Temporal Transcription Factors
and Their Targets Schedule the End
of Neural Proliferation in Drosophila
Ce ´dric Maurange,1Louise Cheng,1and Alex P. Gould1,*
1MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
The timing mechanisms responsible for terminating
cell proliferation toward the end of development
remain unclear. In the Drosophila CNS, individual
progenitors called neuroblasts are known to express
a series of transcription factors endowing daughter
neurons with different temporal identities. Here we
show that Castor and Seven-Up, members of this
neuroblast lineages during late neurogenesis. First,
they schedule a switch in the cell size and identity of
neurons involving the targets Chinmo and Broad
Complex. Second, they regulate the time at which
neuroblasts undergo Prospero-dependent cell-cycle
exit or Reaper/Hid/Grim-dependent apoptosis. Both
types ofprogenitortermination require thecombined
action of a late phase of the temporal series and indi-
rect feedforward via Castor targets such as Grainy-
head and Dichaete. These studies identify the timing
mechanism ending CNS proliferation and reveal how
aging progenitors transduce bursts of transcription
factors into long-lasting changes in cell proliferation
and cell identity.
During development, neural stem and progenitor cells generate
distinct cell fates in a birth order-dependent manner. In the de-
veloping mammalian cerebral cortex, functionally distinct cell
layers develop via a stereotypical ‘‘inside-out’’ sequence of neu-
rogenesis. The later a particular neuronal subtype is born, the
more superficial a position it occupies (McConnell, 1988; Rakic,
1974). Individual cortical progenitors utilize a lineage-intrinsic
timing mechanism to generate progeny fates in a predictable
order (Hanashima et al., 2004; Shen et al., 2006). Birth order-de-
pendent mechanisms of neural fate specification have also been
observed in the retina, hindbrain, and spinal cord (reviewed by
Donovan and Dyer, 2005; Kessaris et al., 2001; Livesey and
Cepko, 2001). To maintain the correct proportions of early-
and late-born subtypes in all of these developmental contexts,
it is essential to regulate precisely the time at which neurogene-
sis ceases. Hence, manipulations of cell-cycle genesthat induce
the premature or delayed cell-cycle exit of cortical progenitors,
lead respectively to loss or overproduction of neurons/glia with
late fates (Caviness et al., 2003; Roy et al., 2004). Although sev-
eral factors influencing neural proliferation have been identified,
the underlying molecular timer scheduling the end of progenitor
divisions remains enigmatic.
In Drosophila, neural stem cell-like progenitors called neuro-
blasts divide asymmetrically to self-renew and to generate
differentiated neurons/glia (Betschinger and Knoblich, 2004;
Wang and Chia, 2005; Yu et al., 2006). The homeodomain pro-
tein Prospero (Pros) is kept inactive in neuroblasts, in part
by cortical tethering to Miranda (Mira) such that it becomes par-
titioned at telophase into the smaller daughter—the ganglion
mother cell (GMC) (Hirata et al., 1995; Ikeshima-Kataoka
et al., 1997; Knoblich et al., 1995; Shen et al., 1997; Spana
and Doe, 1995). Prospero enters the nucleus of the GMC,
where it triggers cell-cycle exit after one division has been com-
pleted (Choksi et al., 2006; Li and Vaessin, 2000). Neuroblasts
divide asymmetrically throughout neurogenesis in the embryo
and also, following a temporary intervening quiescent period,
during a second phase of neurogenesis that spans larval and
pupal (postembryonic) stages and contributes ?90% of cells
to the adult CNS (Prokop et al., 1998; Prokop and Technau,
1991; Truman and Bate, 1988). Neuroblasts are known to
generate their repertoire of different neural fates in an invariant
temporal sequence during embryonic neurogenesis (Bossing
et al., 1996; Higashijima et al., 1996; Novotny et al., 2002; Pear-
son and Doe, 2003, 2004; Schmid et al., 1999; Schmidt et al.,
1997). Several studies have now identified five components of
a temporal series responsible for linking neuronal fate with birth
order during early neurogenesis in the embryo. Four of these
components correspond to temporal transcription factors ex-
pressed in a characteristic sequence (Hunchback / Kruppel
/ Pdm / Castor) in neuroblasts (Brody and Odenwald,
