RESEARCH ARTICLE

983

Development 138, 983-993 (2011) doi:10.1242/dev.058370© 2011. Published by The Company of Biologists Ltd

Concentric zones, cell migration and neuronal circuits in the
Drosophila visual center
Eri Hasegawa1,*, Yusuke Kitada1,2,3,*, Masako Kaido1, Rie Takayama1, Takeshi Awasaki4, Tetsuya Tabata3 andMakoto Sato1,5,†
SUMMARY
The Drosophila optic lobe comprises a wide variety of neurons, which form laminar neuropiles with columnar units andtopographic projections from the retina. The Drosophila optic lobe shares many structural characteristics with mammalian visualsystems. However, little is known about the developmental mechanisms that produce neuronal diversity and organize the circuitsin the primary region of the optic lobe, the medulla. Here, we describe the key features of the developing medulla and reportnovel phenomena that could accelerate our understanding of the Drosophila visual system. The identities of medulla neurons are
pre-determined in the larval medulla primordium, which is subdivided into concentric zones characterized by the expression offour transcription factors: Drifter, Runt, Homothorax and Brain-specific homeobox (Bsh). The expression pattern of these factors
correlates with the order of neuron production. Once the concentric zones are specified, the distribution of medulla neurons
changes rapidly. Each type of medulla neuron exhibits an extensive but defined pattern of migration during pupal development.
The results of clonal analysis suggest homothorax is required to specify the neuronal type by regulating various targets including
Bsh and cell-adhesion molecules such as N-cadherin, while drifter regulates a subset of morphological features of Drifter-positive
neurons. Thus, genes that show the concentric zones may form a genetic hierarchy to establish neuronal circuits in the medulla.

INTRODUCTION
In many animals, visual information is processed by visual centersin the brain that are composed of laminar structures and columnar
units with topographic input from the retina. The neuronal circuitsin the visual centers process various types of visual informationsuch as motion, color and shape. As noted by Ramón y Cajal, thefly optic lobe shares structural characteristics with mammalianvisual systems (Sanes and Zipursky, 2010), and Drosophila
genetics allow high resolution genetic manipulations (Gao et al.,2008; Rister et al., 2007). The Drosophila visual system may
provide a powerful model to investigate the mechanisms
underlying the formation of visual processing circuits. However,
little is known regarding the developmental processes that create
neuronal diversity and organize these circuits in the primary regionof the optic lobe, the medulla.
The Drosophila retina is composed of ~800 ommatidial units,each containing eight types of photoreceptor neurons (R1-8). R16 detect moving stimuli (Katsov and Clandinin, 2008; Rister et al.,1

Frontier Science Organization, Kanazawa University, 13-1 Takaramachi Kanazawashi, Ishikawa 920-8641, Japan. 2Graduate Program in Biophysics and Biochemistry,
Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo
113-0033, Japan. 3Institute of Molecular and Cellular Biosciences, University ofTokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. 4University of
Massachusetts Medical School, Worcester, MA 01605, USA. 5PRESTO, JST, 4-1-8
Honcho Kawaguchi, Saitama 332-0012, Japan.
*These authors contributed equally to this work
Author for correspondence (makotos@staff.kanazawa-u.ac.jp)†This is an Open Access article distributed under the terms of the Creative Commons Attribution
Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), which
permits unrestricted non-commercial use, distribution and reproduction in any medium provided
that the original work is properly cited and all further distributions of the work or adaptation are
subject to the same Creative Commons License terms.

Accepted 1 December 2010

2007; Zhu et al., 2009), while R7-8 are involved in color vision(Gao et al., 2008; Morante and Desplan, 2008). Visual information
received in the retina is transmitted to the optic lobe composed oflamina, medulla, lobula and lobula plate. The medulla, lobula andlobula plate are subdivided into ten (M1-10), six (Lo1-6) and four
(Lop1-4) strata, respectively.
The medulla is the largest component of the Drosophila optic
lobe, containing ~40,000 neurons forming 10 layers. In Golgi
studies, 60 types of medulla neurons with different morphologicalcharacteristics have been identified (Fischbach and Dittrich, 1989;
Hofbauer and Campos-Ortega, 1990). Because R7-8 axons
terminate in the medulla and the visual information carried by R16 axons is sent to the medulla through a relay by lamina neurons,
the medulla might process all of the visual information received in
the retina (Takemura et al., 2008). In spite of the importance of the
medulla, the developmental mechanisms that produce neuronal
diversity and organize medulla circuits remain largely unknown. In
the larval brain, the primary source of medulla neurons is thought
to be neuroblasts (NBs) situated in the outer proliferation center(OPC) (Hofbauer and Campos-Ortega, 1990; Yasugi et al., 2008).
The differentiation of NBs from neuroepithelial cells (NEs)progresses from the medial to the lateral region of the OPC, andeach NB produces ganglion mother cells (GMCs) that subsequently
divide to become neurons (Egger et al., 2007; Toriya et al., 2006).
A key goal in developmental neurobiology is understanding themolecular mechanisms by which each of 40,000 neurons in the
medulla establishes an appropriate identity among 60 different
possibilities and connects to appropriate targets to form neuronal
circuits.
Here, we describe the key features of the developing medulla andreport novel observations that could accelerate our understanding of
the Drosophila visual system. During larval development, themedulla is subdivided into concentric zones that are characterized by

DEVELOPMENT

KEY WORDS: Drosophila, Concentric zone, Medulla, Neuronal circuit, Neuronal migration, Optic lobe

RESEARCH ARTICLE

the expression of four conserved transcription factors: Drifter (Drf),
Runt (Run), Homothorax (Hth) and Brain-specific homeobox (Bsh)(Anderson et al., 1995; Gergen and Butler, 1988; Jones andMcGinnis, 1993; Kurant et al., 1998; Pai et al., 1998; Rieckhof et al.,1997). The expression patterns of these factors correlate with theorder of neuron production. Once the concentric zones are specified,the distribution of medulla neurons changes rapidly, and theconcentric zones are disrupted early in pupal development owing to
the extensive migration of cell bodies. The projection patterns of
medulla neurons are established through a series of steps – axonal
projection, cell body migration and dendritic arborization – that are
stereotypically defined according to the neuronal type most likely
governed by genes that show the concentric zones.
MATERIALS AND METHODS
Generation of Gal4 strains

Following DNA fragments were PCR amplified using primers listed inTable S1 in the supplementary material.
drf-Gal4

The drfL, Gal4 and drfR fragments were inserted to pW25 to have
drfGal/W25, which was used for homologous recombination (Gong andGolic, 2003).
Although drfR was precisely inserted into the drf locus, as revealed by
PCR, drfL was not. As Drf expression was detected in drf-Gal4
homozygous clones, it does not eliminate the drf ORF (not shown).
hth-Gal4

The hthL, Gal4 and hthR fragments were inserted to pW25 to have
hthGal/W25, which was introduced to the hth locus by converting to thehth-lacZ insertion using 2-3 (Sepp and Auld, 1999). hth-Gal4 wasinserted between exons 9 and 10. Hth expression was significantly reduced
in hth-Gal4 homozygous clones (not shown).
bsh-Gal4

The bshLz, Gal4, Kmr and bshRz fragments were inserted to pW25 to havebshLRzGal4Kmr, which was inserted to a BAC containing the bsh locus(Kimura et al., 2006; Venken et al., 2006). The bshLB and bshRB
fragments at the boundary of the bsh locus were inserted to attB-PacmanApR. The bsh locus was retrieved from the BAC to obtainbshGalBAC/Pacman, which was introduced to the fly genome by Pelement mediated transformation (third chromosome).
Fly strains

Fly strains used were: repo-Gal4, AyGal4, Actin>stop>lacZ,
UAS>CD2>CD8GFP, UAS-flp, hs-flp, elav-Gal4, UAS-CD8GFP, tubGal80, FRT40A, FRT2A, FRT82B, M(3R)w, UAS-GFP, UAS-nlsGFP, UASSytHA, UAS-tau-myc, drfE82, drfH76, UAS-drf, hthP2, UAS-hth, hth-lacZ,
NCadM19, NCadomb405, NP2646 (CG11873) and NP6013 (amnesiac). dpnGal4 was provided by Ethan Bier (UC San Diego, La Jolla, CA, USA).
Clonal analysis

