Signals transmitted along retinal axons in Drosophila

Development 125, 3753-3764 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
Signals transmitted along retinal axons in Drosophila: Hedgehog signal
reception and the cell circuitry of lamina cartridge assembly
Zhen Huang and Samuel Kunes*
Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA
*Author for correspondence (e-mail:
Accepted 19 July; published on WWW 7 September 1998
The arrival of retinal axons in the brain of Drosophila
triggers the assembly of glial and neuronal precursors into
a ‘neurocrystalline’ array of lamina synaptic ‘cartridges’.
Hedgehog, a secreted protein, is an inductive signal
delivered by retinal axons for the initial steps of lamina
differentiation. In the development of many tissues,
Hedgehog acts in a signal relay cascade via the induction
of secondary secreted factors. Here we show that lamina
neuronal precursors respond directly to Hedgehog signal
reception by entering S-phase, a step that is controlled by
the Hedgehog-dependent transcriptional regulator Cubitus
interruptus. The terminal differentiation of neuronal
precursors and the migration and differentiation of glia
appear to be controlled by other retinal axon-mediated
signals. Thus retinal axons impose a program of
developmental events on their postsynaptic field utilizing
distinct signals for different precursor populations.
‘neurocrystalline’ field of precisely repeating ‘cartridge’ units
(Braitenberg, 1967; Meinertzhagen and O’Neil, 1991;
Meinertzhagen and Hanson, 1993).
In Drosophila and other arthropods, the development of the
visual portion of the brain is tightly regulated by the ingrowth
of axons from the compound eye (Power, 1943; Macagno,
1979; Fischbach and Technau, 1984; Selleck and Steller, 1991;
reviewed by Kunes and Steller, 1993). As R-cell differentiation
and ommatidial assembly progress in a posterior-to-anterior
order across the eye disc epithelium, the arrival of retinal axons
in the brain triggers cartridge neuron precursors (LPCs) to
complete a final cell division and commence neural
differentiation (Fig. 1; Selleck and Steller, 1991). Retinal axons
also elicit the migration of glial precursors into the lamina
target field, where they mature into glia of several distinct types
(Fig. 1; Winberg et al., 1992; Perez and Steller, 1996). These
events result in the assembly of a retinal axon fascicle and a
set of neuronal and glial precursors into a stereotyped cellular
ensemble known as a ‘lamina column’ (Fig. 1C,D;
Meinertzhagen and Hanson, 1993). The choreography of axon
fascicle, target cell interactions yields a one-to-one
correspondence of ommatidial and cartridge units, and sets the
stage for the subsequent establishment of precise synaptic
One of the signals that retinal axons deliver to the lamina is
the secreted product of the hedgehog gene (hh; Huang and
Kunes, 1996; Nusslein-Volhard and Wieschaus, 1980; Lee et
al., 1992; Mohler and Vani, 1992; Tabata et al., 1992; Tashiro
et al., 1993). Hedgehog (Hh) is expressed by differentiating
photoreceptor cells immediately posterior of the
The survival and differentiation of postsynaptic cell
populations often depends on the arrival of afferent axons in
their target fields. For example, arriving retinal axons are
necessary for the establishment of the retinorecipient layers of
the tectum (Kelly and Cowan, 1972; Yamagata et al., 1995). In
Manduca, the establishment of glomeruli units of the olfactory
bulb depends on sensory innervation (Oland and Tolbert,
1996). Thus growing axons not only detect environmental cues
for guidance and target recognition, but may emit signals that
elicit the survival or maturation of cells in their prospective
target fields. The molecular identities of such signals have in
some cases been determined. Agrin (Rupp et al., 1991; Smith
et al., 1992; Tsim et al., 1992) and Aria (Falls et al., 1993), for
example, are transported to motor axon termini where they
mediate the assembly of the postsynaptic apparatus at the
neuromuscular junction.
The visual system of Drosophila offers a unique opportunity
to investigate these kinds of axon-target interactions. The
Drosophila visual system is a complex and finely ordered
structure composed of tens of thousands of neurons and glia
(reviewed by Meinertzhagen and Hanson, 1993). It consists of
the compound eyes and the optic ganglia, which are the visual
processing centers of the brain. Each of the approximately 800
ommatidial units of the compound eye contains 8
photoreceptor neurons (R-cells) that project retinotopically
into distinct ganglion layers of the brain. The R1-R6
photoreceptors send their axons to destinations in the lamina,
where they establish synaptic connections in a
Key words: Drosophila, Lamina, Photoreceptor axon, hedgehog,
Cubitus interruptus
3754 Z. Huang and S. Kunes
morphogenetic furrow in the eye imaginal disc (Lee et al.,
1992) and is a signal to more anterior cells to enter the pathway
of photoreceptor cell determination (Ma et al., 1993; Heberlein
et al., 1993, 1995; Dominguez and Hafen, 1997). We have
recently provided evidence that Hh is transported from the eye
into the brain along retinal axons and triggers the initial steps
of LPC maturation (Huang and Kunes, 1996). Hedgehog
activity in the eye is both necessary and sufficient for the early
events of lamina neurogenesis, including the terminal cell
division of LPCs and the onset of their expression of early
differentiation markers. Thus Hh acts as a dual signal that
couples neural development between the eye and brain.
In the development of many Drosophila tissues, Hh acts in
a signal relay cascade via the induction of secondary secreted
factors, such as decapentaplegic and wingless (Basler and
Struhl, 1994; Capdevila and Guerrero, 1994; Tabata and
Kornberg, 1994). In the lamina, Hh might be transmitted
directly by retinal axons to G1-phase LPCs to trigger their cell
cycle progression and subsequent differentiation. Alternatively,
LPC maturation might depend all or in part on secondary
signals delivered by an intermediate Hh target. To better
resolve these events, we have examined the Hh-dependence of
glia cell maturation and the autonomy of Hh signaling pathway
components in different lamina cell populations. Here we show
that LPCs respond directly to Hh signal reception by entering
S-phase, and that this step is controlled by the Hh-dependent
transcriptional regulator Cubitus interruptus. Glia cell
precursors, though Hh responsive, apparently depend on some
other retinal axon-mediated signal for their migration and
differentiation in the lamina target field. Thus retinal axons
communicate directly to lamina precursors by utilizing
different signals for distinct precursor populations.
