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Proc. Arthropod. Embryol. Soc. Jpn. 48, 1–11 (2017)Ⓒ 2017 Arthropodan Embryological Society of Japan
ISSN 1341–1527[REVIEW]

Proximodistal Development of the Leg in Drosophila–Creation of New Developmental Fields by Gene Regulatory
Interaction and Tissue Growth–*
Tetsuya KOJIMA
Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, 5–1–5 Kashiwanoha, Kashiwa,
Chiba 277–8562, Japan
E-mail: tkojima@k.u-tokyo.ac.jp

Abstract
The leg development in Drosophila melanogaster has been studied extensively at a molecular level during past dicades andgiving many important insights into development and evolution of appendages. In this review, recent advances that greatly improve
our understanding of the molecular mechanism of leg development are briefly described.

Introduction
Arthropods have legs consisting of several segments
connected by flexible joints along the proximodistal (PD) axis,
as the phylum name stands for. The number and arrangementof segments are basically conserved within a subphylum andsimilar even between subphyla, in spite of the fact that theoverall morphology is sometimes highly diverse between
species (Snodgrass, 1935). This makes arthropod legs haveattracted a considerable attention from a viewpoint of
evolutionary biology. Furthermore, legs are also a good model
system in the view of developmental biology, since interplay
between tissue growth and patterning, which is a fundamental
process of tissue development, can be illustrated.
Generally, it is thought that a tissue is patterned, or
subdivided into several distinct regions, by region-specificexpression of patterning genes according to the positional
information determined by morphogens (Lawrence andStruhl, 1996). However, the tissue pattering merely occurs at
once but rather, new regions are added progressively withgrowth of the tissue. In addition, expression pattern ofpatterning genes is changed dramatically as development
proceeds. Thus, it is important to know how pattering andtissue growth are integrated, in other words, how patterning
gene expression influences tissue growth and how tissue
growth regulates patterning gene expression.
The leg development has been studied extensively at amolecular level using a well-established model insect,
Drosophila melanogaster, and a substantial amount ofknowledge has been accumulated during past decades(previously reviewed in detail by Kojima, 2004 and Estella etal., 2012). Especially, several recent researches clearly
demonstrate that although morphogens are indeed importantto set the initial state of patterning gene expression, additionof new regions occurs de novo through changes in thepatterning gene expression by the regulatory interaction
between them and the interplay between patterning gene
expression and tissue growth. In this review, these new
findings are briefly described.
Overview of the Drosophila leg development
The Drosophila leg has six segments, which are, thecoxa, trochanter, femur, tibia, tarsus and pretarsus, in a
proximal to distal direction, as with other insect species (Fig.
1, lower panel). These segments are called “true” segments
and their number and arrangement are generally conserved inalmost all insect species. Unlike the true segments, the tarsus
is often subdivided further into several tarsal segments. The
number of tarsal segments is diverse from species to species,
with the number generally ranging from one to five
(Snodgrass, 1935). The Drosophila leg has five tarsal
segments, which are designated as tarsal segment 1 (ta1) totarsal segment 5 (ta5) (Fig. 1, lower panel).
Drosophila is a complete holometabolous insect andadult structures, such as antennae, wings and legs, arederived from primordial tissues called imaginal discs (eyeantennal discs, wing discs and leg discs, respectively). Cells
for an imaginal disc are determined in the epithelium of theembryo during embryogenesis and invaginate from the* This article, which was accepted in 2013 and should have been published in 2014, was printed in 2017 being much delayed due to various circumstances.