Following genetic crosses and heat shock conditions were used: Fig. 2, hsflp; UAS-CD8GFP was crossed to AyGal4 (32°C, 15 minutes at early 3rd
instar, dissected 36 hours after heat shock); Fig. 3, elav-Gal4 UAS-CD8GFP
hs-flp; tub-Gal80 FRT2A was crossed to FRT2A (37°C, 60 minutes at 2nd
instar); Fig. 4, hs-flp; UAS>CD2>CD8GFP was crossed to hth-Gal4,
NP6013 and bsh-Gal4 flies. hs-flp; FRT82B tub-Gal80 was crossed to UASSytHA; FRT82B bsh-Gal4 UAS-CD8GFP (32-33°C, 60 minutes at 3rd
instar); Fig. 5, y+ FRT40A; hth-Gal4 UAS-CD8GFP was crossed to hs-flp;
tub-Gal80 FRT40A; UAS-CD8GFP. hs-flp; FRT82B tub-Gal80 was crossed
to FRT82B hth-Gal4 UAS-CD8GFP. hs-flp; UAS-hth; FRT82B tub-Gal80
was crossed to FRT82B hth-Gal4 UAS-CD8GFP (32-33°C, 60 minutes at
3rd instar). hs-flp; FRT82B tub-Gal80 was crossed to FRT82B bsh-Gal4
UAS-CD8GFP or FRT82B bsh-Gal4 UAS-CD8GFP hthP2.
hs-flp; UAS-hth; FRT82B tub-Gal80 was crossed to FRT82B bsh-Gal4
UAS-CD8GFP hthP2 (32-35°C, 60 minutes at 3rd instar). hs-flp; tub-Gal80
FRT40A; UAS-CD8GFP was crossed to NP6013; NCadM19 or NCadomb405
FRT40A; UAS-CD8GFP or NP6013; y+ FRT40A; UAS-CD8GFP (32°C,

Development 138 (5)60 minutes at 2nd to early 3rd instar). hs-flp; tub-Gal80 FRT40A; UASCD8GFP was crossed to NCadM19 or NCadomb405 FRT40A; bsh-Gal4 UASCD8GFP or y+ FRT40A; bsh-Gal4 UAS-CD8GFP (32-33°C, 60 minutes
at 3rd instar). hs-flp; AyGal4 UAS-GFP was crossed to UAS-hth (34°C, 3060 minutes at early 3rd instar). hs-flp; FRT82B hthP2 was crossed to
FRT82B ubi-GFP M(3R)w flies (37°C, 60 minutes at 1st instar).
In Fig. 6, hs-flp; tub-Gal80 FRT40A; drf-Gal4 UAS-CD8GFP wascrossed to UAS-SytHA; FRT40A flies (31°C, 30 minutes at 3rd instar); in
Fig. 7, UAS-SytHA; tub-Gal80 FRT2A was crossed to hs-flp; FRT2A UASCD8GFP NP2646 or hs-flp; drfE82 or drfH76 FRT2A UAS-CD8GFPNP2646. hs-flp; UAS-drf; tub-Gal80 FRT2A was crossed to hs-flp; drfE82
FRT2A UAS-CD8GFP NP2646 (33°C, 20 minutes at 3rd instar).
Histochemistry

Rabbit anti-Hth [Adi Salzberg (Israel Institute of Technology, Haifa,
Israel); 1:1000], guinea pig anti-Bsh (Chi-Hon Lee, NIH Bethesda, MD,
USA; 1:500), guinea pig anti-Run (Asian Distribution Center, 1:500), rat
anti-Drf [Sarah Certel (University of Montana, Missoula, MT, USA);
1:3000], guinea pig anti-Dpn [James Skeath (Washington University, St
Louis, MO, USA); 1:1000), mouse anti-HA (Covance, 1:1000), mouseanti-lacZ (Promega, 1:250), rat anti-CD8 Alexa488-conjugated (Caltag,
1:100) and rabbit anti-GFP Alexa488-conjugated (Invitrogen, 1:2000) wereused. Mouse anti-Chaoptin (1:10), mouse anti-ChAT (1:300), mouse antiCut (1:20), mouse anti-Dac (1:20), rat anti-Elav (1:100), mouse anti-Myc(1:20), rat anti-NCad (1:20), mouse anti-Pros (1:10) and mouse anti-Repo(1:10) were from DSHB. Custom-made antibodies (rabbit anti-Hth, guinea
pig and rat anti-Bsh, Run, Drf antibodies) were raised as described
previously (Jones and McGinnis, 1993; Kosman et al., 1998; Kurant et al.,
1998; Llimargas and Casanova, 1997). Details are available upon request.
In situ hybridization was performed as described previously (Sato et al.,2006). Images were processed using Zeiss LSM image browser, Adobe
Photoshop and Bitplane Imaris.

RESULTSThe larval medulla is subdivided into concentriczones expressing conserved transcription factors
To identify genetic subdivisions in the medulla primordium of thelarval brain, the expression of conserved transcription factors was
examined. We identified concentric zones characterized by theexpression of four conserved genes that encode transcription factors:
Drifter (Drf) from the Brn family, Runt (Run) from the Runx family,
Homothorax (Hth) from the Meis family and Brain-specifichomeobox (Bsh) from the Bsx family (Fig. 1D,E) (Anderson et al.,
1995; Gergen and Butler, 1988; Jones and McGinnis, 1993; Rieckhof
et al., 1997). All of these genes, which we collectively call‘concentric genes’, were expressed in differentiated neurons (Fig.
1F); however, weak Hth signals were also found in NBs and NEs(Fig. 1D!,E!). The concentric zones expressing Drf, Run and Hthwere adjacent but did not overlap (Fig. 1D). Bsh expression was
found in the outer subdomain of the Hth domain (Fig. 1E). Medulla
neurons projected axon-like structures toward the center of the brain
hemisphere to form concentric neuropiles (Fig. 1A,B,F).
Accumulations of tau-Myc suggest that the axon-like structures areindeed axons (Fig. 1J) (Thor et al., 1999). Large glial cells were
found on the surface of the medulla, while small glial cells were in
the innermost area of the medulla cortex adjacent to the Hth domain(Fig. 1G), which most likely become the medulla neuropile glia
(MNG; see Fig. 3B) (Tix et al., 1997). The processes of glial cells
were found in the larval medulla cortex (Fig. 1G).
The birth order of medulla neurons correlateswith concentric gene expression
The NBs are formed from NEs in the OPC during early third larval
instar. NB produces GMCs that subsequently divide to produce
medulla neurons (Colonques et al., 2007; Egger et al., 2007; Nassif

DEVELOPMENT

984

The Drosophila visual center development

RESEARCH ARTICLE

985

et al., 2003; Toriya et al., 2006; Yasugi et al., 2008). Many neurons
are produced from a single NB with a linear and radial orientation
towards the center of the developing medulla (Fig. 1A,B). A weak
expression of concentric genes appeared around mid third instar(not shown).
Each clone of neurons produced from a single NB formed a linethat intersected the concentric zones (Fig. 1A,B,H,I); thus, theexpression of concentric genes may not be lineage dependent. Each
NB may produce several types of medulla neurons that are
distinguished by the expression of concentric genes. To address thispossibility, a small group of cells were labeled with constitutive
expression of UAS-CD8GFP (Fig. 2A,B) (Ito et al., 1997). Mild
induction of FLPase triggered GFP expression in a single NB, as
identified by Deadpan (Dpn) expression, and in daughter cells of
the NB (Fig. 2B) (Bier et al., 1992). The GFP-positive neurons arethus thought to derive from a single NB. Our observations suggestthat various combinations of neurons can be produced from asingle NB (Drf/Bsh, Drf/Run, Bsh/Hth, Run/Hth; Fig. 2B and notshown). Because we could not restrict the timing of clone induction

in newborn NBs, a subset of daughter cells produced from a NB
could be labeled with GFP. However, a single NB clone tended tocontain two or three Drf-positive, one Run-positive and one Bshpositive neurons (Fig. 2C), suggesting that many of the NBs haveessentially the same potential to produce a series of medulla
neurons (Fig. 2D).
Because NBs produce neurons directed towards the center of the
medulla, it is likely that neurons born early in development arelocated in inner concentric zones, while late-born neurons arefound in outer zones. To confirm this theory, we used a neuroblast
specific Gal4 driver (dpn-Gal4) that specifically expresses Gal4mRNA in NB cells (Fig. 2G,H). Differentiated neurons inherit theGal4 protein, even after Gal4 transcription is terminated; thus, astrong GFP signal was detected in neurons in the outer concentriczones (Fig. 2E). However, the GFP signal was decreased in neurons
distant from NBs and was not detectable in the innermost zones. A
prominent GFP signal was observed in the innermost, non-neuronalglial cells (Fig. 2F), a result supporting the hypothesis that NBs
produce glial cells that subsequently migrate toward the medulla