2000; Grosskortenhaus et al., 2006; Isshiki et al., 2001; Kamba-
dur et al., 1998). In principle, progression through this series al-
lows one neuroblast to generate embryonic progeny with at
least four different temporal fates. A fifth member of the tempo-
ral series is the nuclear receptor Seven-up (Svp), transiently
expressed in early embryonic neuroblasts and required for the
Hunchback (Hb) / Kruppel (Kr) switch (Kanai et al., 2005;
Mettler et al., 2006). Loss of Svp activity blocks the Hb / Kr
transition such that excess early Hb-dependent fates are
Cell 133, 891–902, May 30, 2008 ª2008 Elsevier Inc. 891
generated at the expense of later embryonic fates (Kanai et al.,
2005; Mettler et al., 2006). Although the molecular basis of tran-
scription factor switching is not yet clear, multiple crossregula-
tory interactions have been observed between temporal tran-
scription factors (Grosskortenhaus et al., 2006; Isshiki et al.,
2001; Kambadur et al., 1998). Consequently, either loss of func-
tion of temporal factors (such as Cas) or their persistent expres-
sion (for example, Hb, Kr, or Pdm) can block progression of the
temporal series (Cleary and Doe, 2006; Grosskortenhaus et al.,
2006; Isshiki et al., 2001; Pearson and Doe, 2003). Intriguingly,
for neuroblast 7-3, it has been observed that artificially main-
taining Hb or Kr expression increases the number of postmitotic
cells within this lineage, at least by the end of embryogenesis
(Isshiki et al., 2001). Together, the temporal series studies raise
three fundamental questions. First, does the temporal series
continue into postembryonic neurogenesis? Second, does it
regulate the proliferative properties of progenitors? And third,
what are its genetic targets and how do they function? To
address these questions in full necessitates analyzing the roles
of the temporal system during the postembryonic phase of
Postembryonic neurogenesis in Drosophila is highly patterned
along the anterior-posterior axis (reviewed by Maurange and
Gould, 2005). In the central brain and thorax, neuroblasts con-
tinue dividing into pupal stages, generating about 100 progeny
each (Bello et al., 2003; Truman and Bate, 1988). Within the cen-
tral abdomen, however, neuroblasts stop dividing in the larva
some 2 days earlier, producing less than 12 progeny each (Bello
the end of neurogenesis is not yet clear in the brain and thorax,
but in the central abdomen it is known to involve a larval burst
of expression of the Abdominal-A (AbdA) Hox/homeotic protein.
This activates the proapoptotic proteins Reaper, Hid, and/or
Grim (RHG), thus inducing death of the neuroblast (Bello et al.,
2003; Peterson et al., 2002). The competence of the neuroblast
from the Grainyhead (Grh) transcription factor (Almeida and
Bray, 2005; Cenci and Gould, 2005).
During postembryonic neurogenesis, temporal neuronal iden-
tities and their upstream regulatory mechanisms have yet to be
characterized in most regions of the CNS. In the mushroom
body and antennal lobe, however, it is known that multiple differ-
ent neuronal identities are generated in sequence during both
embryonic and postembryonic neurogenesis (Jefferis et al.,
movies to dissect the temporal specification mechanism during
postembryonic neurogenesis. We provide evidence that the
onic neurogenesis, where it schedules two important progenitor
transitions in mostif notallneuroblast lineages. First, itpromotes
a temporal switch in the cell size and identity of neuronal prog-
eny. And second, it shuts down progenitor divisions, thus pre-
venting ectopic cell proliferation in the adult CNS. By identifying
four pan-lineage rather than lineage-specific targets of the tem-
the aging of progenitors to changes in proliferation and neuronal
Neuroblasts Sequentially Generate Large Chinmo+
and Small Br-C+Neurons
We first investigated whether distinct temporal subsets of neu-
rons are generated throughout the larval CNS (Figure 1A). We
observed that Chinmo and Broad Complex (Br-C), two BTB-
zincfinger proteins known to beexpressed in thepostembryonic
CNS (Emery et al.,1994; Zhu et al., 2006), aredistributedin com-
neuromeres at the larval/prepupal transition stage at 96 hr (tim-
ings are relative to larval hatching at 0 hr). Chinmo is expressed
by early-born neurons located in a deep layer, whereas Br-C
marks later-born neurons in a largely nonoverlapping and more