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epidermis to form a sac-like structure composed of a sheet ofmono-layered epithelial cells. Imaginal discs proliferate
within a larva during three larval stages without contributing
to larval structures and terminally differentiate into adultstructures during the pupal stage (Cohen, 1993; Fristrom andFristrom, 1993).
By the genetic analysis, the leg disc is thought to consistof only about 10–30 cells at its birth. Subsequently, cells in theleg disc proliferate to contain over 10,000 cells by the late
third instar stage (Cohen, 1993). Morphologically, two types ofcells are recognized in the leg disc: large, flat and squamous
cells of the peripodial membrane that contributes to the adultbody wall and thick, columnar cells of the disc epithelium thatdifferentiate into the leg proper (Fig. 1, upper panel; Fristrom
and Fristrom, 1993). The disc epithelium is morphologically a“flat” sheet by the beginning of the third instar stage, but
subsequently folded over concentrically several times andbecomes no more “flat” in its appearance, although it remains
to be a mono-layered sheet. By the late third instar stage,
three major foldings and folds are recognized (in this review,the “fold” refers to a protruding-, or mountain-, part of thedisc). The pretarsus and ta5–ta3 come from the most central
fold, ta2–distal part of the tibia from the second one (mid fold),the proximal tibia and the femur from the third fold (peripheralfold), and the more proximal parts are derived from the more
peripheral parts (Fig. 1; Kojima et al., 2000). After the
puparium formation, the disc epithelium begins to telescopeout from its center and elongates by cell rearrangement
during the pupal stage to form the adult leg (Fig. 1; Condic etal., 1991; Fristrom and Fristrom, 1993). Since distal segmentsof the adult leg are derived from central portions of the leg
disc and more proximal segments from more peripheral
portions, each leg segment is determined concentrically by
the concentric expression of patterning genes on the leg disc.
Specification of regions corresponding to each segment iscompleted by the late third instar stage and each leg segment
becomes visible morphologically by the puparium formation.
Determination of precursor cells for the adult leg during
embryogenesis
It has been suggested that arthropod legs are primarily
subdivided along the PD axis to the coxopodite, the proximal
portion that is a simple outgrowth of the body wall, and thetelopodite, the distal portion that are movable (Snodgrass,
1935; reviewed by Boxshall, 2004). In insects, the coxopodite
includes the coxa and the telopodite corresponds to all themore distal leg segments. Studies on Drosophila legs andappendages in other arthropod species indicate that thetelopodite is specified by a homeobox gene, Distal-less (Dll),and proper formation of the coxopodite requires two
homeobox genes, homothorax (hth) and extradenticle (exd), andtwo genes encoding zinc-finger-containing transcription
factors, escargot (esg) and teashsirt (tsh) (Cohen et al., 1989;
Cohen and Jürgens, 1989; Cohen, 1990; Fasano, et al., 1991;