DEVELOPMENT

Fig. 1. Larval medulla primordium
is subdivided into concentriczones. (A-C)Schematics of the
Drosophila visual system in third
instar larva (A,B) and adult (C).
Lateral (A) and horizontal (B,C) views.
(D-G!) The larval medulla in lateral(D-G) and horizontal (D!-G!) views.
(D,D!) Hth (green), Run (magenta)
and Drf (blue) expression. (E,E!) Hth(green) and Bsh (magenta) expression
and NBs (Dpn; blue). Weak Hthexpression in NE and NB cells(arrowhead in D! and arrows in D!
and E!, respectively). Bsh expression
in lamina neurons (arrowhead in E!).
(F,F!) Hth (green) and Drf (blue)
expression and neuronal projections(elav-Gal4 UAS-CD8GFP; white). Only
GFP signal is shown in the right half
(F). Primordia for medulla and lobula
complex (arrows and arrowheads,
respectively). (G,G!) hth-lacZ (green),
glial cell bodies (Repo; magenta) andglial processes (repo-Gal4 UASCD8GFP; white). Large and small glial
cells in medulla cortex (arrows andarrowheads). Putative MNGs (large
arrowheads). (H,I)Schematics of theconcentric zones in larval medulla inlateral (H) and horizontal views (I).
(J)Clones of larval medulla neurons
expressing CD8GFP (green) and tauMyc (magenta) under the control of
AyGal4. Putative axons are indicatedby arrows. Lo, lobula; Lop, lobula
plate; M, medulla.

986

RESEARCH ARTICLE

Development 138 (5)neuropile (Colonques et al., 2007). The signal intensity of GFP
roughly correlated with concentric gene expression; strong GFP
expression was found in Drf-positive neurons but gradually
decreased in Run and Bsh-positive zones (Fig. 1D!, Fig. 2E,I).
These results are consistent with the idea that the birth order of
medulla neurons may control neuronal type via concentric gene
expression.
Medulla neurons migrate extensively and theconcentric zones are disrupted during pupal
development
The expression of concentric genes in the larval medulla
primordium may regulate neuronal identities and contribute to theformation of neuronal circuits in the adult medulla. We thus
examined concentric gene expression during pupal development(Fig. 3A). The concentric zones found in the larval medulla were
essentially conserved during early pupal development until 8 hours
after puparium formation (APF; Fig. 3A, part a). However, theconcentric zones began to collapse at 12 hours APF (Fig. 3A, partb). The distribution of neurons continued to change until 24 hours
APF, becoming completely disorganized (Fig. 3A, parts c-e). The
same disorganized pattern was found in the adult brain (Fig. 3A,part f). Because the expression of concentric transcription factors
appeared stable and strong, it is likely that cell bodies migrate
extensively between 12 and 24 hours APF without changing gene
expression. Hth- and Drf-positive cells were found in the innermostand outer zones of the larval medulla, respectively. However, both
Hth- and Drf-positive cells were found in the inner and outer zones
in adults (Fig. 3A, part f). This result suggests that the cell bodies
of medulla neurons may be migrating in a radial pattern.
In order to address whether they are indeed migrating, clones of
medulla neurons were constitutively labeled with GFP (Lee andLuo, 1999). The relative distributions of GFP-positive cells before
and after migration were compared (Fig. 3C). Because the NBs
situated in the outermost region of the larval medulla primordium
produce neurons with a radial orientation, GFP-positive neurons

were linearly arranged in the larval brain (Fig. 3C, part a). Similar
distributions of GFP-positive cells were found at the beginning of
migration at 12 hours APF (Fig. 3C, part b). However, after thecompletion of migration at 24 hours APF, GFP-positive cells werestochastically distributed (Fig. 3C, part c). Therefore, medulla
neurons changed their relative locations between 12 and 24 hours
APF. Because a radial migration would not disrupt the radialarrangement of cells, medulla neurons may also migrate with atangential orientation, perpendicular to the linear arrangement of
GFP-positive cells in the larval brain (Fig. 3C, part a).
To determine whether the expression of concentric genes persists
during metamorphosis, Drf-positive neurons were constitutively
labeled with lacZ (see Fig. S1 in the supplementary material). A
Gal4 driver that accurately mimics drf expression (drf-Gal4) wasused to express FLPase to constitutively label Drf-positive cellswith lacZ (see Fig. S1 in the supplementary material) (McGuire etal., 2003; Struhl and Basler, 1993). If drf expression persists duringmetamorphosis, all of the lacZ positive neurons in the adult brain
should express GFP, which is under the control of drf-Gal4. Indeed,
our data suggest that drf expression persists during metamorphosis.
Changes in distributions of Hth-, Run- and Drf-positive neurons
were seemingly random. However, Bsh/Hth double positive
neurons displayed a clear pattern of migration (Fig. 3B). Because
Bsh is expressed in a subset of Hth-positive neurons, Bsh/Hthpositive cells were initially situated in the inner area of the medulla
primordium (Fig. 1E, Fig. 3B, part a). However, they always
moved outward during metamorphosis and were eventuallysituated in the outermost area of the adult medulla (Fig. 3B, partsb-f). This finding suggests that the migration of medulla neurons is
indeed an organized and patterned process, but that labeling several
types of neurons may obscure the migration pattern. Because theprojection pattern of Bsh/Hth-positive neurons could becontinuously traced from the larval to adult medulla (Fig. 5), we
assume that the expression of Bsh and Hth in these neurons does
not change throughout development. The locations of Drf-positive
neurons in the adult brain could also be classified according to

DEVELOPMENT

Fig. 2. Birth order of medulla neurons
correlates with concentric gene
expression. (A,B)Clones of cells expressing
CD8GFP (green). Drf (blue; arrowheads) andBsh (magenta; arrowheads) expression and
NBs (Dpn; magenta; arrows). Clones
containing multiple NBs (A) or a single NB(B). (C)Number of cells expressing Drf, Bsh
and Run in single NB clones that intersect the
Drf-Bsh or Drf-Run domains. Clones in theposterior edge of the OPC are notconsidered. (D)A schematic of NBs
producing multiple types of medulla neurons.
(E,F)Neuronal birth order visualized using
dpn-Gal4 UAS-GFP (green). Later-bornneurons show stronger GFP signals. (E)Drf(blue) and Bsh (magenta) expression. (F)Glial
cells (Repo; magenta; arrowheads).
(G,H)Gal4 mRNA (magenta) expression in
NBs (Dpn; blue) in dpn-Gal4 flies. Lateral (G)and horizontal (H) views. (I)A schematic of
the GFP gradient produced by NB-specific
Gal4 expression.

