
. The Developing Eye and Brain at the Third Instar Larval Stage (A and B) The visual system from the lateral perspective. Photoreceptor cells in the eye disc (ed) commence neural differentiation at the morphogenetic furrow (mf), which moves in a posterior-to-anterior direction at this stage. The eight axons of an ommatidial R cell cluster navigate as a fascicle through the optic stalk (os) into the brain. Shown in (A) are 3 of the approximately 500 R cell clusters present at the late third instar. The R1-R6 axons terminate at retinotopic destinations in the lamina (lam, colored blue in [A] ). At the crescent-shaped lamina furrow (lf), recently arriving retinal axons come into close proximity of G 1-phase LPCs. They trigger the LPCs to divide at the posterior margin of the lamina furrow (S-phase cells in red), initiating the events that give rise to lamina neurons. The LPCs are generated by the actively dividing OPC. The confocal micrograph in (B) reveals S-phase cells labeled by the incorporation of BrdU (colored red), LPC progeny in the lamina labeled to detect dac expression (by mAbdac1-1; colored blue), and photoreceptor cells, their axons, and other neuronal cell membranes (labeled by ␣HRP [Jan and Jan, 1982] ; colored green). The OPC, a broad ring of BrdU-positive cells on the periphery, gives rise to an inner ring of LPCs. A set of LPCs enters S phase at the lamina furrow (lf; purple cells that are both BrdU-positive [red] and Dac-positive [blue] ). These cells lie just anterior of the retinal axons that innervate the crescent-shaped Dac-positive lamina. Anterior is to the left, dorsal up. lob, lobula complex. Scale bar in (B), 15 m. (C and D) The visual system from the horizontal perspective. The eye disc and lamina display a temporal gradient of development on their anteroposterior axes. In (C), three ommatidia represented at different anteroposterior positions in the eye disc (ed) send their axons through the optic stalk to corresponding anteroposterior positions in the lamina, where the R1-R6 axons terminate (R1-6 ter in [D] ). The R7 and R8 axons continue to deeper targets in the medulla (medn). The OPC gives rise to G 2-phase LPCs (dark gray; see inset in [C] for the cell-cycle events of lamina neurogenesis) that undergo mitosis at the anterior margin of the lamina furrow (lf). The G 1-phase LPCs (light gray) contact the most recently arriving retinal axons (arrowhead in [D] ) at the trough of the lamina furrow. The LPCs enter S phase and divide at the posterior margin of the lamina furrow (LPC progeny in blue). In the lamina, the LPC progeny are organized into columns in precise register with the axons that regulate their differentiation. LPC progeny and glia of several types assume stereotyped positions on the lateral medial axis. The glia include the medulla (Md-glia), marginal (Ma-glia), epithelial (E-glia), and satellite (S-glia). The confocal micrograph in (D) shows BrdU-positive cells in the OPC (colored red) and at the posterior margin of the lamina furrow, where an LPC has entered S phase (purple, due to BrdU-positive [red] and Dac-positive [blue] labeling). Dac-positive LPC progeny (blue) in the lamina are visible in columns sandwiched between the retinal axon fascicles (labeled by ␣HRP; colored green). Anterior is to the left, lateral up. Scale bar in (D), 5 m. a temporal progression on the anteroposterior axis of at a morphological indentation known as the lamina furrow, where the axons come into the close proximity the lamina primordium ( Figures 1C and 1D ). Each new row of retinal axons that enters the brain takes up more of LPCs (Selleck et al., 1992) . Lamina neurogenesis can be monitored by a number of cell type-specific markers anterior retinotopic positions in the lamina and induces a more anterior population of LPCs to enter S phase that are not expressed in the precursor cells. The expression of