Whiteley et al., 1992; Rauskolb et al., 1993, 1995; González-Crespo and Morata, 1995, 1996; Gorfinkiel et al., 1997; Gotoand Hayashi, 1997, 1999; Panganiban et al., 1997; Reickhof etal., 1997; Campbell and Tomlinson, 1998; González-Crespo etal., 1998; Azpiazu and Morata, 2002; reviewed by Pnaganiban
and Rubenstein, 2002).
The first sign of the leg development is the expression of
Dll in ventrolateral regions at each side of thoracic segments
at the mid embryonic stage, starge 11 (Fig. 2, upper-left panel;
Cohen, 1990; Vachon et al., 1992; González-Crespo et al.,1998). Genetic evidence indicated that at this stage, Dll
expression domain contains precursor cells for the wing or
haltere disc and for the larval sensory organ called the Keilinʼsorgan, in addition to those for the leg disc, and thus, the initial
Dll domain is called the limb primordium (Wieschaus andGehring, 1976; Cohen et al. 1993; Kubota et al. 2000). By stage
14, cells dorsally situated in the initial Dll domain migrate
dorsally, cease Dll expression and eventually form the wing or
haltere disc (Cohen et al., 1993; Fuse et al., 1996; Kim et al.,1996). Before the appearance of the initial Dll expression, hthis expressed in almost all cells in the thoracic segments(González-Crespo et al., 1998). After that, the central region of
Dll domain gradually ceases hth expression, leaving cells with
Dll but no hth expression, by stage 14 (Kubota et al., 2003;
González-Cresop et al., 1998). Since Dll and hth specify the
telopodite and coxopodite, respectively, it was thought
previously that this central, Dll-only domain corresponds tothe telopodite and surrounding hth-expressing cells, which
also express esg, to the coxopodite. This view was revised byrecent findings, however, from analyses of regulatory regionsof Dll and the cell lineage tracing experiments (Estella et al.,2008; McKey et al., 2009; Galindo, et al., 2011).
Extensive studies on regulatory regions of Dll has
revealed several enhancers existing upstream anddownstream of its coding region, which drive leg-related Dll
expression (Vachon et al., 1992; Estella et al., 2008; Galindo etal., 2011). These enhancers are activated differentially andsequentially (Fig. 2). First to be activated is an enhancer called
Dll304 (Fig. 2, left panel), which regulates Dll expression in
the limb primordia (Vachon et al., 1992). This enhancer iscontrolled by a combination of Wingless (Wg), Decapentaplegic(Dpp) and epidermal growth factor receptor (EGFR)
signalings. Wg is a Wnt family member and Dpp is a TGF-betafamily member (for review of these signalins, see Barolo andPosakony 2002). At the mid embryonic stage, Dll304 is
activated by Wg but repressed dorsally by Dpp signaling andventrally by EGFR signaling (Cohen, 1990; Goto and Hayashi,1997). Thus, Dll304 activity is restricted to the ventrolateralregion. In addition, Hox genes determining identities ofabdominal segments, Ultrabithorax (Ubx), abdominal-A(abd-A) and Abdominal-B (Abd-B), repress Dll304 and thus,
its activity is restricted to thoracic segments (Vachon et al.,
1992; Castelli-Gair and Akam, 1995; Gebelein et al., 2002,
2004). Dll304 activity is transient and with its disappearance,
an enhancer called DllLP (Leg Primordium) is activated in the
precursor cells for the Keilinʼs organ and leg disc throughLEG DEVELOPMENT IN DROSOPHILA