superficial layer (Figure 1B). The deep Chinmo+layer comprises
generated postembryonically (Figures 1C and S1A). Thoracic
postembryonic neuroblasts undergo the Chinmo / Br-C switch
at ?60 hr such that they have each generated an average of 15
Chinmo+cells expressing little or no Br-C and 39 Chinmo?Br-C+
cells by 96 hr (Figures S1B and 2D). The Chinmo+and Br-C+
neuronal identities can berecognized as distinct cell populations
on the basis of an ?2-fold difference in cell-body volume. This
equates to an average cell-body diameter for Chinmo+neurons
of 4.5 mm, compared to only 3.6 mm for Br-C+neurons (Figures
1D and 2A). Plotting cell diameter versus deep-to-superficial
position within postembryonic neuroblast clones reveals an
abrupt decrease in neuronal size at the Chinmo / Br-C transi-
tion (Figure 2B). Together, these results provide evidence that
most,ifnotall,postembryonic neuroblastssequentially generate
at least two different populations of neurons. First they generate
large Chinmo+neurons and then they switch to producing
Cas and Svp Regulate the Chinmo+to Br-C+Transition
in Neuronal Size
To begin dissecting the neuronal switching mechanism, we ex-
amined the functions of Chinmo and Br-C but found that neither
factor is required for the transition in cell identity and cell size
(Figures S1C and S1D; data not shown). We next explored
whether a temporal transcription factor series related to the
embryonic Hb / Kr / Pdm / Cas sequence might be in-
volved. Cas is known to be expressed in the larval CNS (Almeida
and Bray, 2005), and we now observe that many different tho-
racic neuroblasts progress through a transient Cas+phase dur-
ing the 30–50 hr time window (Figure S2). We also find that tho-
racic neuroblasts transiently express another member of the
embryonic temporal series, Svp, during a somewhat later time
window, from ?40 to ?60 hr (Figure S2). These results indicate
that postembryonic Cas and Svp bursts are observed in many,
but probably not all, thoracic progenitors and that their timing
varies from neuroblast to neuroblast.
To determine Svp function, we generated thoracic neuroblast
clones homozygous for svpe22, an amorphic allele. In ?53%
of svpe22neuroblast clones induced in the early larva (at
12–36 hr), the Br-C+neuronal identity is completely absent, all
neurons express Chinmo, and there is no sharp decrease in neu-
ronal size (Figures 2A–2E). The proportion of lineages failing to
892 Cell 133, 891–902, May 30, 2008 ª2008 Elsevier Inc.
generate Br-C+neurons rises to ?70% when clones are induced
in the embryo and falls to only ?7% with late-larval (65–75 hr)
induction (Figure 2E). This is consistent with our previous finding
that Svp bursts are asynchronous from neuroblast to neuroblast.
The expression and clonal analyses together demonstrate that
a progenitor-specific burst of Svp is required in many lineages
for the switch from large Chinmo+to small Br-C+neurons.
Thoracic neuroblast lineages homozygous for a strong cas al-
lele, cas24, show no obvious defects in the Chinmo / Br-C tran-
sition when induced at 12–36 hr (Figure 2E). However, as Cas is
expressed in many postembryonic neuroblasts before their first
larval division, it can only be removed by inducing clones during
embryonic neurogenesis. Such cas24clones generate supernu-
merary Chinmo+neurons and completely lack Br-C+neurons
at 96 hr, although this switching phenotype is restricted to only
?16% of thoracic neuroblasts (Figure 2E). Constitutively
expressing Cas blocks the Chinmo / Br-C switch in a similar
manner, with a frequency dependent upon whether thoracic
UAS-cas clones are induced during embryogenesis (?47%), at
early-larval (?10%) or at late-larval (0%) stages (Figures 2C–
2E). This indicates that the response to Cas misexpression
decreases as neuroblasts age. Together, the expression and
loss- and gain-of-function analyses demonstrate that Chinmo
Generate Large Chinmo+and Small Br-C+Neurons
(A) Schematic representation of neuroblasts (circles) in the larval
CNS. There are only three per hemisegment in the central abdo-
men (vm, vl, and dl). The parasagittal plane of section is indicated
(B) Single parasagittal confocal section of the larval CNS at 96 hr
shows that Chinmo+and Br-C+cells occupy largely nonoverlap-
ping deep and superficial layers, respectively.