shortsighted (shs; Treisman et al., 1995) , and proceed to neural differentiation. The interaction between newly arriving retinal axons and LPCs occurs dachshund (dac; Mardon et al., 1994) , fasciclin II (fasII; Grenningloh et al., 1991) , and elav (Bier et al., 1988;  imaginal disc. The animals remained at the restrictive temperature until the late third instar (24-36 hr later), Robinow and White, 1988 ) is found in LPC progeny posterior of the lamina furrow ( Figures 1B, 1D, 2D ; data not when their visual ganglia were examined for lamina differentiation. Lamina neurogenesis was monitored by the shown). In addition, a number of enhancer-trap lacZ insertions have been identified that are expressed in a presence of Dac-positive and Elav-positive cells. dac expression is an early marker of lamina induction and lamina-specific fashion (Selleck and Steller, 1991; Winberg et al., 1992; Mozer and Benzer, 1994) . The expresis first detected in all LPCs at the time they enter S phase at the posterior margin of the lamina furrow (see sion of these markers is innervation-dependent and not detected in eyeless mutants, such as eyes absent (eya) Figure 1D ). Elav is detected in the posterior of the lamina among the subset of LPC progeny that differentiate as and sine oculis (so; see Figure 5B ; data not shown; Selleck and Steller, 1991) . The progression of G 1 -phase L neurons ( Figure 2D ; Selleck and Steller, 1991) . Since both markers are expressed continuously after their inLPCs into S phase is easily visualized by the incorporation of the thymidine analog, bromodeoxyuridine (BrdU), duction, they provide an indelible record of all inductive events. into replicating DNA (Figure 1 ; Truman and Bate, 1988) . LPCs that have entered S phase are detected as a disAs expected, the hh ts2 animals shifted to the nonpermissive condition displayed the "furrow-stop" phenocrete band of labeled cells at the posterior margin of the lamina furrow. These S-phase cells are absent in type, indicating an arrest of morphogenetic furrow progression in the developing eye (Ma et al., 1993 ; Heberlein eyeless mutants, reflecting the failure of LPCs to enter their terminal division (Selleck and Steller, 1991). et al., 1993) . The eye discs of these animals contained between 6 and 12 columns of ommatidial R cell clusters, Markers that denote the differentiation of lamina glia are also regulated by the ingrowth of retinal axons about a third of the number observed in a wild-type animal in the late third instar. The most anterior R cell (Winberg et al., 1992) . We observed that patched (ptc; Nusslein- Volhard and Wieschaus, 1980 ) is expressed in clusters had the appearance of mature clusters normally found in posterior columns. These observations indicate two classes of lamina glia and in subretinal glia cells in the optic stalk and eye disc ( Figure 2G ; K. Kaphingst that these ommatidia were induced by Hh prior to the shift to the nonpermissive condition. In the lamina, the and S. K., unpublished data), cell populations that are in close proximity to retinal axons. ptc expression is axon fascicles from these R cells were found in their proper dorsoventral positions posterior of the lamina absent from the visual ganglia of eya animals, which lack retinal innervation ( Figure 2H ). ptc is regulated by furrow ( Figures 2B and 2C ). However, viewed on the anteroposterior axis ( Figure 2E ), these fascicles apthe activity of the hh gene in a number of tissues (Hidalgo and Ingham, 1990; Ingham et al., 1991; Tabata and Korn- peared collapsed upon one another, as though the cells that normally populate the lamina were missing (see berg, 1994; Heberlein et al., 1995) . This suggests that Hh might be a component of the below). In animals with fewer than eight columns of developing ommatidia (33 of 58 brains examined), no inductive signal that regulates lamina development.