Mosaic analysis
The appropriate crosses were set up as described below and, following
egg collection, larvae were heat shocked at about 6 hours posthatching for 70 minutes at 37˚C to induce hsFLP transgene
expression. The larvae were grown at 25˚C (unless otherwise noted)
before dissection at late third instar stage. Brain samples from these
larvae were then stained with the appropriate antibodies. When
mosaic analysis was done in the hhts2 background, egg collection was
carried out at 18˚C. The larvae were shifted to 28˚C after heat shock
in the first instar to block later photoreceptor cell development in the
eye. The following crosses were used for analyzing the corresponding
• smo: y, hsFLP122; P[arm-lacZ]28A, P[FRT]40A/P[y+], CyO ×
yw/Y; smo3, P[FRT]40A/P[y+], CyO
• pka-DC0h2: y, hsFLP122; P[arm-lacZ]28A, P[FRT]40A/P[y+],
CyO; hhts2/TM6B, Tb × y/Y; DC0h2, P[FRT]40A/P[y+], CyO;
hhts2/TM6B, Tb
• ptc6p: y, hsFLP122; P[FRT]43D, P[arm-lacZ]51A/P[y+], CyO;
hhts2/TM6B, Tb × y/Y; P[FRT]43D, ptc6p/P[y+], CyO; hhts2/TM6B, Tb
Ectopic gene expression
Misexpression of hh with a flp-out hh+ transgene (Basler and Struhl,
1994) was performed in animals made ‘eyeless’ by the use of the hhts2
strain background, as described above (see also Huang and Kunes,
1996). Misexpression of Ci was accomplished by using a P[UAS-Ci]
line (Alexandre et al., 1996) and a ‘flp-out’ GAL4 line,
P[Tub>CD2>GAL4] (Pignoni and Zipursky, 1997). This experiment
was also performed in the ‘eyeless’ condition achieved by the use of
the hhts2 strain background. Crosses were set up at 18˚C, and larvae
were shifted to 28˚C after hatching to prevent R-cell formation. A 30
minute heat shock at 37˚C during the early second instar stage induced
the expression of the hsFLP transgene, and activated GAL4 expression
in somatic clones. The dominant-negative truncated form of Ci (Ci76)
was misexpressed using a P[UAS-Ci76] line (Aza-Blanc et al., 1997)
and the ‘flp-out’ GAL4 driver. The corresponding crosses are listed
• hh: y, hsFLP122; hhts2/TM6B, Tb × y/Y; P[Tub>y+>hh]/+;
hhts2/TM6B, Tb
• Ci: y, hsFLP122; hhts2, P[UAS-Ci]/TM6B, Tb × yw/Y;
P[Tub>CD2>GAL4]/P[y+], CyO; hhts2/TM6B, Tb
• DN-Ci: y, hsFLP122; P[UAS-Ci76]/TM6B, Tb × yw/Y;
P[Tub>CD2>GAL4]/P[y+], CyO
The CNS was dissected from late third instar larvae, fixed and stained
as described previously (Kunes et al., 1993). The ombP1 enhancer trap
line (Sun et al., 1995) and Omb antibody (gift of G. O. Pflugfelder)
were used interchangeably, with similar results. patched gene
expression was monitored with the ptc promoter-lacZ fusion line, FE3
(Alexandre et al., 1996). Anti-Repo polyclonal antibody (Halter et al.,
1995), anti-OMB antibody (Grimm and Pflugfelder, 1996) and antiCi antibody (Motzny and Holmgren, 1995) were used at dilutions of
1:200, 1:400 and 1:20, respectively. S-phase cells were labeled by 5bromo-2′-deoxy-uridine (BrdU) incorporation (Truman and Bate,
1988) and detected with anti-BrdU antibody (1:50, BectonDickinson). Conditions for other primary and secondary antibodies
were described previously (Kaphingst and Kunes, 1994; Huang and
Kunes, 1996). Confocal microscopy was performed on a Zeiss LSM
410 microscope equipped with a Krypton/Argon laser.
Formation of precartridge ensembles in the
developing lamina field
As an ommatidial axon fascicle arrives in the lamina target
field, it assembles with neuronal and glial precursors into a
stereotyped precartridge ensemble known as a lamina column
(Fig. 1; Meinertzhagen and Hanson, 1993). The neuronal and
glial precursors have distinct origins within the optic lobe.
Most neuronal precursors (LPCs) are incorporated into the
lamina target field at the anterior margin of the lamina furrow
(Fig. 1A,C). The precursors of several glia cell types, on the
other hand, arise in anlagen adjacent to the dorsal and ventral
margins of the lamina (Fig. 1A,B) and migrate into the lamina
along an axis perpendicular to that of LPC entry (Perez and
Steller, 1996). The neuronal and glia precursor populations are
interwoven into cell-type specific layers at the anterior margin
of the lamina, so that a lamina column harbors a particular
neuronal or glial cell-type precursor at a specific mediolateral
position (Fig. 1C,D). Lamina neurons L1-L4 form a stack in a
superficial layer, while L5 neurons reside in a medial layer near
the R1-R6 axon termini. Epithelial and marginal glia are
located above and below the R1-R6 termini, respectively.
Satellite glia are interspersed among the neurons of the L1-L4
A number of markers distinguish glial and neuronal precursor
cells from the corresponding mature cell types. The expression
of optomotor-blind (omb; Heisenberg, 1972; Pflugfelder et al.,
1990) labels both glial precursors in the dorsal and ventral
Axonal cues for lamina cartridge assembly 3755
anlagen and mature glia that have migrated into the lamina
target field (Fig. 1B,D; Poeck et al., 1993). The glia cell marker
Repo (Fig. 2A,D; Xiong et al., 1994; Halter et al., 1995) and
the enhancer-trap lacZ insertion 3-109 (not shown; Winberg et
al., 1992) are expressed by glia once they have entered the
lamina target field. Cubitus interruptus (Ci), a transcriptional
mediator of Hh signaling (Eaton and Kornberg, 1990; Orenic et
al., 1990; Domiguez et al., 1996; Alexandre et al., 1996; Hepker
et al., 1997) is expressed by LPCs anterior of the lamina furrow
and by the postmitotic neuronal precursors within the lamina
(see Fig. 4D,F). The nuclear protein Dachshund (Dac; Mardon
et al., 1994) is expressed only by neuronal precursors that have
begun terminal differentiation and lie posterior of the lamina
furrow (Fig. 1B,D; Huang and Kunes, 1996). Thus, Omb and
Ci label the glial and neuronal precursors, respectively, while
the mature cells, following their interaction with retinal axons,
Fig. 1. Lamina development at the late third instar larval stage. (A,B) The lamina as viewed from the lateral perspective. Retinal axons arriving
from the eye imaginal disc trigger the assembly of neuronal and glial precursors into precartridge ensembles in the crescent-shaped lamina
target field. (A) As shown schematically, photoreceptor cells assemble into ommatidial clusters behind the anteriorly moving morphogenetic
furrow (mf). Two such clusters (green color) in the retina are shown to highlight the topographic projection of their axon fascicles into the
crescent-shaped lamina (lam; blue color). As indicated by the arrows, neuronal precursor cells of the lamina (LPCs) are incorporated into the
axon target field at its anterior margin, which is demarcated by a morphological depression known as the lamina furrow (lf in B and D). Glia
precursor cells (GPCs) are generated in two domains (red color) that lie at the dorsal and ventral margins of the prospective lamina.