(C) Single confocal section of a wild-type (wt) thoracic MARCM
clone, induced at 12–36 hr and marked with elav > mCD8::GFP
(mGFP), containing deep Chinmo+and superficial Br-C+neurons
at 96 hr. Note that a subpopulation of Chinmo+neurons also
weakly expresses Br-C and that elav-GAL4C155is not only ex-
pressed in neurons but also in postembryonic neuroblasts (NB)
and GMCs (Bello et al., 2003; Cenci and Gould, 2005).
(D) Histogram showing the distribution of cell body diameters
(f, in mm) for Chinmo+and Br-C+neurons (n = 307 neurons). On
the right is a schematic representation of a postembryonic clone
at 96 hr, containing large early-born Chinmo+neurons (red), small
late-born Br-C+neurons (orange) and a large neuroblast.
and Br-C are negative and positive targets, respec-
tively, of Cas and Svp. They also strongly suggest
that progression through transient Cas+and Svp+
states permits many postembryonic neuroblasts to
switch from generating large to small neurons.
Most Neuroblasts Shut Down via Pros-
Dependent Cell-Cycle Exit, Not Apoptosis
To investigate whether Cas and Svp regulate neural
proliferation as well as neuronal fates, we first identi-
fied the effector mechanism ending neurogenesis in
the central brain and thorax. In these regions, most
man and Bate, 1988). Correspondingly, neurogenesis in all re-
gions of the wild-type CNS ceases before the adult fly ecloses
such that no adult neuroblasts are detected (Ito and Hotta,
1992). In contrast to the central abdomen, blocking cell death
by removing RHG activity in the central brain and thorax does
not prevent or delay pupal neuroblast disappearance (Fig-
roblasts at?120hrreveal an atypical mitosis thatis muchslower
than at ?96 hr, producing two daughters of almost equal size
(Figure 3A; Movies S1 and S2). This is largely accounted for by
a reduction in the average diameter of neuroblasts from 10.4 mm
at96hrto 7mmat120hr,asGMCsizedoes notvarysignificantly
during this time window (Figure 3B). The end of this atypical
progenitor mitosis temporally correlates with reduced numbers
of Mira+cells and disappearance of the M phase marker phos-
division of the neuroblast (Figure S3B).
We next addressed whether late changes in basal complex
components might underlie loss of neuroblast self-renewal. At
120 hr, we find that Mira becomes delocalized from the cortex
to the cytoplasm and nucleus of many interphase neuroblasts
(Figure 3C). In metaphase neuroblasts, Mira fails to localize to
the basal side of the cortex, although it does selectively partition
Cell 133, 891–902, May 30, 2008 ª2008 Elsevier Inc. 893
into onedaughterduringtelophase (Figures3Cand3D).Thislate
basal restoration resembles the ‘‘telophase rescue’’ associated
with several apical complex mutations (Peng et al., 2000). Pros,
the Mira-binding transcription factor and GMC-determinant, is
not detectable in the neuroblast nucleus at 96 hr, but at 120 hr
we observe a burst of Pros in the nucleus of many Mira+cells of
intermediate size indicative of neuroblasts in the interphase pre-
ceding the terminal mitosis (Figures 3C and 3D). Clones lacking
Pros activity contain multiple Mira+neuroblast-like cells (Bello
et al., 2006; Betschinger et al., 2006; Lee et al., 2006). We now
find that they do not respect the ?120 hr proliferation endpoint
and even retain numerous dividing Mira+progenitors into adult-
hood (Figure S3C). In addition, we used GAL80tsinduction
(McGuire et al., 2003) to induce transiently the expression of
a YFP-Pros fusion protein (Choksi et al., 2006) well before the
normal ?120 hr endpoint. Under these conditions, YFP-Pros
can be observed in the nucleus of neuroblasts, most Mira+pro-
genitors disappear prematurely, and neural proliferation ceases
much earlier than normal (Figure 3E; data not shown). Together,
these results provide evidence that most neuroblasts terminate
activity in the pupa via a nuclear burst of Pros that induces cell-
blasts to distinguish them from the much smaller population of
The Temporal Series Schedules Pros-Dependent
Cell-Cycle Exit of Type I Neuroblasts
cell-cycle exit. Remarkably, we observe that many svpe22clones
Transition from Small Chinmo+to Large
(A) Histogram comparing the average cell-body
In wt clones, early- (f = 4.50, SD = 0.26) and late-
or Br-C, respectively, whereas, in svpe22clones,
both populations express Chinmo (f = 4.57, SD =
early versus late neurons in wt clones and also for
late neurons in wt versus svpe22clones.