In a wild-type animal, Hh immunoreactivity is found Dac-positive cells were found in the lamina primordium ( Figure 2B ). In animals with more than eight columns in the most anterior 5-7 columns of R cells adjacent to the morphogenetic furrow ( Figure 3 , left panel). Imporof developing ommatidia (25 of 58 brains examined), a reduced number of Dac-positive cells were found at tantly, Hh immunoreactivity is found on retinal axons in the optic stalk and in the brain, including the dorsal and the posterior of the lamina ( Figure 2C ), the region that receives innervation from the earliest arriving retinal axventral tracts along which retinal axons approach the lamina furrow to make contact with LPCs. Hh was not ons. We suppose that in these animals, the earliest axons arrive prior to the loss of hh ϩ activity. Elav expression detected in any other cell population in the visual system, including the brain, at this stage. Nor was hh exwas likewise absent or restricted to a few cells at the posterior of the lamina ( Figure 2E ; 66 of 66 brains exampression detected in the visual ganglia in either of two hh enhancer-trap lacZ strains, P30 and J413, that faithfully ined). Finally, hh loss of function resulted in the absence of ptc-expressing glia cells from the optic stalk and report hh expression (Lee et al., 1992; Ma et al., 1993) . Our failure to detect hh expression in the developing lamina ( Figure 2I ; 50 of 50 brains examined). Aside from these effects on the expression of innervation-depenvisual ganglia suggested that Hh might be delivered to the brain by the retinal axons. dent markers, no other defects in optic lobe development were evident in hh Ϫ animals at this stage. To test the notion that Hh might be the inductive signal, we sought to generate photoreceptor cell axons Animals harboring a regulatory allele, hh 1 (Lee et al., 1992; Heberlein et al., 1993) , that specifically affects hh lacking hh ϩ activity. hh is required in the developing eye for the induction of R cell differentiation. Since it takes expression in the visual system ( Figure 3 ) displayed a phenotype similar to that observed in hh ts2 animals. In hh 1 about 9 hr (at 25ЊC) for retinal axons to traverse the path between the eye and the brain, we supposed that a animals, the morphogenetic furrow ceased its anterior progression after the induction of about 11 columns of conditional hh allele might be used to inactivate hh function so that retinal axons would arrive in the brain lacking R cell clusters. hh 1 photoreceptor cells and axons lacked Hh immunoreactivity in the mid-to-late third instar stage hh ϩ activity. To this end, hh ts2 animals were shifted to the nonpermissive condition in the early third instar (90 (Figure 3 , right panel), times at which the progression of the morphogenetic furrow had ceased in these animals. hr after egg laying [AEL] ; times normalized to growth at 25ЊC), a time at which the first several columns of Normal Hh expression was observed in the posterior compartments of other imaginal tissues (e.g., the leg ommatidia have been induced at the posterior of the eye animals (E) (shifted to the nonpermissive condition at 90 hr AEL) and hh 1 animals (F), despite the presence of retinal axons in the proper region (indicated by arrowhead). In the lobula (lob) and medulla cortex (medc), Elav expression is unaffected by the loss of hh function. In the hh Ϫ animals, retinal axons appear collapsed upon one another in the lamina, as though lamina cell populations are missing. R1-6ter, the R1-R6 axon termini. L1-4, L5; lamina neuron subtypes. Anterior is to the left, lateral up. (G-I) Horizontal view of wild-type (G), eya (H), and hh ts2 (I) specimens stained to reveal patched expression at the late third instar stage. In the wild type, strong lacZ expression (colored red) by the ptc-lacZ reporter is detected in the satellite glia (S-glia), medulla glia (Md-glia), and subretinal glia (not shown). In the eya animal lacking retinal innervation (H), lacZ expression is absent. In the hh ts2 animal (I) (shifted to the nonpermissive condition at 90 hr AEL), lacZ expression is absent despite the presence of retinal axons (arrowhead indicates the most anterior axons). Anterior is to the left, lateral up. at any particular stage (data not shown). Thus, we con-specimens were additionally stained with propidium iodide to visualize the nuclear DNA of all cells. As can be seen in Figure 4 , the failure of retinal axon fascicles to separate normally on the anteroposterior axis of the lamina in hh Ϫ animals was associated with a large reduction in the number of cells in the lamina (compare the region between brackets in Figure 4CЈ and 4DЈ; 54 of 54 brains examined). Anterior of the lamina furrow, cells expressing high levels of Cyclin A, apparently G 2 -phase LPCs, were present in approximately normal numbers in hh Ϫ animals (compare Figures 4C and 4D) , while the cells among the retinal fascicles expressed low levels of Cyclin A, consistent with their residing in the G1 phase (or G0 phase). Mitotic figures were observed at the boundary between these high and low Cyclin A-express- (Selleck et al., 1992) . The reduction of cell number obfrom the wild-type (A/P marks the anterior, posterior compartment border). The visual system is shown anterior to the left, dorsal up.
served in the lamina of hh Ϫ animals is thus due, at least
Scale bar, 15 m.
in part, to the failure of LPCs to undergo their terminal division at the lamina furrow.
clude that the absence of lamina differentiation in hh Ϫ animals is due to the loss of hh function, rather than to Ectopic hedgehog Expression Induces Lamina Differentiation in Eyeless Animals a reduction in the number of retinal axons.