activation by all of the Wg, Dpp and EGFR signalings (Fig. 2,
middle pannel; Galindo et al., 2011). Since DllLP is not
activated in precursor cells for the wing and haltere discs,
they lose Dll expression with the disappearance of Dll304
activity. Shortly after the DllLP activation, two otherenhancers called DllLT (Leg Trigger) and DKO (Distal-less
Keilinʼs Organ) becomes active in cells with DllLP activity(Fig. 2, right panel; Estella et al., 2008; Galindo et al., 2011).
DKO is activated in the central cells destined to the Keilinʼsorgan and DllLT in cells surrounding it (Mckay et al., 2009).
DllLT is activated by positive inputs from Dll protein itself andhigh levels of Wg and Dpp signalings (Estella et al., 2008).
Although DllLP activity declines during first to second instar
stages, DllLT activity persists and with autoregulatory
modules called DllM (Maintenane) and DllLL (Leg Late),
regulates all aspects of Dll expression during all remaining
stages (Estella et al., 2008; Galindo et al., 2011).
Cell lineage analysis of cells with Dll304, DllLT or DKO
activity showed a fine-scale fate map of the limb primordium(Fig. 2; McKay et al., 2009). Cells in which Dll304 is activatedat mid embryonic stage (stage 11) actually contributed to alldescendants of the limb primordium, that is, the wing or
haltere disc, the Keilinʼs organ as well as the proximal anddistal portions of the leg disc (Fig. 2, left panel). DKO is
activated in the Dll-only domain and these cells indeed
differentiated into the Keilinʼs organ (Fig. 2, right panel),consistent with the finding from the detailed analysis of the
Keilinʼs organ development (Bolinger and Boekhoff-Falk,2005). Most strikingly, DllLT is first activated in cells
surrounding the Dll-only domain and cells with DllLT activity
contributed to the entire telopodite region of the leg disc (Fig.
2, right panel). Initially, these cells also express hth and esg but
lose their expression later and proliferate to form thetelopodite region without their expression. In addition, thecoxopodite region of the leg disc was demonstrated to derivefrom cells in the vicinity of the Dll-only domain but without
DllLT activity (only expressing hth and esg but not Dll) (Fig. 2,
right panel; McKay et al., 2009).
These results provide an important implication that
although Dll is continuously expressed in precursor cells forthe telopodite during embryogenesis, enhancers regulating
its expression change from moment to moment. In otherwords, the precursor cells for the telopodite are determinedthrough several changes in their state.
Subdivision of the telopodite along the PD axis during
larval stages
As described above, the leg disc consists of only two
regions at the initial stage of the leg disc determination: theDll-expressing telopodite and the hth-expressing coxopodite.
After that, however, a new region expressing dachshund (dac)but neither Dll nor hth emerges between Dll and hth domainsby the beginning of the third instar stage (Abu-Shaar andMann, 1998). dac encodes a transcription factor required forthe formation of the medial portion of the leg (Mardon et al.,31994). Subsequently, Dll and dac expression becomesoverlapping with each other and an additional new region
expressing both Dll and dac is formed (Lecuit and Cohen,
1997; Abu-Shaar and Mann, 1998). Consequently, thetelopodite region of the leg disc is now subdivided into daconly domain, Dll + dac domain and Dll-only domain, in a
proximal (peripheral) to distal (central) direction (Fig. 3).
To achieve this subdivision, Wg and Dpp have beenthought to act cooperatively as morphogens (Diaz-Benjumea
et al., 1994; Lecuit and Cohen, 1997). Since Wg and Dpp is
expressed in the ventral and dorsal sides of the leg disc,respectively (Fig. 3, left panel), it seems that the central
portion, near the contact point between their expression
domains, receives high levels of Wg + Dpp signaling andprogressively more peripheral regions are exposed to lower
levels of Wg + Dpp signaling. It has been thought that highlevels of Wg + Dpp signaling induce Dll expression but
repress dac expression and intermediate levels activate dac
but not Dll expression, whereas lower levels activate neither
gene and allows expression of the coxopodite genes, such as
hth and esg (Lecuit and Cohen, 1997; Abu-Shaar and Mann,
1998; González-Crespo et al., 1998; Wu and Cohen, 1999).
Recently, however, this model has been revised remarkably.
The analysis on an enhancer called dacRE (Ring
Enhancer), which regulates dac expression in the leg disc,indicated that although high levels of Wg + Dpp signaling
activity repress dac expression as has been thought, dac
expression is not activated by intermediate levels of Wg +Dpp signaling. Rather, Dll activates dac expression by directly
binding to dacRE. In addition, dacRE is repressed also bytranscription factors expressed more distally than dac, such as
BarH1 and BarH2 (collectively referred to as Bar hereafter;
see below) (Giorgianni and Mann 2011). According to theseresults and those from the analyses of enhancers for Dll
expression, the following scenario is proposed (Fig. 3;
Giorgianni and Mann, 2011; Estella et al., 2012):
By the early second instar stage, Dll is expressed
through the activation of DllLT by high levels of Wg + Dpp
signaling, which counteracts the activation of dacRE by Dll
and remains dac unexpressed (Fig. 3, left panel). Towards thelate second instar stage, growth of the disc drives some Dllexpressing cells outside the region with high levels of Wg +Dpp signaling. This leads to the activation of dacRE by Dll andthe initiation of dac expression. Since dac has a repressive
activity on Dll expression (Abu-Shaar and Mann, 1998), thesecells lose Dll expression (Fig. 3, middle panel). After thefading of Dll expression, dac expression may be maintained by
the autoregulation and thus, the dac-only domain is formed,
maintained and enlarged with the growth of the disc (Fig. 3,
right panel). At the early third instar onwards, further growthof the disc makes more cells move out of the central region. In
these cells, levels of Wg + Dpp signaling are not high enoughand DllLT loses its activity. Instead, however, autoregulatory
elements, DllM and DllLL, maintain Dll expression in thesecells. Since Dll activates dac expression through the activation

