cell body and axon (if still alive), a thicker arrow indicates a pCC cell body and axons (if still alive), and an arrowhead indicates U axons. (A) Blue staining indicates live cells due to mosaic action of flp activity. At bottom left, two blue cell bodies are aCC (top) and pCC (bottom). An aCC axon extends laterally to the left (thin arrow) and is followed by the U axons (arrowhead). At top left, aCC is ablated and U axons extend posteriorly within the CNS. At bottom right, U axons branch into multiple pathways as they extend laterally. (B) Arrowhead shows branched U axons. (C) Arrowhead shows branched U axons that have extended posteriorly within the CNS; at least one axon has turned laterally. (D) In middle, two blue cell bodies are aCC (top) and pCC (bottom). pCC's axon is seen extending anteriorly as normal in the next anterior segment. Arrowheads on left and right show U axons extending posteriorly within CNS. (E) Arrowheads show branched and highly abnormal U axons. (F) Arrowheads show U axons extending posteriorly within CNS. Bar, 10 p.m. Neuron 712

Table 3. The Effect of Ablating the First LongitudinalAxons on the Formation of LongitudinalAxon Pathways Control Embryos Injected Experimental Embryos in which flp Sense RNA Is with flp antisense RNA Injected into ftz,g-GAL4/UAS-Blue Death Embryos Stage Stage Stage Stage 14 14 15 16 Longitudinal pathways present 268 (/>99%) 39 (300/0) 49 (64%) 130 (80°/0) Pathways highly abnormal (total) 1 91 (70o/0) 27 (36o/o) 33 (200/o) Gaps in longitudinalpathways 1 56 (43%) 21 (28°/o) 18 (11%) Other abnormalities 0 35 (27%) 6 (80/0) 15 (9%) in longitudinalpathways n (# segments) 269 130 76 163 All percentages are of total number of segments examined.

Formation of Longitudinal Axon Pathways in the Discussion Absence of the First Longitudinal Axons The first two longitudinal axon pathways to form in the We developed a novel, rapid, and efficient cell ablation CNS of the Drosophila embryo are the vMP2 and MP1 method and have used it here to study the role of pioneer fascicles (Lin et al., 1994; Fetter et al., unpublished data). axons in the formation of both PNS and CNS axon path- The vM P2 fascicle is pioneered at stage late 12 by the pCC ways in the Drosophila embryo. We examined the behavior growth cone, with vMP2 following immediately behind it. of the U growth cones in the absence of the aCC axon, The MP1 fascicle is pioneered by the dMP2 growth cone, which pioneers the ISN. The aCC axon appears to facili- with MP1 following right behind it. By early stage 14, two tate the rapid development of the ISN pathway, but in most pathways have formed within each segment: the medial cases it is not absolutely essential. In its absence, in the vMP2 fascicle and the lateral MP1 fascicle. As develop- short term, the follower U axons are delayed and make ment proceeds, many other axons join these pathways, pathfinding errors in 73% of segments (Table 2). In the and additional longitudinal fascicles form. The vMP2 and long term, however, they ultimately correct their mistakes MP1 pathways can be visualized with MAb 1D4. At stage and do form the ISN pathway in 88% of segments. 16, MAb 1D4 stains three large longitudinal axon fascicles: We also ablated the axons that pioneer the first longitudi- the inside MP1 pathway, the middle FN3 pathway, and a nal pathways (pCC, vMP2, dMP2, and MP1; see Lin et third outside pathway (Lin et al., 1994). The ftzng control al., 1994) within the CNS and observed similar short-term element can drive expression of a heterologous gene in versus long-term results. In the short term, the formation many of the neurons whose axons fasciculate in these of longitudinal pathways is delayed and disorganized in three pathways (Lin et al., 1994). 70% of segments (Table 3). However, in the long term, the Because ftzng-GAL41UASo-Blue Death leads to the longitudinal tracts ultimately do form in 80% of segments. death of the pCC, vMP2, dMP2, and MP1 neurons at or Thus, the pioneers appear to play a major role in facilitat- prior to axonogenesis, the pioneers of both the vMP2 and ing the rapid and robust development of PNS and CNS MP1 pathways can be ablated. To follow the development axon pathways. In the absence of the pioneers, followers of these and other longitudinal axon pathways, we stained are delayed and make frequent errors. In the short term, ftzng-GAL41UASo-Blue Death embryos with MAb 1D4. In we observed major abnormalities in the formation of the control antisense flp RNA-injected ftz,g-GAL41UASG-Blue ISN and CNS longitudinal tracts in 73% and 70% of seg- Death embryos, the MP1 pathway forms normally in i>99% ments, respectively. Given that our ablation method ap- of segments (Table 3). pears to be effective in about 85% of segments (Table 1), In flp sense RNA-injected embryos, the longitudinal this increases the real short-term percentage of abnormali- axon pathways are either absent or highly abnormal in ties to more like 82% of segments in which the cells are 70% of segments at stage 14 (Table 3). In 43% of seg- actually ablated. However, in spite of this dramatic short- ments at stage 14, these pathways are simply missing term effect, in the long term, the pioneers are not abso- (Figures 4A and 4B). In 27% of segments, these pathways lutely required, in that these embryos display a remarkable appear abnormal in shape, and the longitudinal pathways ability to correct and compensate for the loss of these in general appear disorganized (Figure 4). The effects of early pioneering neurons, a higher degree of plasticity than ablating the vMP2 and MP1 pathway pioneers decrease might have been anticipated. but are still apparent at stage 16. In 11% of segments at These results suggest that the first axons, the pioneers, stage 16, the longitudinal pathways are missing (Figure do not necessarily represent a special class of neurons 4C). In another 9% of segments, the longitudinal pathways that play an exclusive and necessary role for the formation are present but appear highly disorganized (Figure 4D). of the major axon pathways in the CNS and PNS. Rather, In other segments, the longitudinal pathways appear par- the importance of each pioneer depends upon the context tially disorganized. However, as with the ISN, the propor- of the pathway it establishes in relationship to when and tion of segments displaying abnormal phenotypes drops from where the next axons arrive. significantly from 70% at stage 14 to 36% at stage 15 to It is interesting to compare the results reported here for only 20% by stage 16 (Table 3). the formation of the ISN in the Drosophila embryo with Targeted Neuronal Cell Ablation in Drosophila 713