If Hh is the sole inductive signal delivered to the lamina by retinal axons, we would expect that ectopic hh ϩ exCell-Cycle Arrest of LPCs in Animals Lacking hh Function pression in the brains of animals lacking photoreceptor cells would be sufficient to induce lamina differentiation. The axon fascicles from each column of ommatidia are normally separated by the LPC progeny and glia that
To this end, we used the flp-out strategy of Basler and Struhl (1994) to generate somatic clones of hh ϩ -expresspopulated the nascent cartridge units ( Figures 1C and  1D ). In hh Ϫ animals, the fascicles were compressed one ing cells in the brains of eyes absent (eya) animals, which lack differentiating photoreceptor cells. In the first exupon another (compare Figures 2D and 2E ), suggesting that these lamina cell populations are largely absent.
periments, hh ϩ clones were generated under conditions that resulted in a high frequency of large clones, so that One explanation for this apparent reduction in cell number might be that LPCs fail to undergo their terminal cell multiple hh ϩ clones would be present in the brains of most animals. We found that in these animals, the LPCs division at the lamina furrow.
To determine whether this is the case, S-phase cells completed their terminal division at the lamina furrow and expressed lamina-specific markers despite the abin hh 1 animals were labeled by BrdU incorporation at several timepoints throughout the early-to-mid-third insence of retinal axons (90 of 105 brains examined). For example, in the specimen shown in Figure 5C , ectopic star, times at which retinal axons enter the brain and would be expected to deliver the inductive signal. As hh ϩ expression induced a field of Dac-positive cells in the lamina similar to that observed in a wild-type animal shown in Figure 4B , the number of S-phase cells at the posterior margin of the lamina furrow was greatly (compare Figures 5A and 5C ). The incorporation of BrdU into S-phase cells in this animal revealed a discrete band reduced in hh 1 animals (compare with the wild-type in Figure 4A ; 47 of 50 brains examined). The small number of labeled cells at the anterior border of the Dac-positive cell population. In eya animals, both the BrdU-positive of BrdU-positive cells remaining in hh 1 animals is also observed in animals lacking retinal innervation (data not and Dac-positive populations were absent ( Figure 5B ). In addition to dac, ectopic hh ϩ expression in animals shown; Selleck et al., 1992) . Other proliferation centers, such as the outer proliferation center (OPC), were not lacking photoreceptor cells induced the expression of all other early lamina markers tested. These included affected by the loss of hh function (compare Figures 4A  and 4B ).
FasII (data not shown) and the lamina-specific lacZ reporter gene construct, P[S14:lacZ] ( Figures 5D and 5E ). 5D ). This temporal relationship was also observed for in the lamina. These observations suggest that Hh alone is not sufficient for the later events of lamina develop-P[S14:lacZ] expression induced by ectopic hh ϩ expression ( Figure 5E ). Cells expressing both dac and lacZ ment that include the specification of LPC progeny as L neurons. These later events would appear to require were found to the posterior of cells which expressed dac alone. Finally, ectopic hh ϩ expression in eya animals at least one additional signal that can be provided by retinal axons. induced the expression of ptc in lamina glia ( Figure 5F ).