(B) Confocal micrograph shows the photoreceptor cells and their axons (green color) as revealed by staining with anti-horseradish peroxidase
(anti-HRP) antibody. Postmitotic LPCs within the lamina axon target field express the nuclear protein Dac, as revealed by anti-Dac antibody
staining (blue color). Glial cells in the lamina as well as their precursors in the two posterior domains are detected by lacZ expression (red
color) from the ombP1 enhancer-trap line (Sun et al., 1995). An arrow and adjacent bar in A and a bar in B mark the dorsoventral midline. In A
and B, anterior is to the left, dorsal up. Scale bar in B, 40 µm. (C,D) Lamina cartridge assembly from the horizontal perspective. Like the eye,
lamina differentiation occurs in a temporal progression on the anterioposterior axis. (C) As shown schematically, axon fascicles from ‘new’
ommatidial R-cell clusters arrive at the anterior margin of the lamina (adjacent to the lamina furrow; lf in C and D) and associate with neuronal
(blue colors) and glia precursors (red color) in a vertical lamina column assembly (two such columns are shown in C). At the anterior of the
lamina, LPCs await a retinal axon-mediated signal in G1-phase (gray cells at the trough of the lamina furrow (lf)) and enter their terminal Sphase (black cell) at the posterior margin of the furrow. Postmitotic (Dac-positive; blue color) LPCs assemble into columns at the posterior
margin of the furrow. In older columns at the posterior of the lamina, a subset of postmitotic LPCs express definitive neuronal markers (dark
blue color) as they become specified as the lamina neurons L1-L5. These neurons arise at cell-type specific positions along the column’s
vertical axis. Lamina glial cells (red color) take up cell-type positions in the precartridge assemblies. The marginal (Ma-glia) and epithelial glia
(E-glia) layers sandwich the R1-R6 axon termini, whereas the satellite glia (S-glia) are interspersed within the neuronal precursor layer. The
medulla neuropil (medn), which serves as the target for R7/8 axons, is separated from the lamina by the medulla (Md) glia (D). The features of
lamina cartridge assembly described schematically in C are revealed in the confocal micrograph shown in D. Neuronal precursors (blue color),
glial precursors (red color), and neuronal membranes, including photoreceptor cell axons (green color; arriving via the optic stalk (os)) are
stained as described for B. In C and D, anterior is to the left, lateral is up. Scale bar in D, 15 µm.
3756 Z. Huang and S. Kunes
additionally express Repo and Dac. In the lamina target field of
‘eyeless’ mutants, such as eyes absent (eya) or sine oculis (so),
Dac expression is not detected and Repo expression is greatly
diminshed (Fig. 3E; Perez and Steller, 1996; Huang and Kunes,
expression in the dorsal and ventral glia anlagen is normal in
eya animals (Fig. 3B), consistent with previous observations
using other glia precursor markers and ‘eyeless’ mutant strains
(e.g. sine oculis1; Perez and Steller, 1996).
We determined whether glia precursor migration is Hhdependent by examining the distribution of Omb-positive cells
in hh1 animals. hh1 is a regulatory mutation that specifically
affects hh expression in the visual system. In hh1 animals,
approximately 12 columns of ommatidia initiate differentiation
in the eye imaginal disc before the anterior progression of the
morphogenetic furrow ceases (Heberlein et al., 1993). hh1
retinal axons lack Hh immunoreactivity by the time they reach
the lamina target field and thus the Hh-dependent steps of LPC
maturation fail to occur in hh1 animals (Huang and Kunes,
1996). As shown in Fig. 3C, Omb staining reveals a relatively
normal number of glia precursors in the lamina target field of
hh1 animals, despite the absence of Dac induction (see also Fig.
3F). The Omb-positive cells are distributed uniformly along the
dorsoventral axis among the retinal axon fascicles, but appear
more closely spaced than in the wild type. A likely explanation
for this spacing defect is the absence of the neuronal precursors
(Huang and Kunes, 1996) that would constitute the majority of
lamina cells at this point of development.
The migration and early differentiation of lamina glia
are independent of Hh
Enhanced transcription of the putative Hh receptor, patched (ptc;
Nusslein-Volhard and Wieschaus, 1980; Hooper and Scott, 1989;
Nakano et al., 1989; Ingham et al., 1991; Marigo et al., 1996a;
Stone et al., 1996) is a universal characteristic of Hh signal
reception (Hidalgo and Ingham, 1990; Phillips et al., 1990;
Capdevila et al., 1994; Tabata and Kornberg, 1994; Heberlein et
al., 1995; Goodrich et al., 1996; Marigo et al., 1996b; Vortkamp
et al., 1996). All classes of glia in the lamina region upregulate
ptc expression in a hh-dependent fashion (Fig. 2; Huang and
Kunes, 1996). These cells are thus Hh-responsive. As shown in
Fig. 2F, all three classes of lamina glia, as well as medulla glia,
that express a ptc-lacZ reporter construct are in close proximity
to Hh-bearing retinal axons. We have previously shown that glia
cell ptc reporter gene expression is not observed in hh− animals
(data not shown; Huang and Kunes, 1996). This raises the
question of whether Hh signal
reception is responsible for the
migration and/or subsequent
maturation of glia cells.
To determine whether the
migration of glial precursors
into the lamina target field is
Hh-dependent, we examined
the distribution of Ombpositive cells in hh− animals.
In the wild type, a ‘trail’ of
Omb-positive cells delineates
a path of glia migration from
the dorsal and vental anlagen
(arrowheads in Fig. 3A). The
clonal relationship between
the Omb-positive cells in the
lamina and in the glial anlage
was confirmed by lineage
studies (S. K., unpublished
clones were marked by a ‘flpout’ lacZ reporter construct
(Basler and Struhl, 1994).