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of dacRE, this leads to the stable formation of the Dll + dac
domain (Fig. 3 right panel). The distal extent of the Dll + dac
domain is determined through the repression of dacRE by
distally expressed transcription factors, such as Bar (Fig. 3,
right panel).
As described above, medial regions in the telopodite isformed mainly by the regulatory interaction between
patterning genes and tissue growth but not by intermediatelevels of morphogen signaling. This means that a major role ofmorphogens is to set the initial state of the regulatoryinteraction cascade and once the cascade is triggered, newregions are formed de novo by interplay between patterning
genes themselves and tissue growth.
Formation and determination of five tarsal segments
and pretarsus
During the third instar stage, the Dll-only domain isfurther subdivided into five tarsal segments and the pretarsus.
Considerable numbers of patterning genes are known to be
expressed within this domain in a region-specific manner(Fig. 4B). At the late third instar stage, the future pretarsal
region expresses aristaless (al) and clawless (cll; also known asC15), encoding homeodomain transcription factors, as well as
Lim1, encoding a LIM-homeodomain transcription factor(Campbell et al., 1993; Schneitz et al., 1993; Lilly et al., 1999;
Pueyo et al., 2000; Tsuji et al., 2000). Just proximal to thepretarsal region, another genes encoding homeodomaintranscription factors, Bar, is expressed strongly in the future
ta5 region and weakly in the ta4 region (Kojima et al., 1991;
Higashijima et al., 1992; Kojima et al., 2000). The ta5 region
also expresses nubbin (nub), which encodes a POUhomeodomain transcription factor, while the ta4 region
expresses apterous (ap), a LIM-homeodomain transcription
factor (Billin et al., 1991; Cohen et al., 1992; Rauskolb andIrvine, 1999; Kojima et al., 2000). trachealess (trh), encoding abHLH-PAS type transcription factor, is expressed in bothpretarsal and ta5 regions (Isaac and Andrew, 1996; Wilk et al.,
1996; Tajiri et al., 2007). Furthermore, dac is expressedstrongly in the ta1 region and weakly in the ta2 region (Mardon
et al., 1994; Lecuit and Cohen, 1997; Natori et al., 2012). In
addition, bric à brac 1 (bab1) and bric à brac 2 (bab2), which
encodes BTB/POZ domain containing transcription factors,
are expressed from the distal region of ta1 to the ta4 region
with their expression stronger towards ta2 and ta3 regions(Godt et al., 1993; Couderc et al., 2002). The process of tarsal
segment and pretarsus formation has been illustrated in great
detail in relation to the regulation and function of thesepatterning genes.
Morphogen signaling for the tarsal and pretarsal
development
In the process of tarsal and pretarsal development,
EGFR signaling is acting as a morphogen regulating
patterning gene expression (Fig. 4A). Ligands for EGFR are
produced by cells at the most central region of the leg discaccording to high levels of Wg + Dpp signalings. Theyemanate from the most central region and set a concentric
gradient of EGFR signaling activity in a central (distal) to
peripheral (proximal) direction (Campbell, 2002; Galindo etal., 2002; Galindo et al., 2005).
Precise establishment of the pretarsal region
At the early third instar stage, when al, cll and Bar
expression initiates, there is an overlap between theirexpression domains. But later, by the mid third instar stage,
the overlap is resolved and a sharp boundary is formed
between them (Kojima et al., 2000). During this refinement
process, al and cll repress Bar expression cooperatively in thepretarsal region, whereas their expression is repressed by
Bar in the ta5 region (Fig. 4B; Campbell, 2005; Kojima et al.,2005). The expression of Lim1, which is required for thesufficient levels of al and cll expression, is activated by thecooperative function of al and cll, while it is repressed by Bar(Tsuji et al., 2000). The expression of Bar in the ta5 isstrengthened by autoactivation (Kojima et al., 2000). In
addition, trh potentiates the repressive activity of al and cll on
Bar expression in the pretarsal region, while it also facilitates
the autoactivation of Bar expression in the ta5 region (Tajiri etal., 2007). In this manner, the pretarsal region is precisely
determined through the induction of patterning genes in an
approximate region by the morphogen signaling, followed by
the refinement of their expression domains throughregulatory interaction between them (Fig. 4B). Interestingly,
it was reported that al expression in the pretarsal region and
Bar expression in the ta5 region is unchanged by the loss of
EGFR signaling after the mid third instar stage (Campbell,2002). This suggests that their expression no more requires
the input from the morphogen signaling. Therefore, themorphogen only determines initial, rough expression domainsof patterning genes and once rough domains are set, theregulatory interaction between them establishes andmaintains their precise expression domains.
Formation of five tarsal segments
After the puparium formation, the leg disc is partiallyelongated and each tarsal segment becomes morphologicallyvisible (Fig. 1 middle panel). These five segments are
prefigured by the expression of several patterning genes by
the late third instar stage (Fig. 4B). As described below,
however, the expression of the patterning genes changes
dramatically during the third instar stage (Fig. 5). For example,
Bar is strongly expressed in the ta5 region and weakly in theta4 region, while dac is expressed weakly in the ta2 region andstrongly in the ta1 region. Neither gene is expressed in the ta3
region (Mardon et al., 1994; Lecuit and Cohen, 1997; Kojima etal., 2000; Natori et al., 2012). At the early third instar stage,
however, their expression is homogeneous within eachdomain and abuts each other (Fig. 5, upper-left panel). Then, agap region devoid of both Bar and dac expression emerges
between their domains (Fig. 5, upper-middle panel). After theLEG DEVELOPMENT IN DROSOPHILA