The Drosophila visual center development

RESEARCH ARTICLE

987

neuronal subtypes (see Fig. S4 in the supplementary material),
again suggesting that each type of medulla neuron exhibits astereotyped migration pattern.
Hth is expressed in four types of medulla neurons,
while co-expression of Hth and Bsh is found in asingle subtype
To investigate projection patterns of Hth-positive neurons, a Gal4
driver that mimics hth expression was generated. The resulting hthGal4 allele widely induced UAS-GFP expression in the larval
medulla probably due to residual expression of Gal4 produced in
NBs. In the adult brain, hth-Gal4 largely recapitulated Hthexpression (Fig. 4A). We also searched for Gal4 drivers that mimic
Hth expression. NP6013 induced UAS-CD8GFP expression in a
subset of Hth-positive neurons in the larval brain (Fig. 4B). The
projections of Hth-positive medulla neurons were already present
in the larval brain; their terminals were restricted to the medulla
neuropile and were not found in the lobula complex (lobula andlobula plate; Fig. 4B).
Generating single cell clones expressing CD8GFP under thecontrol of hth-Gal4 and NP6013 (Wong et al., 2002), we identified
at least four types of Hth-positive neurons in the adult brain (Fig.
4C-F; see Fig. S2B in the supplementary material). As observed in

the larval medulla, none of these neurons projected to the lobula
complex (Fig. 4B). Neuronal types were classified according to the
combinations of layers to which they project, as confirmed by NCadherin expression (NCad; Fig. 6E, part c) (Fischbach andDittrich, 1989; Morante and Desplan, 2008). The neuronal typemost frequently observed was Mi1, which shows small
arborizations at M1, M5 and M9-10 (Fig. 4C; see Fig. S2B in the
supplementary material). Neurons that show stratifications at M1
and M4 in the medulla and projections to the lamina were
temporarily named lamina wide field-like1 (Lawfl1; Fig. 4D)(Fischbach and Dittrich, 1989) as arborizations in the lamina werebarely detectable owing to weak GFP expression. The other Lawflike neuron, Lawfl2, showed arborizations at M1 and M8-10 in the
medulla and projections to the lamina (Fig. 4E). Lawfl2 has notbeen described in the literature. Pm3 are local neurons restricted tothe proximal medulla (Fig. 4F).
Bsh is expressed in a subset of Hth-positive neurons from larval
to adult stages (Fig. 1E, Fig. 3B). We found that Bsh was only
expressed in Mi1 type neurons in the adult medulla (see Fig. S2Bin the supplementary material). We thus generated a bsh-Gal4
strain by inserting the Gal4-coding sequence into the genomic
fragment of the bsh gene that contains the entire bsh locus. The
expression of bsh-Gal4 was similar to Bsh in adults (Fig. 4G).

DEVELOPMENT

Fig. 3. Medulla neurons migrate
during pupal development.
(A)Hth- (green), Run- (magenta)
and Drf- (blue) positive neurons in
8, 12, 16, 20, 24 hours APF pupal(a-e) and adult (f) brains. Runpositive cells in the outer zone
(arrowhead in b). Concentric zones
are disrupted in 24 hours APF andadult brains (e,f). (B)Hth- (green)and Bsh- (magenta) positive neurons
and glial cells (Repo; blue) in 8, 12,
16, 20, 24 hours APF pupal (a-e)
and adult (f) brains. Hth/Bsh doublepositive neurons migrate outwards(arrows). Putative MNGs areindicated (arrowheads). (C)Clonesof medulla neurons visualized byelav-Gal4 UAS-CD8GFP (white;arrows) at late third instar (a), 12
hours APF (b) and 24 hours APF (c),showing tangential migration
between 12 and 24 hours APF.
Lateral views.

988

RESEARCH ARTICLE

Development 138 (5)Indeed, mosaic analyses using bsh-Gal4 specifically indicated thepresence of Mi1 neurons in the medulla. Presynaptic sites wererevealed using synaptotagmin-HA (SytHA) (Robinson et al., 2002).
The SytHA signal was very strong at M9-10 but weak or hardly
detectable at M1/5, suggesting that Mi1 neurons have postsynaptic
sites at M1/5 and a presynaptic site at M9-10 shaped like a fish
hook (Fig. 4H).
In addition to the morphological features, neurotransmitters areimportant characteristics of neurons. We found that choline
acetyltransferase (ChAT) was always upregulated in Bsh-positive
neurons in adults (Fig. 4I). Therefore, Bsh expression alone canidentify a specific type of medulla neuron, Mi1, which
stereotypically migrates outward during metamorphosis and ischolinergic.
Hth regulates Bsh expression and neuronal
identities
The roles of hth in medulla neuron formation were examined bygenerating hth-Gal4-homozygous mutant clones and visualizing
their neuronal projection patterns (Fig. 5). The loss of hth function
completely changed the morphology of hth-Gal4-positive medulla
neurons, making cells apparently switch neuronal types (Fig. 5;
Fig. S3A in the supplementary material). In a hth-Gal4heterozygous background, control clones exhibited the normal
morphology of Hth-positive neurons, primarily Mi1 (Fig. 5D). In
hth-Gal4-homozygous clones, normal Mi1 neurons were notfound; instead, we frequently observed neurons very different from
Mi1, neurons that were Hth-negative on a wild-type backgroundsuch as Tm13-like and other Tm-type lobula projection neurons(Fig. 5B,D; see Fig. S3A in the supplementary material). Mi1-like
abnormal neurons, which were similar to Mi1 but exhibitedirregular arborizations, were occasionally found (Fig. 5A,D). The
fish hook-like shape at M9-10 was occasionally disrupted in the
Mi1-like abnormal neurons. The phenotype could partially be

rescued by introducing exogenous hth (Fig. 5D; see Fig. S3A in the
supplementary material). Thus, the loss of hth function transforms
Hth-positive neurons into diverse types of medulla neurons that areprobably specified by genes other than hth.
Using bsh-Gal4, consistent phenotypes were observed in clones
homozygous for a strong hypomorphic allele, hthP2. In control
experiments, bsh-Gal4 exclusively labeled Mi1 neurons (Fig. 5D).
However, Tm1-like rather than Mi1 neurons were found in hthP2homozygous clones (Fig. 5C,D; see Fig. S3A in the supplementarymaterial). The formation of Mi1 was partially rescued byintroducing exogenous hth (Fig. 5D; see Fig. S3A in the
supplementary material). In contrast to the distal localization of
Mi1 cell bodies in the wild-type medulla cortex, the cell bodies ofhth-mutant Tm-like neurons were found in the proximal tointermediate cortical zone (n11/16; Fig. 5C). Therefore, hth isessential to determining the identity of Mi1 neurons, including thelocation of cell bodies in the adult brain. Cell bodies of non-Mi1
type Hth-positive neurons tended to be found in more proximal
cortical zones in the wild-type medulla cortex (Fig. 3Bf). Thus, hthand bsh may cooperate to determine the location of Mi1 cellbodies.
Because Hth was weakly expressed in NE and NB cells in thelarval brain (Fig. 1D!,E!), the hth-mutant phenotypes shown above
may be caused by general defects in neurogenesis resulting fromabnormalities in neuronal precursor cells. However, we did notdetect such defects in production of GMCs and neurons, as
determined by Prospero and Elav expression in hth-mutant clones
in the larval medulla (see Fig. S3B in the supplementary material).
Hth may regulate expression of downstream target genes tocontrol specification and formation of Hth-positive neurons. bsh isa potential candidate of Hth-target gene because Bsh expression
was frequently reduced in hth mutant clones (Fig. 5L). However,residual Bsh expression was detected (Fig. 5L). This may be due
to hypomorphic nature of hthP2, or hth function may not be

DEVELOPMENT

Fig. 4. Hth is expressed in four types of medulla
neurons. (A)hth-Gal4 UAS-nlsGFP (green) overlaps Hth(red) in adult medulla. (B)NP6013 UAS-CD8GFP (white)overlaps Hth (green) in larval medulla. NCad (blue). Hthpositive neurons project to medulla (M; arrow) but not tolobula complex (Lo/Lop; arrowhead). (C-F)Clones of Hthpositive neurons in adult medulla. NCad (blue) and
UAS>CD2>CD8GFP (green) under the control of hth-Gal4(C,D,F) and NP6013 (E). (C)Mi1, (D) Lawfl1, (E) Lawfl2and (F) Pm3. A cell body expressing a low level of Hth(arrow). Lamina projections (arrowheads). (G) bsh-Gal4
UAS-CD8GFP (green) overlaps Bsh (red) in adult medulla.
NCad (blue). (H) Mi1: a clone expressing UAS-CD8GFP(green) and UAS-SytHA (red) under the control of bshGal4. (I)Co-expression of ChAT (white) and Bsh(magenta) in adult medulla cortex. Lo, lobula; Lop, lobula
plate; M, medulla; M1-M10, layers of the medulla.