In contrast, ectopic hh expression was not sufficient to induce a late marker, Elav, which is expressed among Clonal Analysis of hh Function: Hh Expressed in the Eye Can Act in the Brain the subset of LPC progeny that differentiate as L neurons ( Figures 5G and 5H) . Similar results were obtained To examine the spatial relationship between hh ϩ -expressing cells and the induction of lamina differentiawith either of two genetic backgrounds, using eya (data not shown) or the early inactivation of hh function (aption, we employed a construct in which hh ϩ clones were marked by the loss of sequences encoding the memproximately 34 hr AEL in hh ts2 animals; Figure 5H ) to prevent the differentiation of photoreceptor cells. On brane protein CD2 during the flp-out recombination event (Zecca et al., 1995 ; Dunin-Borkowski and Brown, the other hand, when retinal axons were present (in hh ts2 animals maintained at the permissive condition until 90 1995). hh ts2 animals harboring this construct were subjected to a mild FLP recombinase-induction to generate hr AEL to allow some R cell induction), ectopic hh ϩ expression resulted in the induction of Elav-positive rare hh ϩ clones. These animals were shifted to the nonpermissive condition at an early time (approximately 34 cells in approximately normal numbers and position ( Figure 5I ; 10 of 11 brains examined). In the specimen hr AEL) to prevent photoreceptor cell differentiation. Rare hh ϩ clones in the LPC area were often associated shown in Figure 5I , Elav was expressed among the Dacpositive cells induced by ectopic hh ϩ in the vicinity of with the induction of Dac-positive cells (57 of 77 clones examined; Figures 6A and 6C) . In the specimen shown the reduced number of retinal axons in this animal. These axons would normally induce neither Dac nor Elav in Figure 6A , a CD2-negative clone that includes the Animals harboring hh ϩ clones in the developing eye five no such clones were found adjacent to Dac-positive cells. These observations indicate that Hh expressed in were examined to determine whether Hh expressed in the eye could be transmitted into the brain. These hh ts2 the eye can act in the brain to induce the initial steps of lamina differentiation. animals were shifted to the nonpermissive condition at 90 hr AEL, which would normally generate photoreceptor cell axons lacking hh ϩ activity. In a small fraction of Discussion specimens (7 of 46 brains), small CD2-negative clones in the eye resulted in a subset of retinal axons that could It has long been known that in Drosophila and other arthropods, the development of the visual ganglia deharbor hh ϩ activity from the flp-out construct. As shown in Figures 6E-6H , these retinal axons induced Dac-posipends on innervation from the compound eye (Power, 1943; Fischbach and Technau, 1984; Macagno, 1979 ; tive cells in the proper retinotopic position in the lamina, while the axons from neighboring hh Ϫ (CD2-positive) reviewed by . In Drosophila, the role of ingrowing retinal axons in the first optic ganglion, photoreceptors did not. In two of these specimens (including the one shown in Figure 6 ), no hh ϩ (CD2-negathe lamina, is to induce the precursors of their synaptic target cells, the LPCs, to complete a terminal division tive) clones were detected in the brain, and in the other and initiate neural differentiation (Selleck and Steller, in the G1 phase following mitosis at the anterior margin of the lamina furrow. Glia precursor cells that lie immedi-1991). Here, we have shown that the primary inductive signal delivered by retinal axons is the product of the ately anterior of the lamina furrow (Perez and Steller, 1996) , fail to induce lamina neurogenesis on entering the brain. Despite their presence in the proper target field, and then transmit a second inductive cue to the LPCs. these axons fail to induce LPCs to divide (Figure 4) or to express neural or lamina-specific markers (Figure 2) .
Hedgehog and Pattern Formation in the This is the same phenotype observed in mutants lacking Developing Visual Ganglia photoreceptor cells. Second, ectopic hh ϩ expression in In the visual ganglia, the role of hh appears restricted the brain is sufficient to initiate lamina neurogenesis in to the regulation of lamina development. hh expression animals lacking photoreceptor cells ( Figure 5 ). In these is not detected in this tissue during the larval stages animals, the LPCs complete their terminal division and (Figure 3 ; data not shown). Moreover, loss of hh function express markers that denote the early steps of lamina or ectopic hh ϩ expression yields no discernible defect development. The expression of a late marker, Elav, in optic lobe organization at the late third instar stage normally in the subset of LPC progeny specified as L (K. Kaphingst and S. K., unpublished data). These are neurons, requires both Hh and at least one additional striking observations in light of the role of hh in the retinal axon-mediated cue. Third, we show that in moregulation of the TGF-␤ family member decapentaplegic saic animals, hh ϩ expressed in the eye can induce lamina (dpp; Spencer et al., 1982; Padgett et al., 1987) in other neurogenesis when autonomous hh ϩ activity is absent imaginal tissues, including the developing eye (Basler from the brain (Figure 6 ). Last, the pattern of Hh expresand Struhl, 1994; : Capdevila and sion is consistent with the notion that it is the inductive Tabata and Kornberg, 1994 ; Ma et al., cue delivered by retinal axons. Hh immunoreactivity is 1993; Heberlein et al., 1993; Zecca et al., 1995) . In the detected on photoreceptor cells in the eye disc and their brain, dpp is expressed in dorsal and ventral domains axons within the optic stalk and brain (Figure 3) . We of the visual primordia and is regulated by wingless, thus conclude that Hh is the inductive cue provided by which is expressed in immediately adjacent domains retinal axons to initiate lamina neurogenesis. A later (Kaphingst and Kunes, 1994) . We find that neither loss step, the specification of LPC progeny as L neurons, of hh function nor ectopic hh ϩ expression alters the apparently requires at least one additional retinal axonpattern of dpp expression (our unpublished data). The mediated cue.