appearance of the gap, the central folding, or the central foldformation, occurs just outside the Bar domain (Kojima et al.,
2000; Natori et al., 2012). As the central folding deepens, or thecentral and second folds (i.e. the tarsal region) grow, the gap
region continues to expand into the proximal part of thecentral fold due to the progressive repression of Bar
expression (Fig. 5, upper-right panel). Around the mid third
instar stage, Bar expression in the ta5 region is strengthened

5(Fig. 5, lower-left panel; Kojima et al., 2000) and at very late
third instar stage, weak dac expression appears in the ta2
region (Fig. 5, lower-right panel; Natori et al., 2012).
Eventually, the tarsal region is subdivided into five regions
corresponding to each tarsal segment. Therefore, at mostonly two regions exist in the tarsal region initially andsubdivision into five segments occurs de novo afterward. The
complex but ingenious regulatory interaction between

Fig. 1 Schematic representation of the adult leg and leg disc development. Dorsal is to the top. Distal is towards right except for the leftmost figure in the top panel. Regional relationships are represented by colors.
de: disc epithelium, pm: periodial membrane.

Fig. 2 Formation of the leg disc during embryogenesis. Upper panel represents changes in the usage of Dll enhancers and their regulation.
Thin arrows and T-bars indicate activation and repression, respectively. Genes expressed in the telopodaite and coxopodite region
are shown below the leg disc. Progenitors of each Dll-expressing cells are shown in lower panel. Lineage relationships are indicatedby colors. Dorsal is to the top except for the Keilinʼs organs and wings.

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Fig. 3 Telopodite subdivision along the PD axis during larval stages. Arrows and T-bars indicate positive and negative regulation,
respectively. Gray indicates inactive state. Size and color depth of letters represent a combined activity of Wg and Dpp signalings.
Dorsal is to the top.