Omb-positive cells of the
lamina, the glia anlagen and
Fig. 2. Derepression of ptc transcription in glia and neuronal precursors. Upregulation of ptc expression in
the lamina, as viewed from a lateral perspective (A-C) or a horizontal perspective (D-F). The specimens are
co-labeled with anti-Repo antibody to visualize glia (blue color; shown separately in A,D), anti-βpathway were included
galactosidase antibody to visualize ptc-lacZ (FE3) reporter expression (red color; shown separately in B,E)
within the same clones. In an
and anti-HRP to visualize neuronal membranes, including retinal axons (green color; all panels).
eya1 animal, Omb-positive
(A-C) The focal plane is through the epithelial glial layer, though some neuronal precursor cells are present.
cells are largely absent from
In C, purple cells are expressing both ptc and Repo, while the β-galactosidase-positive, Repo-negative cells
the lamina target field (Fig.
are red. (D-F) As seen from the horizontal perspective, the satellite (S-glia), epithelial (E-glia), marginal
3B), with the exception of the
(Ma-glia) and medulla glia (Md-glia) express the ptc-lacZ reporter. Subretinal glia, located on the lateral
medulla glia, which are
surface of the lamina (see D), do not express the ptc-lacZ reporter. Cells of the neuronal precursor layer
distinguished by their larger
(LPCs) are also β-galactosidase-positive. Significant levels of ptc-lacZ expression are first detectable in
size and medial location
neuronal precursors at the trough of the lamina furrow (lf; see E), where G1-phase LPCs apparently receive
adjacent to the medulla
a retinal axon-mediated signal that triggers entry into S-phase. In A-C, anterior is to the left, dorsal is up. In
D-F, anterior is to the left, lateral is up. Scale bars, 30 µm in A for A-C; 10 µm in D for D-F.
neuropil (not shown). Omb
Axonal cues for lamina cartridge assembly 3757
To determine whether the glial precursors that enter the
in Ingham, 1995; Hammerschmidt et al., 1997). Mutations at
lamina target field in hh− animals express a retinal innervationthese loci have been shown to either mimic or block Hh signal
dependent marker, we examined their expression of Repo. As
reception in a cell-autonomous fashion (see below). Examining
noted above, Repo-positive cells are largely absent in eya
the cellular requirements for these genes in mosaic animals
animals (Fig. 3E). Occasionally, a small number of Reposhould help illuminate the cellular circuitry that mediates the
positive cells are observed at ventral or dorsal lamina positions
Hh-dependent events of lamina development.
(the arrowhead in Fig. 3E). In hh1 animals, the Omb-positive
The seven-pass transmembrane protein encoded by
cells within the lamina also express Repo (Fig. 3F). Moreover,
smoothened (smo; Nusslein-Volhard et al., 1984; Alcedo et al.,
the Repo-positive cells occupy proper layers above and below
1996; van den Heuvel and Ingham, 1996) acts downstream of
the R1-R6 axon termini expected for satellite, marginal and
the Hh receptor Patched as a positive effector of Hh signal
epithelial glia, though the lack of markers specific for these
reception (Chen and Struhl, 1996). If Hh exerts its effects
three glia types precludes an unambiguous determination of
directly on LPCs, we would expect that loss of smo function
glial cell type. The presence of marginal and epithelial glia is
should block the entry of G1-phase LPCs into S-phase and/or
consistent with the observation that R1-R6 growth cones
prevent the expression of Hh-dependent markers of lamina
terminate in their proper positions between these layers in hh−
differentiation such as Dac. We used the FLP/FRT system
animals (not shown; Huang and Kunes, 1996).
(Golic and Lindquist, 1989; Xu and Rubin, 1993) to generate
The ectopic expression of Hh in the brains of ‘eyeless’
mosaic animals harboring somatic clones homozygous for the
animals is sufficient to induce
the initial steps of LPC
maturation in the absence of
retinal axons (Huang and
Kunes, 1996). For example,
photoreceptor differentiation
can be prevented by shifting a
nonpermissive condition at an
early larval timepoint. hh+
somatic clones generated by a
‘flp-out’ hh construct (Zecca et
al., 1995) in the brains of these
animals induce the LPC
terminal division and the onset
of Dac expression. To
determine whether either Hh
or the Hh-mediated events of
LPC maturation are sufficient
for glia cell migration and
maturation, we examined such
animals for the presence of
Repo-positive cells in the
vicinity of hh+ clones within
the lamina target field. Ectopic
hh+ clones in various positions
Fig. 3. A Hh-independent retinal axon-mediated signal is required for glia migration and differentiation.
throughout the lamina target
(A-C) Migration of glial precursor cells into the lamina was monitored by anti-Omb antibody staining
region, as well as other sites
(red color) in wild type (A), eya (B) and hh1 (C) animals. In the wild type (A), Omb-positive glia
within the optic lobe, failed to
precursors migrate from anlagen at the prospective dorsal and ventral margins of the lamina (see
induce Repo expression (data
schematic in Fig. 1A) along paths indicated by the arrowheads and disperse among the retinal axons
not shown). Thus neither Hh
(green color; stained by anti-HRP antibody) in the crescent-shaped lamina target field (i.e., the region
nor a secondary signal
posterior of the lamina furrow (lf). (Some cells in the lobula (lob) also express Omb). In the absence of
provided by the maturation of
retinal innervation, as in eya animals (B), Omb-positive cells seem to remain in the glial progenitor
domains. The few cells that enter the lamina field appear to stall along the migratory pathway (arrowhead
LPCs is sufficient to induce
in B). In hh1 animals (C), where retinal axon fascicles reach the brain but lack active Hh, Omb-positive
glia cell maturation.
LPCs require
smoothened for cell cycle
progression and neuronal
The activities of a number of
pathway components are now
well characterized (reviewed
glia migrate into the lamina in large numbers, despite the absence of LPC maturation (as shown in F).
(D-F) The differentiation of lamina glia was examined by staining with anti-Repo antibody in wild type
(D), eya (E) and hh1 (F) animals. In the wild type (D), Repo-positive glia (red color) are dispersed among
Dac-positive neuronal precursors (blue color) in the lamina target field. In eya animals (E), which lack
retinal axons, only a few Repo-positive cells are found in the lamina target field (as indicated by the
arrowhead) and Dac-positive LPCs are completely absent. In hh1 mutants (F), despite the complete
absence of Dac-positive LPCs, retinal axons are surrounded by a large number of Repo-positive glia (red
color). The close apposition of these Repo-positive cells is likely due to the absence of the neuronal
precursors. Arrows in C and F indicate the lamina furrow. In all panels, anterior is to the left, dorsal is up.
Scale bars, 40 µm in A for A-C; 40 µm in D for D-F.