(B) Graph showing how the diameter (f) of neuro-
nal cell bodies varies according to birth order
within a postembryonic clone. Cells are numbered
in ascending order from deep (first born) to super-
ficial (last born) and diameters averaged from 4 wt
(blue) and 6 svpe22(yellow) clones. A steep de-
crease in diameter, correlating with the Chinmo+
to Br-C+transition in wt clones (red vertical line)
is absent in svpe22clones.
(C) Single confocal sections of thoracic svpe22and
UAS-cas MARCM clones, induced at 12–36 hr. At
96 hr, Br-C+cells are absent and all cells express
(D) Histogram of average numbers of Chinmo+and
Br-C+cells in thoracic wild-type (wt), svpe22, and
UAS-cas MARCM clones, induced at 12–36 hr.
Wild-typeclonescontain anaverage of15Chinmo+
progeny (including some weak Br-C+cells, number
of clones = 14; SD = 7.7) and 39 Br-C+Chinmo?
progeny (n = 14; SD = 10.3). svpe22and UAS-cas
clones that fail to express Br-C contain an average
of 45 (n = 10, SD = 8.35) and 40 (n = 7, SD = 7.5)
Chinmo+neurons respectively. Note that, in both
cases, the total number of neurons per lineage is
sent 1 SD; asterisks indicate P < 0.001 for Chinmo+
cells in wt versus svpe22and wt versus UAS-cas.
(E) Histogram of % of MARCM clones expressing
Br-C at 96 hr when induced during embryogenesis,
at 12–36 hr or at 65–75 hr. Numbers of clones
counted for each of the three inductions were 135,
47, and 14 for svpe22; 76, 69, and 10 for UAS-cas;
51, 40, and 40 for cas24,, and for wild-type > 30
clones were examined for each.
2. Casand Svp Regulate the
894 Cell 133, 891–902, May 30, 2008 ª2008 Elsevier Inc.
induced at early-larval stages retain a single Mira+neuroblast at
7 days into adulthood (Figures 4A, 4C, and 4D). The persistent
adult neuroblasts in a proportion of these clones also express
the M phase marker, PH3, indicating that they remain engaged
in the cell cycle and, accordingly, they generate approximately
4B–4D). Furthermore, superficial last-born neurons in these over
proliferating svpe22adult clones are Chinmo+Br-C?indicating
a blocked Chinmo / Br-Ctransition (Figures4C and4D).Similar
ures S4A–S4D). This analysis demonstrates that stalling the tem-
Figure 3. Pros-Dependent Cell-Cycle Exit
Terminates Type I Neuroblasts
(A) Frames from Movies S1 and S2 of larval (96 hr)
and pupal (120 hr) thoracic neuroblasts expressing
His2AvDGFP (HisGFP). At 96 hr, a large neuroblast
?16 min to complete anaphase and telophase.
(B) Histogram depicting the mean telophase diam-
eters (f) of thoracic and central brain neuroblasts
(Mira?daughters) and their progeny (Mira+daugh-
ters) at 96 hr (f = 10.4 mm, SD = 0.85 versus
f = 5.2 mm, SD = 0.94) and 120 hr (f = 7.0 mm,
SD = 0.23 versus f = 5.6 mm, SD = 0.7). Error
bars represent 1 SD; asterisk Asterisk indicates
p < 0.001 for 96 hr versus 120 hr Mira?daughters.
(C) Interphase neuroblasts express nuclear Pros at
120 hr but not at 96 hr. Metaphase neuroblasts
show Mira enrichment at the basal cortex at 96 hr
but not at 120 hr. Mira becomes asymmetrically
partitioned at telophase at both 96 hr and 120 hr.
(D) Graph showing the frequency of neuroblasts
at 96 hr and 120 hr that express nuclear Prospero
at interphase and nonbasal (delocalized) Mira at
(E) Transient induction of YFP-Pros in larval neuro-
vae) from 55–75 hr or from 66–96 hr leads tolossof
Mira+and cessation of proliferation (loss of PH3+
cells, compare to wt) in the central brain, thorax, and
abdomen (dotted outline) by 96 hr. The 55–75 hr
induction window shows that Mira+neuroblast
loss is not reversed, even 21 hr after YFP-pros in-
protein is observed in the neuroblast nucleus and
that neuroblast loss does not appear to involve
caspase-dependent cell death (data not shown).
nal identity but also prevents the pupal
cell-cycle exit of type I neuroblasts.