patterning genes and growth of the tarsal region again play
important roles in this process (Figs. 4B and 5).
In addition to the patterning genes described above,
genes transiently expressed in the medial tarsal region, suchas rotund (rn), spineless (ss) and tarsal-less (tal; also known aspolished rice, pri) play key roles. rn and ss encode a zinc-finger
transcription factor and a bHLH-PAS transcription factor,
respectively, while tal encodes four short peptides known toact cell-non-autonomously (Duncan et al., 1998; St Pierre etal., 2002; Kozu et al., 2006; Galindo et al., 2007; Kondo et al.,2007; Pueyo and Couso, 2008). Bar activates tal expression
cell-non-autonomously, whereas attenuates it cellautonomously (Pueyo and Couso, 2008). Through thisregulation, tal expression starts weakly in nearly the same
domain as Bar at early stages but becomes strongly expressedaround the Bar domain later (Fig. 5; Pueyo and Couso, 2008;
Natori et al., 2012). Since ss expression is activated by tal cellnon-autonomously, ss is expressed in the same timing as, but
in a larger domain than, tal (Fig. 5; Pueyo and Couso, 2008;
Natori et al., 2012). rn expression is activated by tal directly
and also indirectly through the activation of ss expression(Pueyo and Couso, 2008; Natori et al., 2012). By the late third
instar stage, ss and rn expression fades with the cessation oftal expression (Fig. 5, lower panels; Duncan et al., 1998; St
Pierre et al., 2002; Pueyo and Couso, 2008).
With the initiation of its expression, Bar represses dac
expression to limit the distal extent of the dac domain (Kojima
et al., 2000; Giorgianni and Mann, 2011). Conversely, theproximal extent of the Bar domain is determined by theconcerted action of dac and tal (Pueyo and Couso, 2008). Thus,
Bar and dac domains come to abut each other (Fig 5, upperleft panel). Since tal and ss are already expressed at this earlystage (Pueyo and Couso, 2008; Natori et al., 2012), rn is readyto be expressed. However, rn expression is blocked by nub,which is expressed by EGFR signaling in a broad domain thatspans all the tarsal and pretarsal regions (Fig. 5, upper-leftpanel; Natori et al., 2012). Then, the growth of the tarsal region
possively drives some cells outside the levels of EGFR
signaling required for the activation of nub expression, leading
to an emersion of a region not expressing nub. This results in
the initiation of rn expression in the region devoid of nub
expression (Fig. 5, upper-middle panel; Natori et al., 2012).
Afterwards, by the late third instar stage, the region without
nub expression expands continuously with the growth of thetarsal and pretarsal regions, restricting nub expression
distally to the ta5 region and proximally to the distal tip of thetibia (Figs. 4B and 5, lower panel; Rauskolb and Irvine, 1999;
Natori et al., 2012). In association with this, rn expression
domain also expands. Since rn has an activity of repressing
Bar, the expansion of the rn domain into the proximal part ofthe central fold results in the repression of Bar expression
there, leading to the formation of the ta3 region (Fig. 5, upperright panel; Natori et al., 2012).
From the mid third instar stage onward, the Bar domain
starts overlapping with the expanding rn expression. This isdue to the initiation of ap expression in cells at the proximal
part of the Bar domain (Fig. 5, lower-left panel). ap expressionis activated by the concerted action of the cell-autonomous
function of Bar and cell-non-autonomous function of tal, while
repressed in the distal region of the Bar domain by Notch
signaling (Campbell, 2005; Pueyo and Couso, 2008; Natori etal., 2012). ap renders Bar refractory to the repression by rn,
so that Bar expression is no more repressed. This leads to theformation of the ta4 region, in which weak Bar expression andap expression is observed at the late third instar stage (Fig. 5,
lower panels; Natori et al., 2012).
The strong expression of Bar in the ta5 region at the late
third instar stage is regulated by a dedicated enhancer calledLEG DEVELOPMENT IN DROSOPHILA

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Fig. 4 Subdivision of the Dll-only domain into the pretarsus and five tarsal segments during the third instar stage. A. From the onset of thethird instar stage, ligands for EGFR signalins are produced and secreted at the most centar of the disc by Wg and Dpp signalings, seta center to peripheral gradient of EGFR signaling activity, and induce the region-specific expression of patterning genes, such as al,cll, Bar. B. Relationship between adult leg segments and expression domains of several patterning genes at the late third instar
stage (upper panel) and regulatory interaction between them (lower panel). Arrows and T-bars indicate positive and negative
regulation, respectively. Distal is to the right.