The Drosophila visual center development

RESEARCH ARTICLE

989

essential for Bsh expression (e.g. hth and an unidentified gene may
act partially redundantly). Interestingly, the transformation of Mi1
neurons to Tm1-like neurons, as revealed by bsh-Gal4, wasobserved in both Bsh-positive (n4/65) and Bsh-negative(n61/65) clones that were hth mutants. In most cases, Mi1-like
abnormal neurons were positive for Bsh (n7/8), suggesting that
Bsh expression alone cannot determine the fate and morphology of
Mi1 neurons.
NCad may act downstream of Hth to regulate
projection of Mi1 neurons
The development of Mi1 neurons was traced from the larval toadult brain using NP6013 and bsh-Gal4 (Fig. 5E-H). At the end oflarval development (0 hours APF), Bsh-positive neurons form twoarborization sites along the neuropile structures strongly expressing
NCad (proximal and distal NCad domains; Fig. 5E). Found in theproximal NCad domain, the arborization at the tip of axonal shafts
most likely corresponds to the terminal at M9-10 in adults (Fig.
5E,H). In the distal NCad domain, the third arborization site, whichmost likely becomes M1 arborization in adults, was occasionallyfound immediately distal to the pre-existing M5 arborization at 24hours APF (Fig. 5G). At 20 hours APF, typical Mi1 neurons lacked
the third arborization while cell bodies were located in the distal

cortical area, suggesting that the M1 arborization is formed around20-24 hours APF coincidently with the completion of cell body
migration (Fig. 5F,G).
Although NCad was widely expressed in the cell bodies of larval
medulla neurons, its expression was upregulated in the Hth-positive
innermost concentric zone (Fig. 5J). Because NCad was ectopicallyupregulated in hth-expressing clones and was autonomously reduced
to the basal level in hth mutant clones (Fig. 5K,L), NCad
upregulation is most likely controlled by hth in the larval brain.
NCad is a homophilic cell adhesion molecule; therefore, neurites of
Mi1 neurons that strongly express NCad may innervate targets also
expressing high levels of NCad (Iwai et al., 1997). NCad is known
to regulate the target layer specificity of R-axons and lamina neurons(Lee et al., 2001; Nern et al., 2008; Ting et al., 2005). Thus, NCad
upregulation in Mi1 may influence the arborization sites of Mi1
neurons located in the proximal and distal NCad domains in thelarval medulla neuropile (Fig. 5E,J). Only the proximal arborizations
were occasionally eliminated in clones homozygous for NCadM19(n6/10; Fig. 5I) and NCadomb405 (n3/6; not shown). The defects
found in the adult brain were diverse and may not specifically derivefrom larval defects. However, the loss of or disorganized M9-10
terminals can be explained by a lack of NCad upregulation in Mi1
neurons during larval development (Fig. 5I and not shown).

DEVELOPMENT

Fig. 5. hth regulates Bsh and NCad expression and neuronal identities. (A-C)Projections of hth mutant neurons labeled with UAS-CD8GFP(green) under the control of hth-Gal4 (A,B; hth-Gal4 clones) and bsh-Gal4 (C; hthP2 clone) in adults. NCad (blue). Bsh (blue in A,B). (A)Irregulararborizations of Mi1-like neuron (arrowheads). (B,C)Tm13-like and Tm1-like neurons presumably transformed from Hth- and Bsh-positive neurons,
respectively. Putative glial cells are indicated (arrowhead in B). (D)Catalogue of neurons in control, hth mutant and hth rescue clones. (E-I)Mi1
neurons of control (E-H) and NCadM19 (I) clones at 0 (E,I), 20 (F), 24 hours APF (G) and in adult (H) brains. Clones expressing UAS-CD8GFP (green)
under the control of NP6013 (E,I) or bsh-Gal4 (F-H). NCad, magenta; Bsh, red in E,I. Arrows in E,I indicate Bsh-positive cell bodies. (E,F)Two
arborization sites at ‘Distal’ (D) and ‘Proximal’ (P) NCad domains (arrowheads). (G)The arborizations of M1 are formed at 24 hours APF. ‘Distal’ and‘Proximal’ arborizations in E,F correspond to M5 and M9-10, respectively. (I)Proximal arborization is missing in NcadM19 clones. (J)NCad
upregulation (white; arrow) in Hth domain (magenta) in larval medulla. Only NCad is shown in the right half. (K)Ectopic NCad upregulation (white)
in clones expressing hth (GFP; green; arrows). (L) Downregulation (arrows) of NCad (white) and Bsh expression (magenta) in hth mutant clones(dotted lines). Lo1 and 4, lobula layers 1 and 4; M, medulla; M1-M10, layers of the medulla.

990

RESEARCH ARTICLE

Development 138 (5)Drf is expressed in nine types of medulla neurons
To compare neuronal types of Drf- and Hth-positive population, we
generated drf-Gal4 strains that accurately mimic drf expression.
The drf-Gal4 induced GFP expression in a manner identical to Drf
distribution throughout development (Fig. 6A,B). In the adultmedulla, restricted subsets of medulla strata were densely labeledwith GFP (Fig. 6E). In the lobula, axonal terminals were
exclusively found in layers Lo1 and Lo4. Drf-positive neurons thus
project to common target layers in the adult lobula.
To identify neuronal types showing Drf expression, we
performed MARCM analyses using drf-Gal4 to catalog Drfpositive medulla neurons in adults and identified nine subtypes ofDrf-positive neurons (Fig. 6F-M), the majority of which were
lobula projection neurons that projected to the lobula complex(TmY3, Tm27(Y), Tm9 and Tm3; Fig. 6F-J). In the distal medulla,
SytHA was weak or hardly detectable, but was prominent in thelobula complex. Thus, the arborizations in the distal medulla wereprimarily postsynaptic, while terminals in the lobula complex were
presynaptic. We also identified three types of local neurons (Mi10b,
Dm8a and Dm8b; Dm8a is similar to Dm8 in the literature) the
innervations of which were restricted to the medulla (Fig. 6K-M).
A stochastic distribution of SytHA was found along arborizationsof these local neurons. Tm27 and Tm27Y neurons have not beenpreviously described. Tm3b neurons are similar to Tm3 neurons(hereafter Tm3a) with the exception of a small branch at M10
projecting back towards M8 (Fig. 6J).
The Bsh/Hth-positive Mi1 neurons were shown tostereotypically migrate from the inner to outer areas of the medulla
cortex (Fig. 3B). To determine whether a similar migration patternoccurs in Drf-positive neurons, locations of cell bodies in the
medulla cortex were categorized into distal, intermediate andproximal zones (see Fig. S4 in the supplementary material). For

DEVELOPMENT

Fig. 6. Drf is expressed in nine types of medulla neurons. (A)drf-Gal4 UAS-CD8GFP (white) overlaps Drf (red) in larval medulla. Medulla (M,arrows) and lobula complex (Lo/Lop, arrowheads). NCad is in blue. (B)drf-Gal4 UAS-nlsGFP (white) overlaps Drf (red) in adult medulla. Drf
expression in non-neuronal lamina cells (arrowheads). (C-E) drf-Gal4 UAS-CD8GFP (white), NCad (blue) and R-axons (Chaoptin; red) in larva (C),24 hours APF pupa (D) and adult (Ea). Drf-positive neurons project to larval medulla and lobula complex (C). Distribution of Drf-neurons changes by
24 hours APF (D). Elongated cell bodies are indicated by arrowheads in D. Definition of layers (Eb,c). Lateral (A) and horizontal (B-E) views.
(F-M) Nine types of Drf-positive neurons. Clones expressing UAS-CD8GFP (green) and UAS-SytHA (red) under the control of drf-Gal4. NCad (blue).
(F)TmY3, (G) Tm27, (H) Tm27Y, (I) Tm9, (J) Tm3a/b, (K) Mi10b, (L) Dm8a and (M) Dm8b. Three-dimensional reconstructed images (F-M, part a) andsingle confocal sections (F-M, part b; F-J, part c; J, part d). Lo1 and 4, lobula layers 1 and 4; M1-M10, layers of the medulla.