3758 Z. Huang and S. Kunes
Fig. 4. smoothened is required cellautonomously for the Hh-dependent
steps of LPC maturation. Somatic
mosaic clones were generated using
the FLP/FRT system (as described in
Materials and Methods) and marked
by the expression of an arm-lacZ
reporter gene on the smo+
chromosome arm (blue color,
revealed by anti-β-galactosidase
antibody staining). Homozygous
mutant cells lack β-galactosidase
expression. (A-C) Dac expression
(red color) was examined in somatic
clones homozygous for the
smo3mutation. The boxed area
harboring a smo3 clone in A is shown
at higher magnification in B and C. A
smo3 clone that spans the lamina
furrow (lf) and includes the
anteroventral portion of the lamina
lacks Dac expression (red color in A
and C). The smo3 cells, visualized by
the absence of anti-β-galactosidase
staining (shown exclusively in B and
outlined in C), are Dac-negative.
smo3/smo+ cells immediately bordering the clone and within the lamina field are Dac-positive. The dashed portion of the outline at the posterior
of the clone in C indicates uncertainty at the border, which is obscured by lacZ-positive retinal axons in that area. In addition, it is evident that the
anterior progression of the lamina furrow (visualized by anti-HRP staining (green color) as a dorsoventral line running through the clone) is
retarded in the region of the clone. (D-F) Ci immunoreactivity was monitored in somatic clones homozygous for the smo3mutation. Ci
immunoreactivity (red color in D and F) was detected with an antibody against its C terminus (see text) and reflects stabilization of the protein by
Hh signal reception (Aza-Blanc et al., 1997). The boxed area harboring a smo3 clone in D is shown at higher magnification in E and F. smo3 cells
are visualized by the absence of anti-β-galactosidase staining (shown exclusively in E and outlined in F). Two smo3 clones that span the lamina
furrow (lf) and include anteroventral portions of the lamina have lower levels of Ci expression (red color in A and C) than smo3/smo+ cells
outside of the clone. The border between high-level and low-level Ci immunoreactivity precisely coincides with the clonal bordery. Thus smo
acts cell autonomously with regard to the stabilization of Ci. lob, lobula. Scale bars, 40 µm in A for A and D; 20 µm in B for B, C, E and F.
null allele smo3 (Nusslein-Volhard et al., 1984). Homozygous
smo clones were visualized by the absence of an arm-lacZ
marker residing on the smo+ chromosome of a smo3/smo+
animal (blue color in Fig. 4; Vincent et al., 1994). The majority
of smo clones recovered included the region anterior or
immediately posterior of the lamina furrow. smo clones
localized to the anterior of the lamina furrow often appeared
to slow or block its anterior progression, as indicated by
relative posterior position of the lamina furrow in the vicinity
of such clones (Fig. 4A). Clones spanning the lamina furrow
were particularly informative, as smo cells posterior of the
furrow (i.e., within the lamina) were in all cases Dac-negative
(Fig. 4A,C). In all such cases examined, smo+ cells adjacent to
these clones within the lamina were Dac-positive. Thus, with
respect to lamina differentiation, smo acts cell autonomously.
smo clones that extended to the posterior of the lamina were
rare. It is possible that LPCs that cannot respond to Hh are not
readily incorporated into the lamina and displaced by smo+
LPCs. LPCs that are unable to respond to Hh might be
eliminated by cell death, as occurs in ‘eyeless’ mutant animals
(Selleck et al., 1992).
A hallmark of Hh signal reception in many Drosophila
tissues is an increase in immunoreactivity to the C-terminal
portion of the protein Ci, a transcriptional mediator of Hh
signaling (Motzny and Holmgren, 1995; Johnson et al., 1995;
Slusarski et al., 1995). This enhanced Ci immunoreactivity is
due to inhibition of Ci proteolytic processing, a cellular
response to Hh signal reception (Aza-Blanc et al., 1997). LPCs
posterior of the lamina furrow display the enhanced Ci
immunoreactivity that would be predicted for Hh signal
reception by LPCs (see Fig. 4D,F). In animals in which hh−
retinal axons innervate the lamina target field, cells posterior
to the lamina furrow display a level of Ci immunoreactivity
equivalent to the basal level detected anterior to the furrow (not
shown), indicating that the increased Ci observed in the wild
type is Hh-dependent. In smo mosaic animals, smo cells either
anterior or posterior of the lamina furrow display a basal level
of Ci immunoreactivity, (Fig. 4F) while smo+ cells
immediately adjacent to the portion of smo clones within the
lamina display the high Hh-dependent level.
The initial response of LPCs to the arrival of Hh-bearing
retinal axons would appear to be entry into S-phase at the
lamina furrow. To determine whether cell cycle progression is
directly dependent on Hh signal reception, we examined the
incorporation of bromodeoxyuridine (BrdU) into S-phase cells
in smo mosaic animals. In the wild type, LPCs that have
entered their terminal S-phase form a discrete and continuous
band at the posterior margin of the lamina furrow (Fig. 5A). In
‘eyeless’ animals lacking photoreceptor innervation or animals
in which photoreceptor axons lacking functional Hh enter the
lamina target field, only a low background of scattered S-phase
cells are detected (see Fig. 7H; Selleck and Steller, 1991). It is
Axonal cues for lamina cartridge assembly 3759
unclear whether the products of these scattered divisions are
incorporated into the lamina (i.e., that these cells are indeed
LPCs; see Selleck et al., 1992, for further discussion of these
cells). In smo3 mosaic animals, mutant clones that included the
posterior margin of the lamina furrow lacked S-phase LPCs
(Fig. 5B). In contrast, the scattered S-phase cells anterior to the
lamina furrow, and the distribution of S-phase cells in other
proliferation centers, such as the OPC, were unaffected by the
loss of smo function. At the lamina furrow, smo+ cells
bordering smo clones were often found in S-phase. Thus, in
sum, smo+ behaved as a cell-autonomous requirement for
LPCs to initiate the Hh-dependent steps of lamina
Activation of the hh signaling pathway results in
cell-autonomous LPC maturation
The Hh receptor Ptc, a multiple-pass membrane protein, and
the cAMP-dependent protein kinase (PKA) normally maintain
the Hh signal transduction pathway in a repressed state
Fig. 5. smo is required cell-autonomously for LPCs to enter S-phase.