Fig. 5 Expression changes in and some key regulatory interactions betwee patterning genes during formation of five tarsal segments
during the third instar stage. Thin arrows and T-bars indicate positive and negative interaction, respectively. Depth of colors
represents expression levels and light gray indictes a repressed state. Distal to the right.

ta5-enhancer (Kojima et al., 2000; Kozu et al., 2006). Althoughta5-enhancer is activated by the function of Bar itself and trh(Tajiri et al., 2007), and they are already expressed at the early
third instar stage, the activity of ta5-enhancer is repressed
directly by ss at this early stages. The cessation of ss
expression by the late third instar stage releases the activityof ta5-enhancer. This leads to the strong expression of Bar in
the distal part of its domain and thus, the specification of theta5 region (Fig. 5, lower panels; Kozu et al., 2006).

It has been suggested that the ta2 region is derived from
cells in the initial gap between Bar and dac domains before thecentral fold formation and cells in the most proximal part ofthe early central fold (Kojima et al., 2000; Natori et al., 2012).
The initial gap appears to be formed mainly by the cessation of
Bar expression. Although rn is already expressed in the initialgap, however, rn is dispensable for its formation (Natori et al.,2012). At present, the mechanism of Bar repression here is
unknown as well as the mechanism of weak dac expression in8

T. KOJIMA

the ta2 region at the late third instar stage (Fig. 5, uppermiddle and lower-right panels).
Other than the patterning genes described above, lines(lin) and odd-skipped family genes, odd-skipped (odd), brotherof odd with entrails limited (bowl), sister of odd and bowl (sob)and dramstick (drm), are also implicated in the subdivision andspecification of the tarsal region (de Celis Ibeas and Bray
2003; Hao et al., 2003; Greenberg and Hatini, 2009). odd family
genes encode zinc-finger transcription factors and lin encodesa protein that degrades Bowl protein (Green et al., 2002; Hatini
et al., 2005; Greenberg and Hatini, 2009; Del Signore et al.,2012). The activity of Lin is in turn inhibited by Odd, Sob andDrm. Lin protein gradually accumulated in the tarsal region
by the late third instar stage and represses strong dac
expression, while promote ap and weak Bar expression, in the
medial tarsal region through the degradation of Bowl protein(Greenberg and Hatini, 2009). A detailed investigation of theregulatory and functional relationship between this systemand those described above will provide a more perfect pictureof tarsal development.
As described above, the dynamic regulatory interaction
between patterning genes and growth of the tarsal region play
key roles in the subdivision of the tarsal region into each tarsal
segment. This also has an important implication for leg or
appendage evolution and diversity. Even if functions andregulatory relationship between patterning genes areunchanged, small alterations in the timing of patterning gene
expression and in the growth rate of the tissue could lead tochanges in the number and proportion of leg segments. For
example, it is easily imagined that the number and proportionof each tarsal segment can be altered by changes in the timingof the initiation of ap expression and the disappearance of ss
and nub expression. Especially, the alteration in the timing of
nub disappearance might occur by changes in the tissue
growth rate and/or the sensitivity of nub to EGFR signaling,that is, a balance between tissue growth and EGFR signaling
strength. This might be one of mechanisms underlying thediversity in tarsal segmentation among different insect
species.

advances in techniques of gene disruption or genome editing,such as RNAi, TALEN and CRISPER (reviewed by Wei et al.,2013), allows us to analyze not only gene expression but also
gene function in non-model organisms. So, we have a good
opportunity for tackling this issue now.
Acknowledgements: I am deeply thankful to Y. Kobayashi,
K. Tojo, M. Hatakeyama and other core members of
Arthopodan Embryological Society of Japan for giving me an
opportunity to write this review. Several works of T. K.
reviewed here are supported in part by grants from the
Ministry of Education, Culture, Sports, Science andTechnology of Japan and by research grants in natural sciencesfrom the Mitsubishi Foundation to T.K.
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