Figure 4. Abnormal Longitudinal Axon Tracts in Stage 14 and 16 Embryos after Ablation of the vMP2 and MP1 Pathway Pioneers Photomicrographs of filleted, stage 14 (A and B) and stage 16 (C and D), sense flp RNA- injected ftz,g-GAL 41UASo-Blue Death embryos as stained with anti-fasciclin II MAb 1D4 (brown horseradish peroxidase immunocytochemistry) and 13-galactosidaseactivity stained. In all panels, blue cells indicate neurons targeted for death but having insufficient flp activity owing to mosaic nature of method. Even where present, most of the longitudinal pathways appear disorganized. (A) At top, MP1 pathway and other longitudinal pathways are missing on both sides between two segments (asterisks). At bottom left, normal longitudinal pathways are missing, and instead some axons are following intersegmental nerve root pathways (arrowhead with asterisk). (B) At middle left, longitudinal pathways are missing (asterisk). (C) At middle right, longitudinal pathways are missing between two segments (asterisk). (D) At middle left, longitudinal pathways are thin and disorganized. Feathered arrow shows area in which longitudinal pathways extend across the midline in a highly abnormal fashion. Bar, 10 p.m.

previous studies on the formation of the ISN in the grass- As shown in the present paper, in the absence of aCC, hopper embryo. In the grasshopper embryo, the aCC the U growth cones typically make a variety of errors and growth cone is further away from the segment boundary delays (Figures 2A-2D), but usually are ultimately able to glial cell that marks the ISN root (Bastiani et al., 1986). turn laterally and extend into the periphery to help pioneer The U growth cones arrive at the dorsal surface of the a fairly normal ISN pathway. CNS, turn posteriorly to pioneer the U fascicle, and then Thus, the very same pathway (the ISN) and homologous turn laterally to pioneer the ISN. The aCC growth cone neurons (aCC and the three U axons) provide different waits for the U axons, and then follows behind them. When results in different species, presumably reflecting evolu- the U axons are ablated, the aCC growth cone stalls and tionary changes in the pathfinding abilities of particular is unable to pioneer the pathway on its own (Figures 2E growth cones. The aCC is the pioneer in Drosophila, and 2F) (du Lac et al., 1986). whereas the U axons are the pioneers in grasshopper. In contrast, as shown here, one obtains very different One consequence is that the ISN forms relatively earlier results in this divergent insect species. In Drosophila, the during the embryogenesis of Drosophila as compared with aCC growth cone is within filopodial grasp of the segment grasshopper. In Drosophila, the follower U axons can pio- boundary glial cell, and from the outset turns laterally and neer the pathway in the absence of aCC, although they pioneers the ISN. The U growth cones arrive at the dorsal do not do as robust a job as does aCC. In grasshopper, surface of the CNS and then turn and follow aCC's axon. the follower aCC cannot pioneer the pathway on its own. Neuron 714