Cells in S-phase were identified by incubating whole-mount tissues
immediately after dissection in culture medium containing BrdU, as
described in Materials and Methods. (A) In the wild type, LPCs in
their terminal S-phase are visualized as a discrete band (red color;
anti-BrdU antibody staining) that runs along the posterior margin of
the lamina furrow (lf). Retinal axons and other neuronal membranes
are visualized by anti-HRP antibody staining (green color). S-phase
cells of the outer proliferation center (OPC) and of the inner
proliferation center (adjacent to the lobula, lob) are also visible to the
anterior and posterior, respectively, of the lamina. (B-B″) In a smo3
mosaic specimen, homozygous smo3 cells are marked by the absence
of arm-lacZ reporter gene expression (blue color in B′ and B″; see
the legend to Fig. 4 for details). In smo3 clones that span the lamina
furrow, LPCs are not detected in S-phase at the posterior margin of
the lamina furrow (red color in B and B″), in contrast to smo3/smo+
cells immediately bordering the mutant clones. Other cell
proliferation centers, such as the OPC, are not affected by the loss of
smo function. In all panels, anterior is to the left, dorsal is up. Scale
bar, 20 µm in A for all panels.
(reviewed in Ingham, 1995; Hammerschmidt et al., 1997).
Loss-of-function mutations in either of these genes mimic Hh
signal reception and result in the cell autonomous activation of
Hh target genes in many tissues (Ingham et al., 1991; Jiang and
Struhl, 1995; Lepage et al., 1995; Li et al., 1995; Pan and
Rubin, 1995; Strutt et al., 1995). If LPC differentiation is
indeed induced directly by Hh, LPCs harboring mutations for
either pka or ptc should undergo differentiation cellautonomously and independently of retinal innervation. To test
this prediction, we generated somatic clones for pka or ptc in
a hhts2 mutant background, where an early shift to the
nonpermissive temperature was used to block photoreceptor
cell differentiation. Clones homozygous for the pka allele
DC0h2 or the ptc allele, ptc6p, behaved similarly. In both cases,
cells within homozygous clones in the lamina region expressed
lamina differentiation markers despite the absence of retinal
axons, while neighboring pka+ or ptc+ cells did not (Fig. 6AC and 6D-F, respectively). The marked clones often spanned a
region including the OPC and the LPCs, containing
populations both anterior and posterior of the lamina furrow.
In these clones, cell-autonomous induction of the lamina
differentiation marker Dac was observed exclusively in mutant
cells posterior of the lamina furrow. Mutant cells anterior of
the furrow did not differentiate precociously. This observation
is consistent with the consequences of ectopic Hh expression
in an ‘eyeless’ mutant background (Huang and Kunes, 1996).
Hh expression in regions anterior of the lamina furrow did not
induce precocious lamina differentiation, as though
‘competence’ to respond to Hh is acquired by G1-phase LPCs
at the anterior margin of the lamina furrow. Within the lamina
target field, wild-type cells neighboring the DC0h2 or ptc6p
mutant cells were never observed to express Dac. Thus
activation of the Hh pathway by loss-of-function in either gene
results in a strictly autonomous induction of LPC maturation.
These results permit the conclusion that the terminal cell
division and differentiation of LPCs both require the direct
reception of the Hh signal.
LPC cell cycle progression and cell fate
determination are jointly controlled by the
transcriptional regulator, Cubitus interruptus
In a number of instances, pattern formation mediated by Hh is
accompanied by cell division. The well-defined pattern of Hhinduced cell division in the lamina provides an opportunity to
determine the point at which the Hh signal reception engages
the cell cycle machinery. Biochemical and epistasis
experiments have placed the zinc finger molecule Ci
downstream of all other hh signaling pathway components
(Motzny and Holmgren, 1995; Sanchez-Herrero et al., 1996;
Robbins et al., 1997; Sisson et al., 1997; Chen et al., 1998). Ci
has been shown to bind directly to the regulatory sequences of
Hh-responsive genes (Alexandre et al., 1996; Von Ohlen et al.,
1997; Von Ohlen and Hooper, 1997; Aza-Blanc et al., 1997).
Should all Hh-mediated events of LPC maturation be found to
depend on Ci function, we could conclude that, at least with
regard to cell proliferation and the expression of differentiation
markers, there is no ‘branchpoint’ within the signaling
To examine the requirement for Ci, we utilized two
recombinant constructs that result in either dominant Ci gainof-function or loss-of-function phenotypes. Overexpression of
3760 Z. Huang and S. Kunes
the wild-type Ci gene results in a gain-of-function phenotype
photoreceptor cell axons (reviewed in Meinertzhagen and
that mimics activation of the Hh signaling pathway (Alexandre
Hanson, 1993), can be viewed as occurring in two distinct
et al., 1996; Domiguez et al., 1996; Hepker et al., 1997).
stages. Initially, an arriving ommatidial axon fascicle elicits the
Expression of an amino terminal fragment of Ci (hereafter
formation of a precartridge cellular ensemble (a lamina
referred to as DN-Ci) results in a dominant loss-of-function
column; Fig. 1C) by triggering the terminal differentiation of
phenotype, as the normal in vivo function of this portion of the
both glial and neuronal precursors. The activity of each
molecule appears to be transcriptional repression of Hh target
ommatidial fascicle in establishing a cartridge ensemble results
genes (Aza-Blanc et al., 1997; Hepker et al., 1997). Both of
in a one-to-one correspondence of ommatidial and cartridge
these constructs employ the yeast UAS promoter, so that they
units. The second stage of cartridge formation occurs later
can be expressed in somatic clones of GAL4-expressing cells
when the six R1-R6 axons of an ommatidial fascicle separate,
(Brand and Perrimon, 1993) generated by a ‘flp-out’ GAL4
migrate laterally and form their synaptic connections with the
construct (Pignoni and Zipursky, 1997).
neurons of six neighboring cartridges (Trujillo-Cenoz and
With either construct, ectopic expression in the lamina
Melamed, 1973). In the adult lamina a cartridge thus receives
region resulted in the expected phenotype with respect to the
its complement of R1-R6 axons from six different ommatidial
lamina differentiation marker Dac. Dac expression in cells
units, none of which were members of the fascicle that
posterior of the lamina furrow was strongly reduced or
originally established the cartridge cell ensemble. The
undetectable in cells expressing DN-Ci (Fig. 7B). Conversely,
mechanism by which this remarkable feat of ‘axon-shuffling’
the ectopic expression of wild-type Ci resulted in the induction
occurs is unknown.
of Dac-positive cells in the lamina target field of ‘eyeless’
Here we have focused on the signaling events by which an
animals (Fig. 7I). The effects observed with either construct
ommatidial axon fascicle elicits the assembly of a cartridge
were strictly cell autonomous (Fig. 7B,C; data not shown).
ensemble. It is clear that this process involves specific
Thus our results with ectopic Ci and DN-Ci expression are
communication events between the axons and neuronal and
consistent with the expectation that Ci modulates Hh signaling
glial precursors. Retinal axon-mediated signals trigger LPC
activity directly in LPCs.
maturation (Selleck and Steller, 1991; Huang and Kunes,
To determine whether Hh signaling acts via Ci to regulate
1996), the specification of L1-L5 neurons (Huang and Kunes,
the G1- to S-phase transition of LPCs at the lamina furrow, we
1996), and the migration and subsequent maturation of glia in
examined the incorporation of BrdU into S-phase cells in
the lamina target field (Winberg et al., 1992; Perez and Steller,
animals harboring clones expressing either of the
two constructs described above. As shown in Fig.