To test the regulatory relationship be-
tween Pros and the temporal series, we
examined svpe22clones at pupal stages.
We find that mutant interphase neuro-
blasts fail to switch on nuclear Pros at
roblast, whereas those lacking Pros contain multiple neuroblast-
like progenitors. Importantly, these results demonstrate that
roblasts. Together, the genetic and expression analyses of Svp
andProsshowthatthetemporalseriestriggersa burst ofnuclear
Pros in type I neuroblasts, thus inducing their cell-cycle exit.
Embryonic Cas Represses Dichaete and Activates
Grainyhead in Neuroblasts
To determine how the temporal series is linked to the cessation
of progenitor divisions, we examined two transcription factors
Cell 133, 891–902, May 30, 2008 ª2008 Elsevier Inc. 895
expressed in neuroblasts in a temporally restricted manner.
Dichaete (D), a member of the SoxB family, is dynamically ex-
tion (Nambu and Nambu, 1996; Soriano and Russell, 1998; Zhao
and Skeath, 2002). Consistent with the previous studies, we ob-
served that most or all embryonic neuroblasts progress through
nerve cord initiate expression after their medial and intermediate
counterparts (Figure S6A). D subsequently becomes repressed
in ?85% of neuroblasts during late-embryonic and postembry-
onic stages (Figures S6B–S6D). Grainyhead (Grh) is first acti-
vated in neuroblasts in the late embryo and is required for regu-
2005; Bray et al., 1989; Brody and Odenwald, 2000; Cenci and
Gould, 2005). We find that blocking early temporal series
progression in the embryo, either by persistent Hb or loss of
Cas activity, prevents most neuroblasts from downregulating D
and also from activating Grh at late-embryonic and postembry-
onic stages (Figures 5A–5D, S7A, and S7C). As forcing prema-
ture Cas expression leads to precocious D repression and Grh
activation, both factors are likely to be regulated by Cas rather
than by a later member of the temporal series (Figures 5B,
S7B, and S7D). These results demonstrate that transient embry-
onic Cas activity permanently switches the expression of Grh on
and D off. They also identify Grh and D as positive and negative
targets, respectively, of the temporal seriesin many neuroblasts.
Timely Cell-Cycle Exit of Type I Neuroblasts Requires
Early and Late Temporal Series Inputs
neuroblasts) leads to their reduced cell-cycle speed and disap-
pearance during larval stages (Almeida and Bray, 2005; Cenci
and Gould, 2005). We now observe, at 96 hr, that 65% (n = 182)
of grh370type I neuroblasts are smaller than normal (?6.4 mm in
diameter), delocalize Mira from the cortex to the cytoplasm and
nucleus, and strongly express Pros in the nucleus (Figures 5E
and 5F). These events, reminiscent of the 120 hr terminal cell cy-
cle of wild-type progenitors, show that Grh is required to prevent
the premature cell-cycle exit of type I neuroblasts. Given this
finding, and that Cas is required to activate Grh, the question
arises as to how some neuroblasts lacking embryonic Cas activ-
ity are able to continue dividing into adulthood (see Figures S4C
and S4D). However, cas24neuroblasts persisting in adults all re-
tain Grh (data not shown), suggesting that they may derive from
some embryonic neuroblasts (Cleary and Doe, 2006), perhaps
retaining enough Cas activity to support Grh activation but not
later progression of the temporal series.
To determine if late temporal series inputs, after embryonic
Cas, are also required to maintain the long-lasting postembry-
onic expression of Grh, we induced svpe22clones in early larvae.
Figure 4. Postembryonic Loss of Svp Activity Leads to Neuroblast
Divisions in the Adult
(A) Schematic view of the wild-type adult CNS, which, in contrast to the larval
CNS (Figure 1A), contains no neuroblasts.
(B) Thoracic MARCM clones, induced at 0–24 hr and analyzed in 3-day-old
adults. Wild-type and svpe22clones contain an average of 81 (n = 22, SD = 29)
and 142 (n = 12, SD = 56) progeny respectively. Error bars represent 1 SD;
asterisk indicates P < 0.001 for wt versus svpe22clones.