regulatory circuits that pattern the developing visual Retinal innervation also controls the terminal differenganglia are thus clearly distinct from those observed in tiation of lamina glia (Winberg et al., 1992) . Among the other tissues. innervation-dependent markers we find to be regulated by hh ϩ activity is the transmembrane protein Patched Temporal Coordination of Neurogenesis and the (Figure 2 ; Nusslein- Volhard and Wieschaus, 1980;  Assembly of Synaptic Cartridge Units Hooper and Scott, 1989; Nakano et al., 1989) . ptc is In the eye, the temporal pattern of cell recruitment into expressed in three glia cell classes (subretinal, satellite, differentiating ommatidial units plays the central role in and medulla) which make intimate contact with retinal the assignment of cell fate (reviewed by Wolff and axons. ptc is downstream of Hh signaling in both imagiReady, 1993). Each step in the assembly of an ommatidnal and embryonic tissues in Drosophila (Hidalgo and ial unit offers an uncommitted cell a unique set of cell- Ingham, 1990; Ingham et al., 1991; Tabata and Kornberg, cell contacts from which to derive a set of intercellular 1994; Heberlein et al., 1995) . In signals that define a specific cell type (Tomlinson and vertebrates, homologs of hh (reviewed in Perrimon, Ready, 1987) . The role of the hh gene in this patterning 1995) are involved in an analogous regulatory circuit mechanism is to regulate the progression of ommatidial with homologs of ptc (Goodrich et al., 1996; Marigo et assembly along an epithelium of uncommitted cells (Ma al., 1996) . Whether the maturation of other lamina glia et al., 1993; Heberlein et al., 1993) . The absence of specell classes (see Figure 1C ; Winberg et al., 1992 ) is regucific lineage relationships among LPC progeny (Hoflated by Hh is yet to be determined. Thus, Hedgehog bauer and Campos-Ortega, 1990) suggests that cell-cell may be the sole inductive signal controlling the initial interactions may likewise play the dominant role in the events of lamina differentiation, but additional factors assignment of L-neuron identity. Hence, the temporal may be required for later events.
control of lamina neurogenesis by incoming retinal axRetinal axons may transmit Hh directly to the G 1 -phase ons may be a key feature of the mechanism that deterLPCs, with which they appear to make direct contact.
mines the cell types of a lamina cartridge unit ( Figure  Indeed , among optic lobe neuronal precursors, this cell 7). Hh would thus play the dual role of regulating events population appears uniquely programmed to respond to that specify cell identity in both the pre-and postsynapthe inductive signal. Ectopic hh ϩ cell clones that extend tic cell populations. anterior of the lamina furrow into the population of G 2 -
The lamina cartridge consists of five L neurons, L1-5, phase LPCs (Figure 6 ) do not induce their precocious glia cells, and a set of R1-R6 axons organized into a division or expression of lamina-specific markers. The LPCs appear to acquire competence for this response precise synaptic array (see Meinertzhagen and Hanson, Hanson, 1993) . The cell-cell contacts that may program . Primary antibodies were used at the following L-neuron and L-glia identity might include multiple in- is the first in a series of molecular interactions that coorwas as described by Truman and Bate (1988) . Propidium-iodide dinate the specification of pre-and postsynaptic cell staining was as described by Whitfield et al. (1990) . Specimens were viewed on a Zeiss LSM410 confocal microscope equipped with a identities (Figure 7 ).
Krypton/Argon laser.
Inductive Events and the Assembly


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