7E, cells expressing DN-Ci at the posterior margin
of the lamina furrow failed to enter S-phase.
Where clones of DN-Ci-expressing cells traversed
the lamina furrow, S-phase LPCs are absent (Fig.
7E), while S-phase LPCs are observed
immediately outside of the clone (arrowhead in
Fig. 7E). Moreover, the effect on cell division was
limited to the LPCs at the lamina furrow. No
defects were observed in other proliferation zones
such as the OPC or IPC, the other major
proliferation centers of the optic lobe when they
contained DN-Ci-expressing cells. Conversely, the
induction of lamina differentiation by ectopic Ci
expression in a hhts2 (‘eyeless’; as described in
Materials and Methods) background was
accompanied by the entry of LPCs into S-phase at
the lamina furrow (Fig. 7I). As when lamina
differentiation was induced in the absence of
retinal axons by ectopic Hh expression (Huang and
Fig. 6. Activation of the Hh signaling pathway triggers cell-autonomous LPC
Kunes, 1996), ectopic Ci expression triggers a
maturation. Dac expression (red color) was examined in somatic clones
posterior-to-anterior pattern of differentiation such
homozygous for pka-DC0h2 (A-C) or ptc6p (D-F). Mosaicism was induced by the
FLP/FRT system in a homozygous hhts2 background to block R-cell formation (as
that S-phase LPCs are found at the anterior margin
in Materials and Methods). These animals thus lack retinal axons (as
(arrow in Fig. 7I). In sum, these observations
the absence of anti-HRP-positive photoreceptor axons (green color) in
indicate that the induction of cell division by Hh
A, B, D, and E). Mutant somatic clones, marked by the absence of arm-lacZ
occurs via the transcriptional regulation of Hh
reporter expression (blue color) extend into the lamina field and are outlined in B
target genes.
The assembly of a lamina cartridge, an intricate
structure composed of five lamina neurons and six
and E. For animals harboring either pka-DC0 (A-C) or ptc (D-F) clones,
homozygous mutant cells within the lamina target field express Dac (red color;
anti-Dac antibody staining). Homozygous mutant cells outside of the lamina target
are Dac-negative, as explained in the text. The effects of the mutations are strictly
cell-autonomous, as wild-type cells bordering the clones, but within the lamina
target field, do not express Dac. lob, lobula. In all panels, anterior is to the left,
dorsal is up. Scale bar, 20 µm in A for all panels.
Axonal cues for lamina cartridge assembly 3761
1996). Here we have addressed the question of whether these
with the location of G1-phase LPCs (Figs 4, 5). Wild-type
events involve the same or different retinal-axon mediated
LPCs immediately adjacent to those smo cells commenced
signals, and whether these signals act
directly or via the induction of
secondary signals from resident cell
Hedgehog is one of the signals that
retinal axons deliver to the lamina
(Huang and Kunes, 1996). Hh
immunoreactivity is found on retinal
axons at the time of their ingrowth
into the lamina. Hh is required for
lamina differentiation (see Fig. 3, for
example) and is sufficient for the
onset of LPC maturation when
expressed ectopically in the brain.
Nonetheless, in many tissues Hh
exerts at least some of its effects
through the induction or maintenance
of secondary signals. In the embryo,
for example, Hh maintains wingless
expression, thus exerting an indirect
effect on anterior compartment
pattern (Hidalgo and Ingham, 1990;
Ingham and Hidalgo, 1993). In the
wing imaginal disc, Hh-mediated
activation of dpp expression along the
AP compartment border yields a
morphogen gradient with activity in
both compartments (Lecuit et al.,
1996; Nellen et al., 1996; Singer et al.,
1997). Finally, in the eye, ommatidial
development can take place in cells
suggesting that the involvment of Hh
is indirect (Strutt and Mlodzik, 1997).
Fig. 7. Cubitus interruptus (Ci) regulates both LPC cell division and differentiation. (A-C) Dac
This raises the question of whether Hh
expression (red color) in the lamina was examined in the wild type (A) and specimens harboring
is a direct signal for cartridge neuron
somatic clones expressing a dominant-negative Ci (DN-Ci) transgene, UAS-Ci76 (B,C; Azadifferentiation or acts via the
Blanc et al., 1997). (A) In the wild type, Dac expression (red color) is detected in LPCs
induction of a secondary signal,
immediately posterior of the lamina furrow (lf). (B,C) In this specimen, clones expressing DNperhaps in some other cell population.
Ci are marked by the absence of CD2 immunoreactivity (blue color shown in C; region left of
the line drawn in B). The LPCs expressing DN-Ci adjacent to the lamina furrow (arrow) fail to
Our data indicate that neuronal
express Dac, while LPCs that are not included in the DN-Ci-expressing clones are Dac-positive.
precursor (LPC) differentiation in the
DN-Ci thus acts cell autonomously. (D-F) The effect of DN-Ci expression on LPC cell cycle
lamina is due to direct Hh signal
progression was monitored by analyzing the incorporation of BrdU into whole-mount tissues in
reception. A mosaic analysis with four
culture medium. (D) In the wild type, S-phase LPCs form a discrete band on the posterior
Hh signaling pathway components
margin of the lamina furrow (lf), just anterior to the crescent-shaped array of retinal axons
(green color; visualized by anti-HRP staining). (E,F) In somatic clones expressing DN-Ci
Kinase-A and cubitus interruptus)
(clones marked by the lack of CD2 immunoreactivity – blue color, shown in F, to the left of the
revealed in all cases a cellline drawn in E), S-phase LPCs are entirely absent, while cells that do not express the transgene
autonomous requirement for Hh
(arrowhead in E) are BrdU-positive. (G-I) Ectopic expression of a wild-type Ci transgene
signal reception in LPCs. Since
induces LPC maturation in an ‘eyeless’ animal. Dac expression (blue color) and BrdU
incorporation into S-phase cells (red color) were monitored in the wild type (G), an eya animal
lamina neurons and glia derive from
lacking retinal axons (H), and an animal expressing a wild-type Ci transgene (I). In the wild
distinct lineages (Winberg et al. 1993;
type, LPCs that have entered S-phase at the posterior margin of the lamina furrow (lf) are purple
Perez and Steller, 1996), these
(BrdU-positive, red color; Dac-positive, blue color). Postmitotic LPCs within the field of retinal
observations rule out a requirement
axons (green color, anti-HRP antibody staining) are Dac-positive. In an eyeless animal (H), Sfor Hh signal reception in glia for LPC
phase LPCs and Dac expression are completely absent. In an ‘eyeless’ hhts2 animal expressing a
maturation. The requirement for
UAS-Ci transgene (I), Dac expression in the lamina field occurs in the absence of retinal axons.