each subtype of Drf-positive neurons, locations of cell bodies were
examined. As a result, we found that four subtypes of Drf neurons
have cell bodies in the distal zone; three subtypes in theintermediate zone and two subtypes in the proximal to intermediatezones. Thus, cell body location correlated with the neuronalsubtype of Drf-positive neuron, suggesting that each subtype showsa stereotyped pattern of migration during pupal development.
Intriguingly, many of Drf-positive neurons already projected to
the lobula primordium in the larval brain (Fig. 6A,C). After
projecting to the lobula, cell bodies began to migrate between 12
and 24 hours APF (Fig. 3A). At 24 hours APF, Drf-positive cellbodies were found in the inner area of the medulla cortex (Fig. 6D).
Cell bodies were occasionally elongated with a radial orientation
at 24 hours APF (arrowheads in Fig. 6D), which may imply thesecells are migrating in radial orientation.
During pupal development, the medulla and lobula complex
rotated 90° around the vertical axis, and the medulla cortex was
eventually placed between the lamina and the medulla neuropile(Fig. 6D,E) (Meinertzhagen and Hanson, 1993). The migration of
medulla neurons may be a secondary effect of medulla rotation.
However, the relative locations of the medulla cortex and thelamina at 24 hours APF were relatively unchanged when comparedwith the larval brain (Fig. 6C,D), suggesting that the medulla
neurons independently migrate before the rotation that occurs after
24 hours APF.
Drf regulates axonal projections and dendritic
arborizations
Because the drf-Gal4 we used is not a strong loss-of-function allele,
it is not suitable for an analysis of drf mutants. We thus searched forGal4 drivers that could label Drf-positive neurons independently of
drf gene expression. NP2646 expressed Gal4 in some types of
medulla neurons, including Tm27(Y) neurons (see Fig. S5 in the
supplementary material). Tm27 neurons were rare, so we focused
our analyses on Tm27Y. We created drf mutant MARCM clones andexamined their morphology (Fig. 7). Although wild-type Tm27Y
neurons showed extensive arborizations at M4-5 and M8-10 in the
medulla and targeted Lo4 in the lobula, loss of drf function caused
reduced arborization at M8-10, ectopic arborization at M2-3 andaxonal targeting defects in the lobula (Fig. 7 and see Fig. S5C-F in
the supplementary material). These defects are most likely caused by
drf mutation because similar phenotypes were observed after using
two independent drf alleles. Moreover, the defects were partiallyrescued by exogenous drf expression (see Fig. S5C-F in the
supplementary material). The localization of Tm27Y cell bodies in
the proximal to intermediate cortical zone was not noticeably alteredin drf mutant clones (see Fig. S5G in the supplementary material).
Essentially the same results were observed in Tm27 neurons (notshown). In other types of Drf-positive neurons, minor morphologicaldefects were observed by knocking down drf under the control of
drf-Gal4 (not shown).
DISCUSSION
Neuronal identities are pre-determined in thelarval medulla
Concentric genes are expressed in a defined subset of medulla
neurons throughout development, suggesting that a part of neuronal
identities are pre-determined in the larval medulla primordium. Ourdata suggest that Drf-positive neurons produce nine types of medulla
neurons, including lobula projection and medulla intrinsic neurons,
while Hth-positive neurons produce at least four types of neurons,
including lamina projection and medulla intrinsic neurons. In Hth-RESEARCH ARTICLE

991

Fig. 7. drf regulates axonal targeting and dendritic arborizations.
(A,B)Morphology of Tm27Y visualized by NP2646 UAS-CD8GFP (green)in control (A, parts a,b) and drfE82 clones (B, parts a,b) in adults.
(a)Ectopic and reduced arborizations at M2-3 (arrows) and M8-10(arrowheads) in medulla, respectively. (b)Axonal overshooting (arrows)
and ectopic branch (arrowhead) in lobula. Lo4 is defined by reduced
NCad expression (blue; dotted lines).

positive neurons, Bsh is exclusively expressed in medulla intrinsic
Mi1 neurons. A hth mutation caused the neuron to switch type (Fig.
5), while a drf mutation affected subsets of morphological featuresof Drf-positive neurons (Fig. 7). Thus, roles of concentric genes may
be functionally segregated to form a genetic hierarchy. Apparently,
other concentric genes must exist in addition to the four genes
reported in this study. Because there are many neurons outside of the
Drf domain in the larval medulla, some concentric genes may be
expressed in the outer zones. Some transcription factors may haveexpression patterns that differ from those of concentric genes, andtheir combined expression may specify restricted subtypes of
medulla neurons. For example, apterous (ap) and Cut are widely
expressed in medulla neurons (see Fig. S4D in the supplementarymaterial) (Morante and Desplan, 2008). Cut was co-expressed insubsets of Drf-positive neurons, while ap was expressed in all Drfand Bsh/Hth-positive neurons (see Fig. S4B in the supplementarymaterial and data not shown).
Early-born medulla neurons express the inner concentric genes,
while late born neurons express the outer ones. Thus, concentric gene
expression correlates with neuronal birth order (Fig. 2). However, it
is still unknown how concentric gene expression is specified. It
would be possible to speculate that genes controlling temporal
specification of neurons are expressed in NBs to control theconcentric gene expression. However, the genes that are known tocontrol neuronal birth order in the embryonic CNS were notexpressed in larval medulla NBs (not shown) (Isshiki et al., 2001).
In addition to local temporal mechanisms, such as birth order, global
and spatial mechanisms governed by morphogen gradient may also
play a role in determining medulla cell type (Jessell, 2000). In
addition to birth order or a morphogen gradient, mutual repression
among concentric genes may be essential in establishing definedconcentric zones. Except for rare occasions, de-repression of otherconcentric genes was not induced in clones mutant for hth or drf.
Additionally, ectopic hth expression did not compromise Drf and
Run expression (not shown). These results may suggest that

DEVELOPMENTThe Drosophila visual center development

RESEARCH ARTICLE

unidentified genes act redundantly with these genes to repress
expression of other concentric genes and that weak Hth expression
in NBs does not play roles in temporal specification of medulla
neurons.
Regulation of migration and its biologicalsignificanceVarious types of cell migration play important roles during vertebrate
neurogenesis (Hatten, 1999; Nobrega-Pereira and Marin, 2009).
Although Drosophila has been a powerful model of neuraldevelopment, extensive neuronal migrations coupled with layer
formation found in this study have not been previously reported. Ourfindings may establish a model to understand molecular mechanismsthat govern brain development via neuronal migrations.
It is important to know whether the migration of medulla neurons
occurs actively or passively. The distribution of cell bodies in theadult medulla cortex was not random, but organized according to cell
type (Fig. 3; see Fig. S4 in the supplementary material). In particular,
the Mi1 neurons identified by Bsh expression migrated outwards andwere eventually located in the outermost area of the adult medulla
cortex (Fig. 3), which was affected in hth mutant clones (Fig. 5). The
observation that defined localization of cell bodies is under thecontrol of genetic program may not be explained by passive
migration. Repression of apoptosis by expressing p35 under thecontrol of elav-Gal4 did not compromise migration of Bsh- and Drfpositive neurons, suggesting that apoptosis is not a driving force ofthe migration (not shown). If neurons migrate actively in an
organized manner, what regulates the pattern of migration? In manycases, glial cells play important roles in neuronal migration (Hatten,
1999). Indeed, we identified glial cells and their processes in the
medulla cortex (Fig. 1G). Glial cells or other cell types could provide
cues for neuronal migration.
The medulla neurons project axons near their targets forming
subsets of dendrites in the larval brain (Figs 5 and 6); the cellbodies migrated in the presence of preformed neurites during pupal
development (Fig. 3). During or following cell body migration,
additional dendrites were formed along the axonal shafts (Fig. 5).
Therefore, cell body migration may somehow contribute to circuit
formation in the medulla. Indeed, similar strategies have beenreported in sensory neurons of C. elegans and cerebellar granulecells in mammals (Heiman and Shaham, 2009; Solecki et al.,2006). Thus, cell body migration in the presence of neurites may
be a general conserved mechanism of circuit formation. Cell body
migration may also allow developing cells to receive inductive cues
provided by cells in the vicinity of the medulla cortex. For
example, glial cells placed on the surface of the brain may trigger
the expression of specific genes (e.g. ChAT) in Mi1 cells that arelocated in the outermost area of the adult medulla cortex.
Contributions to functional neuronal circuits
In adults, Mi1 neurons have arborization sites at M1 and M5,
which coincide with the L1 lamina neuron terminals. In Golgi
studies, Mi1 neurons were found in all parts of the retinotopic field(Fischbach and Dittrich, 1989). Indeed, the number of Bsh
expressing medulla neurons was about 800, a figure similar to thenumber of ommatidial units (not shown). Therefore, the Mi1
neurons identified by Bsh expression are most probably columnar
neurons with direct inputs from L1 neurons. Because L1 is known
to have inputs from R1-6, which processes motion detection, Mi1
may participate in the motion detection circuit (Fischbach andDittrich, 1989; Joesch et al., 2010; Katsov and Clandinin, 2008;
Rister et al., 2007; Zhu et al., 2009).