These results suggest that changes have occurred during in a humidified, 18 °C incubator. Upon reaching the desired stage, the evolution in the ability of these neurons to pioneer the ISN. embryos were exposed to a gentle stream of water to displace the oil. The water was replaced by fixative (1 x PBS, 3.7% formaldehyde) In one organism (grasshopper), the pioneers (the U axons) and incubated at 18°C for 45 min to 1 hr. After removal of the fixative, of the ISN are absolutely necessary; in the other organism embryos were washed with PBT (1 x PBS with 0.1% Triton X-100), (Drosophila), the pioneer (aCC) of the ISN is not absolutely manually devitellinized, and collected for staining. necessary, but helps facilitate formation of the pathway. The UAS~-B/ue Death reporter was designed to prevent expression of the toxin when inserted into the genome. Less than 5% of all em- It is unclear why these sorts of evolutionary changes oc- bryos from this stock displayed generic defects (as detected with MAb cured in the pathfinding abilities of these homologous neu- 1D4), confirming that the construct functioned as predicted. The ftzn~- rons. One possibility is that different temporal constraints GAL4 stock also had very few background defects. When the two were (particularly the relative early extension of sensory axons crossed together (ftz,g-GAL41UASG-B/ue Death), no increase in the toward the CN S in Drosophila as compared with grasshop- percentage of defects occurred. When the injection procedure was tested by injecting antisense flp RNA, the proportion of severely dis- per) may have helped drive this earlier pioneering function rupted embryos rose to 15%. This indicates that some additional dam- of aCC. age was caused by injection, but not at unacceptable levels. Moreover, these defects are generic and easily distinguishable from toxin- Experimental Procedures induced defects, which are highly specific. Thus, it was easy to elimi- nate these 15% of highly abnormal embryos from the remaining 85% Cloning of Blue Death and Transcription Constructs carrying specific targeted cell ablation defects; these 15% are not Standard molecular techniques (Sambrook et al., 1989) were used to counted in Table 1 and Table 2. create the Blue Death reporter construct and the constructs for in vitro transcription of f/p RNA. HRP Immunohistochemistry and I~-Galactosidase The diphtheria toxin construct was created using the SK402 vector Activity Assay (Kunes and Steller, 1991) as a template. This plasmid contains the A HRP immunocytochemistry was carried out as described (Lin and chain of diphtheria toxin in a standard Bluescript vector. A 5' Xbal Goodman, 1994). The MAb directed against fascielin II (1D4; G. Helt site upstream of the start o1 translation and a Kpnl site immediately and C, S. Goodman, unpublished data) was used at a dilution of downstream of the initiator methionine were cleaved, and two oligos 1:5. ~-Galactosidase activity staining was carried out as described (Lin were inserted. The oligos contain the DT-A ATG and two copies of and Goodman, 1994). Embryos were reacted 20 rain to overnight at the minimal FRTsequence (oriented as direct repeats), as well as Notl 37°C. Embryos were staged according to Campos-Ortega and and Stul restriction endonuclease recognition sequences between the Hartenstein (1985) and Kl~mbt et al. (1991). FRTs. These sites were cleaved, and a NotI-Stul fragment containing a/acZ/hsp70 poly(A) chimeric fusion was inserted. When the toxin is activated, the coding region includes an additional 22 amino acids at Acknowledgments the 5' end derived from the FRT, whose sequence is MRFEVPIPKFLF- SRKYRN FRAL. We thank Paul Sadowski for providing the tip and FRTDNA and Dar- This B/ue Death cassette was excised as a Pstl and Scal (blunted) lene BuenzIow and Robert Holmgren for the anti-~-galactosidase anti- fragment and placed in a UASG reporter construct (R. Kostriken, unpub- body. D. M. L. was supported by a U. C. Berkeley Fellowship, NIH lished data). The UASGvector contains eight copies of the UAS~ GAL4 Training Grant 5T32 GM07232, and NIH grant HD21294. V. A. was binding site, as well as an SV40 polyadenylation sequence. supported by a postdoctoral fellowship from the Canadian Medical For the sense and antisense f/p transcription vectors, a 1.4 kb Bglll Research Council and was also a Fellow with the Howard Hughes fragment from DV56 (provided by Paul Sadowski) containing the f/p Medical Institute. C. S. G. is an Investigator with the Howard Hughes coding region was inserted in either orientation into the pSP64T tran- Medical Institute. scription vector (Krieg and Melton, 1984). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby In Vitro Transcription marked "advertisement" in accordance with 18 USC Section 1734 All transcription reactions used Boehringer Mannheim reagents, ex- solely to indicate this fact. cept for G(5')ppp(5')G (New England Biolabs). The 64T+flp and 64T-flp constructs were linearized by cleavage with Xbal, ethanol precipitated, Received January 5, 1995; revised January 31, 1995. and resuspended in Tris-EDTA (10 mM Tris [pH 7.0], 1 mM EDTA). Standard SP6 transcription reactions were used to produce RNA. References Briefly, each 10 I~1 of reaction contained: 1 i11 of DNA (150 ng of template), 1.5 I11 of ddH20, 1 p_l of G(5')ppp(5')G (10 raM), 1 pJ of 10x buffer Bastiani, M J., Raper, J. A., and Goodman, C. S. (1984). Pathfinding by (BMB), 2 td of restriction buffer A (2 x ), 2 td of dNTPs (10 mM each), neuronal growth cones in grasshopper embryos. II1. 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