smoothened for LPCs to enter S-phase
At the anterior margin of the lamina (indicated by an arrow), LPCs are observed in S-phase
and express lamina differentiation
(purple colored cells). Ectopic Ci expression also results in abnormalities in the organization of
markers was observed in relatively
the OPC, as indicated by the unusual pattern of S-phase cells in that region. lob, lobula. In all
small somatic clones that coincided
panels, anterior is to the left, dorsal is up. Scale bar, in A, 40 µm for all panels.
3762 Z. Huang and S. Kunes
lamina differentiation normally. Conversely, in the absence of
retinal axons, activation of the Hh signaling pathway via loss
of function in patched or Protein Kinase-A triggered LPC
maturation in a cell-autonomous fashion (Fig. 6). These
somatic clones did not induce differentiative events in adjacent
wild-type LPCs, which failed to undergo differentiation due to
the absence of retinal axons. This observations argue strongly
against the notion that Hh signal reception triggers the
expression of a secondary diffusible signal that is sufficient for
LPC maturation.
Hh signal reception is in a number of instances accompanied
by cell proliferation. In the chick, Sonic Hedgehog triggers cell
proliferation in conjunction with its patterning activity in the
somitic mesoderm (Fan et al., 1995). In the mouse, loss of
patched function is associated with enhanced cell proliferation
in the neural tube and a higher incidence of a proliferative
disease, medulloblastoma (Goodrich et al., 1997). In
Drosophila, morphogenesis induced by Hh in the developing
eye includes two waves of cell division, one anterior and one
posterior of the morphogenetic furrow (Ready et al., 1976;
Heberlein et al., 1995). Our data indicate that, at least in the
case of the lamina, Hh acts via the transcription factor Ci in
regulating cell cycle progression (Fig. 7). Both genetic
epistasis and biochemical experiments place Ci at a terminal
step in Hh signal transduction, as a nuclear effector of the
upstream components Ptc, Smo, PKA, and Fused (Motzny and
Holmgren, 1995; Sanchez-Herrero et al., 1996; Robbins et al.,
1997; Sisson et al., 1997; Chen et al., 1998). Thus we conclude
that, at least with regard to cell proliferation and the expression
of differentiation markers, there is no ‘branchpoint’ within the
signaling pathway. Rather, cell proliferation would appear to
be controlled by a transcriptional target of Hh. This might be
a component of the cell cycle regulatory machinery, but less
direct avenues are also possible.
There is evidence for the activity of at least two additional
retinal axon-mediated signals in lamina cartridge assembly.
First, retinal axons appear to provide at least one signal
downstream of Hh that mediates the final differentiation of
cartridge neurons (Huang and Kunes, 1996). Only a subset of
the postmitotic LPCs generated by Hh-dependent events
become cartridge neurons. Five postmitotic LPCs associated
with each retinal axon fascicle begin to express neuronal
markers (Fig. 1C); the remaining LPCs are eliminated by
apoptosis. Hh is not sufficient for the terminal differentiation
of cartridge neurons. Our recent observations (Z. H.,
unpublished data) indicate that a second retinal axon-mediated
signal is required and that it acts via the Drosophila EGF
receptor. A third retinal axon-mediated signal is apparently
required for lamina glial differentiation. Though responsive to
retinal axon-borne Hh (Fig. 2), the satellite, marginal and
epithelial glia depend on retinal axons, but not Hh, in order to
migrate into the lamina target field and undergo subsequent
differentiation (Fig. 3). Because the hh− retinal axons that can
promote these events cannot trigger LPC maturation, we
suppose that this third signal is independent of the Hh-mediated
events of LPC maturation (Fig. 3). These events are also
independent of EGF receptor activity (our unpublished
observations). Thus we suppose that at least three retinal axonmediated signals orchestrate the assembly of a lamina cartridge.
The transmission of signals from the eye to the brain is a
mechanism by which the precision of ommatidial assembly in
the eye is imposed on the developing postsynaptic field. As in
the case of the eye (Ready et al., 1976), the assembly of a
cartridge cellular ensemble proceeds according a stereotyped
choreography of cellular events (Meinertzhagen and Hanson,
1993). At the anterior of the lamina, a retinal axon fascicle is
joined first by a cell destined to become the L1 neuron. Lneurons appear to be added to the fascicle-associated ensemble
in a cell-type-dependent sequence (Meinertzhagen and
Hanson, 1993; S. Kunes, unpublished observations). This
process yields a precise geometric array of retinal axon, target
cell associations that set the stage for the remarkable events of
synaptic cartridge formation that follow. One might suppose
then that to some extent the presynaptic axons serve as a
‘template’ for the establishment of order in the lamina.
Conversely, it is clear that target field cells play some role in
the establishment of precise connectivity, for example in the
initial guidance of photoreceptor axons (see, for example,
Martin et al., 1995) and the maintenance of growth cone, target
cell associations via the synthesis of nitric oxide (Gibbs and
Truman, 1998). Thus, in the Drosophila visual system, there is
a complex interplay between pre- and postsynaptic cell
populations, many aspects of which are as yet unresolved.
We are indebted to M. Mlodzik, Y. H. Sun, R. Holmgren, D.
Kalderon T. Kornberg, G. Pflugfelder, G. Technau, P. Ingham, G.
Mardon, G. Rubin, L. Zipursky, K. Basler, G. Struhl and K. Mathews
(Drosophila Stock Center, Bloomington) for strains and antibody
reagents. We thank members of the Kunes laboratory for critical
reading of the manuscript. This work was supported by a Pew
Scholars Award and NIH-NEI grant EY10112 to S. K.; Z. H. was
supported by a Corning Costar Fellowship Fund award during the
course of this work. We are especially indebted to Mr Jeff Tarr and
the Markey Foundation for their support of the MCB Imaging Center.
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