Development 138 (5)If the genetic codes that specify each type of neuron are found,it may encourage the functional study of defined neurons. In the
medulla, bsh-Gal4 is solely expressed by Mi1 neurons. Although
the expression of Bsh is also observed in L4/5 lamina neurons (Zhu
et al., 2009), intersectional strategies such as split Gal4 may enable
us to specifically manipulate the activity of Mi1 by inducing
expression of neurogenetic tools like shibirets (Kitamoto, 2001;
Luan et al., 2006). Our findings may thus provide insight into highresolution functional neurobiology in the Drosophila visual system.
Similarities with vertebrate neurogenesis
Development of the mammalian central nervous system
reiteratively establishes cell identity, directs cell migration andassembles neuronal layers (Hatten, 1999), processes similar to thepatterns observed during medulla development. In the cerebral
cortex, neurons are generated within the ventricular or
subventricular zones and migrate outwards, leaving their birthplace
along the radial glial fibers. Later-born neurons migrate radiallyinto the cortical plate, past the deep layer neurons and become theupper layers. The layers of the cortex are thus created inside-out(Nobrega-Pereira and Marin, 2009). In the developing spinal cord,
neuronal types are specified according to morphogen gradients(Jessell, 2000). Within each domain along the dorsoventral axis,
neuronal and glial types are specified according to their birth order(Guillemot, 2007). The spinal cord neurons then migrate
extensively along the radial, tangential and rostrocaudal axes
(Leber and Sanes, 1995). Therefore, the initial organization of
spinal cord neurons is disrupted in the mature system.
The medulla shares intriguing similarities with the mammalian
central nervous system. For example, the concentric zones
established in the larval medulla resemble the dorsoventral
subdivisions of the spinal cord (Jessell, 2000). Extensive
migrations of medulla neurons disrupt concentric zones, as
observed in the spinal cord. However, we found that the locationsof cell bodies were organized according to neuronal type, a
distribution that may be similar to the cortical organization of thecerebral cortex (Nobrega-Pereira and Marin, 2009). Thus, thedevelopment of the medulla may share characteristics withvarious forms of neurogenesis found in the mammalian central
nervous system. A comprehensive study of important features ofneurogenesis will now be possible using the Drosophila visual
center and powerful tools of Drosophila genetics. Unveiling all
aspects of development in the medulla will not only shed lightinto the functional neurobiology of the visual system, but also
elucidate the developmental neurobiology of vertebrates andinvertebrates.
Acknowledgements
We thank Daiki Umetsu and Miyako Tai for the note that Bsh and Drf are
expressed in concentric zones. We thank Karl Fischbach, Kei Ito, Tom
Kornberg, Ian Meinertzhagen, Takaki Miyata, Koji Oishi, Michiya Sugimori,Shin-ya Takemura and Larry Zipursky for critical comments. We thank Haruhiro
Higashida for use of facilities. We are grateful to Adi Salzberg, Masahide
Asano, Ethan Bier, Sarah Certel, Takahiro Chihara, Shin-ichi Higashijima,
Takako Isshiki, Kei Ito, Liqun Luo, Richard Mann, Chi-Hon Lee, William
McGinnis, Andreas Prokop, James Skeath, Thomas Schwarz, Gary Struhl,
Tadashi Uemura and Larry Zipursky for antibodies, strains and plasmids. We
thank BestGene for their injection service; Bloomington Stock Center and
DGRC, Kyoto for strains; DSHB and Asian Distribution Center for
Segmentation Antibodies for antibodies; and DGRC, Indiana for vectors. This
work was supported by Program for Improvement of Research Environment for
Young Researchers and PRESTO from JST, Takeda Science Foundation, Brain
Science Foundation, Mitsubishi Foundation, Sumitomo Foundation, Uehara
Memorial Foundation, Nakajima Foundation (to M.S.), Grants-in-Aid from
MEXT (to M.S. and T.T.) and JSPS Research fellowship (to Y.K.).

DEVELOPMENT

992

Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.058370/-/DC1
References
Anderson, M. G., Perkins, G. L., Chittick, P., Shrigley, R. J. and Johnson, W. A.
(1995). Drifter, a Drosophila POU-domain transcription factor, is required forcorrect differentiation and migration of tracheal cells and midline glia. Genes Dev.
9, 123-137.
Bier, E., Vaessin, H., Younger-Shepherd, S., Jan, L. Y. and Jan, Y. N. (1992).
Deadpan, an essential pan-neural gene in Drosophila, encodes a helix-loop-helix
protein similar to the hairy gene product. Genes Dev. 6, 2137-2151.
Colonques, J., Ceron, J. and Tejedor, F. J. (2007). Segregation of postembryonicneuronal and glial lineages inferred from a mosaic analysis of the Drosophila larval
brain. Mech. Dev. 124, 327-340.
Egger, B., Boone, J. Q., Stevens, N. R., Brand, A. H. and Doe, C. Q. (2007).
Regulation of spindle orientation and neural stem cell fate in the Drosophila optic
lobe. Neural Dev. 2, 1.
Fischbach, K. F. and Dittrich, A. P. M. (1989). The optic lobe of Drosophilamelanogaster. I. A Golgi analysis of wild-type structure. Cell Tissue Res. 258, 441475.
Gao, S., Takemura, S. Y., Ting, C. Y., Huang, S., Lu, Z., Luan, H., Rister, J., Thum,
A. S., Yang, M., Hong, S. T. et al. (2008). The neural substrate of spectral
preference in Drosophila. Neuron 60, 328-342.
Gergen, J. P. and Butler, B. A. (1988). Isolation of the Drosophila segmentation
gene runt and analysis of its expression during embryogenesis. Genes Dev. 2,
1179-1193.
Gong, W. J. and Golic, K. G. (2003). Ends-out, or replacement, gene targeting in
Drosophila. Proc. Natl. Acad. Sci. USA 100, 2556-2561.
Guillemot, F. (2007). Spatial and temporal specification of neural fates bytranscription factor codes. Development 134, 3771-3780.
Hatten, M. E. (1999). Central nervous system neuronal migration. Annu. Rev.
Neurosci. 22, 511-539.
Heiman, M. G. and Shaham, S. (2009). DEX-1 and DYF-7 establish sensory
dendrite length by anchoring dendritic tips during cell migration. Cell 137, 344355.
Hofbauer, A. and Campos-Ortega, J. A. (1990). Proliferation pattern and early
differentiation of the optic lobes in Drosophila melanogaster. Rouxs Arch. Dev.
Biol. 198, 264-274.
Isshiki, T., Pearson, B., Holbrook, S. and Doe, C. Q. (2001). Drosophila
neuroblasts sequentially express transcription factors which specify the temporal
identity of their neuronal progeny. Cell 106, 511-521.
Ito, K., Awano, W., Suzuki, K., Hiromi, Y. and Yamamoto, D. (1997). The
Drosophila mushroom body is a quadruple structure of clonal units each of whichcontains a virtually identical set of neurones and glial cells. Development 124, 761771.
Iwai, Y., Usui, T., Hirano, S., Steward, R., Takeichi, M. and Uemura, T. (1997).
Axon patterning requires DN-cadherin, a novel neuronal adhesion receptor, in the
Drosophila embryonic CNS. Neuron 19, 77-89.
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals andtranscriptional codes. Nat. Rev. Genet. 1, 20-29.
Joesch, M., Schnell, B., Raghu, S. V., Reiff, D. F. and Borst, A. (2010). ON and
OFF pathways in Drosophila motion vision. Nature 468, 300-304.
Jones, B. and McGinnis, W. (1993). A new Drosophila homeobox gene, bsh, isexpressed in a subset of brain cells during embryogenesis. Development 117, 793806.
Katsov, A. Y. and Clandinin, T. R. (2008). Motion processing streams in Drosophila
are behaviorally specialized. Neuron 59, 322-335.
Kimura, Y., Okamura, Y. and Higashijima, S. (2006). alx, a zebrafish homolog of
Chx10, marks ipsilateral descending excitatory interneurons that participate in theregulation of spinal locomotor circuits. J. Neurosci. 26, 5684-5697.
Kitamoto, T. (2001). Conditional modification of behavior in Drosophila by targeted
expression of a temperature-sensitive shibire allele in defined neurons. J.
Neurobiol. 47, 81-92.
Kosman, D., Small, S. and Reinitz, J. (1998). Rapid preparation of a panel ofpolyclonal antibodies to Drosophila segmentation proteins. Dev. Genes Evol. 208,290-294.
Kurant, E., Pai, C. Y., Sharf, R., Halachmi, N., Sun, Y. H. and Salzberg, A. (1998).
Dorsotonals/homothorax, the Drosophila homologue of meis1, interacts withextradenticle in patterning of the embryonic PNS. Development 125, 1037-1048.
Leber, S. M. and Sanes, J. R. (1995). Migratory paths of neurons and glia in theembryonic chick spinal cord. J. Neurosci. 15, 1236-1248.
Lee, C. H., Herman, T., Clandinin, T. R., Lee, R. and Zipursky, S. L. (2001). Ncadherin regulates target specificity in the Drosophila visual system. Neuron 30,
437-450.

RESEARCH ARTICLE

993

Lee, T. and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies
of gene function in neuronal morphogenesis. Neuron 22, 451-461.
Llimargas, M. and Casanova, J. (1997). ventral veinless, a POU domaintranscription factor, regulates different transduction pathways required for tracheal
branching in Drosophila. Development 124, 3273-3281.
Luan, H., Peabody, N. C., Vinson, C. R. and White, B. H. (2006). Refined spatial
manipulation of neuronal function by combinatorial restriction of transgene
expression. Neuron 52, 425-436.
McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K. and Davis, R. L. (2003).
Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302, 17651768.
Meinertzhagen, I. A. and Hanson, T. E. (1993). The development of the optic
lobe. In The Development of Drosophila melanogaster (ed. M. Bate and A.
Martinez-Arias), pp. 1363-1491. Cold Spring Harbor Laboratory, NY: Cold Spring
Harbor Laboratory Press.
Morante, J. and Desplan, C. (2008). The color-vision circuit in the medulla ofDrosophila. Curr. Biol. 18, 553-565.
Nassif, C., Noveen, A. and Hartenstein, V. (2003). Early development of the
Drosophila brain: III. The pattern of neuropile founder tracts during the larval
period. J. Comp. Neurol. 455, 417-434.
Nern, A., Zhu, Y. and Zipursky, S. L. (2008). Local N-cadherin interactions mediate
distinct steps in the targeting of lamina neurons. Neuron 58, 34-41.
Nobrega-Pereira, S. and Marin, O. (2009). Transcriptional control of neuronalmigration in the developing mouse brain. Cereb. Cortex 19 Suppl. 1, i107-i113.
Pai, C. Y., Kuo, T. S., Jaw, T. J., Kurant, E., Chen, C. T., Bessarab, D. A., Salzberg,
A. and Sun, Y. H. (1998). The Homothorax homeoprotein activates the nuclear
localization of another homeoprotein, extradenticle, and suppresses eye
development in Drosophila. Genes Dev. 12, 435-446.
Rieckhof, G. E., Casares, F., Ryoo, H. D., Abu-Shaar, M. and Mann, R. S. (1997).
Nuclear translocation of extradenticle requires homothorax, which encodes anextradenticle-related homeodomain protein. Cell 91, 171-183.
Rister, J., Pauls, D., Schnell, B., Ting, C. Y., Lee, C. H., Sinakevitch, I., Morante,J., Strausfeld, N. J., Ito, K. and Heisenberg, M. (2007). Dissection of theperipheral motion channel in the visual system of Drosophila melanogaster.
Neuron 56, 155-170.
Robinson, I. M., Ranjan, R. and Schwarz, T. L. (2002). Synaptotagmins I and IV
promote transmitter release independently of Ca(2+) binding in the C(2)A domain.
Nature 418, 336-340.
Sanes, J. R. and Zipursky, S. L. (2010). Design principles of insect and vertebrate
visual systems. Neuron 66, 15-36.
Sato, M., Umetsu, D., Murakami, S., Yasugi, T. and Tabata, T. (2006). DWnt4
regulates the dorsoventral specificity of retinal projections in the Drosophilamelanogaster visual system. Nat. Neurosci. 9, 67-75.
Sepp, K. J. and Auld, V. J. (1999). Conversion of lacZ enhancer trap lines to GAL4lines using targeted transposition in Drosophila melanogaster. Genetics 151, 10931101.
Solecki, D. J., Govek, E. E., Tomoda, T. and Hatten, M. E. (2006). Neuronalpolarity in CNS development. Genes Dev. 20, 2639-2647.
Struhl, G. and Basler, K. (1993). Organizing activity of wingless protein in
Drosophila. Cell 72, 527-540.
Takemura, S. Y., Lu, Z. and Meinertzhagen, I. A. (2008). Synaptic circuits of the
Drosophila optic lobe: the input terminals to the medulla. J. Comp. Neurol. 509,
493-513.
Thor, S., Andersson, S. G., Tomlinson, A. and Thomas, J. B. (1999). A LIMhomeodomain combinatorial code for motor-neuron pathway selection. Nature
397, 76-80.
Ting, C. Y., Yonekura, S., Chung, P., Hsu, S. N., Robertson, H. M., Chiba, A. and
Lee, C. H. (2005). Drosophila N-cadherin functions in the first stage of the twostage layer-selection process of R7 photoreceptor afferents. Development 132,
953-963.
Tix, S., Eule, E., Fischbach, K. F. and Benzer, S. (1997). Glia in the chiasms andmedulla of the Drosophila melanogaster optic lobes. Cell Tissue Res. 289, 397409.
Toriya, M., Tokunaga, A., Sawamoto, K., Nakao, K. and Okano, H. (2006).
Distinct functions of human numb isoforms revealed by misexpression in theneural stem cell lineage in the Drosophila larval brain. Dev. Neurosci. 28, 142155.
Venken, K. J., He, Y., Hoskins, R. A. and Bellen, H. J. (2006). P[acman]: a BAC
transgenic platform for targeted insertion of large DNA fragments in D.
melanogaster. Science 314, 1747-1751.
Wong, A. M., Wang, J. W. and Axel, R. (2002). Spatial representation of theglomerular map in the Drosophila protocerebrum. Cell 109, 229-241.
Yasugi, T., Umetsu, D., Murakami, S., Sato, M. and Tabata, T. (2008). Drosophila
optic lobe neuroblasts triggered by a wave of proneural gene expression that isnegatively regulated by JAK/STAT. Development 135, 1471-1480.
Zhu, Y., Nern, A., Zipursky, S. L. and Frye, M. A. (2009). Peripheral visualcircuits functionally segregate motion and phototaxis behaviors in the fly. Curr.
Biol. 19, 613-619.

DEVELOPMENTThe Drosophila visual center development