(C and D) Ventral views of central brain (C) and thoracic (D) regions of 3 day
adult CNS. Many svpe22MARCM clones, induced at 0–24 hr and marked
with tub > nGFP, retain a single Mira+neuroblast (a subset are PH3+), fail to
generate Br-C+progeny, but continuously generate Chinmo+neurons. Insets
show single confocal sections enlarged from framed areas with nGFP+clones
containing a Mira+PH3+neuroblast or Chinmo+adult neurons. In (C), the clone
containing the Mira+PH3+neuroblast (arrowhead and enlargement) is merged
with other nGFP+clones. No Mira+or Chinmo+cells are observed in control
adult tissue surrounding the nGFP+clones.
896 Cell 133, 891–902, May 30, 2008 ª2008 Elsevier Inc.
Figure 5. Embryonic Cas Activity Switches Neuroblasts from D+Grh?to D?Grh+and Prevents Premature Nuclear Pros
(A)Histogramdepictingthat Grh isexpressedin 86%ofwild-type (n=26hemisegments, SD= 5.8)comparedtoonly31% ofcas24(n= 20,SD= 11.6) neuroblasts
(NBs) in the thorax and abdomen of stage 14 embryos. Error bars represent 1 SD; asterisk indicates p < 0.001.
(B) Histogram showing that the percentage of lateral Mira+neuroblasts expressing D in T2-A1 falls from 70% to 27% between embryonic stages 14 (n = 12,
SD = 15.2) and 15 (n = 12, SD = 12.6). The percentage of lateral Mira+neuroblasts retaining D expression in cas24mutant embryos at stage 15 is 68%
(n = 18, SD = 16), whereas in wor > cas embryos (UAS-cas) at stage 14 it is only 27% (n = 14, SD = 13.3). Error bars represent 1 SD; asterisks indicate
P < 0.001 for wt versus UAS-cas at stage 14, wt versus cas24at stage 15, and wt stage 14 versus wt stage 15.
(C and D) Thoracic cas24MARCM clones at 96 hr, induced in the early embryo and marked with tub > nGFP.In (C), two PH3+cas24clones (arrows) in T2 (enlarged
express Grh, whereas the lower wild-type Mira+neuroblast is Grh+.
(E) Histogram showing that grh370neuroblasts delocalize Mira and are smaller than normal at 96 hr. Average f = 9.6 mm (SD = 1.1, n = 16) for neuroblasts with
cortical Mira but f = 6.4 mm (SD = 0.7, n = 15) for neuroblasts with cytoplasmic and nuclear Mira. Error bars represent 1 SD; asterisk indicates P < 0.001.
(F) Single confocal sections of wild-type (wt) and grh370thoracic Mira+neuroblasts at 96 hr. Many interphase neuroblasts (arrows) lacking grh activity are smaller
than normal and delocalize Mira and Pros from the cortex to the cytoplasm and nucleus.
Cell 133, 891–902, May 30, 2008 ª2008 Elsevier Inc. 897
Althoughtype Ineuroblastsin thesemutantclones haveastalled
temporal series, they retain postembryonic Grh expression
through to adult stages (Figure S8). Thus, two sequential inputs
from the temporal series are required for type I neuroblasts to
undergo timely Pros-dependent cell-cycle exit. First, embryonic
mature nuclear Pros and permitting continued mitotic activity.
Second, a late postembryonic input, requiring Svp, counteracts
this activity of Grh by triggering a pupal burst of nuclear Pros.
Despite undergoing premature cell-cycle exit, we noticed that
grh mutant neuroblasts can still generate both Chinmo+and
nal Chinmo / Br-C switch. Conversely, neither Chinmo nor Br-C
appears to be required postembryonically for neuroblast cell-cy-
cle exit (data not shown). In summary, the properties of both neu-
roblasts and neurons are regulated by downstream targets of the
AbdA and the Temporal Series Specify the
Combinatorial Code for Type II Neuroblast Apoptosis
We next tested if the temporal series and its targets also function
in type II neuroblasts, which terminate via RHG-dependent apo-
ptosis rather than Pros-dependent cell-cycle exit. We focused
on one identified type II neuroblast in the central abdomen,
called dl, which undergoes apoptosis at 70–75 hr and produces
only a small postembryonic lineage of ?10 neurons (Bello et al.,
expresses bursts of Cas (?45 to ?60 hr) and Svp (?62 to ?65 hr)
and sequentially generates Chinmo+(?7 deep) and Br-C+(?3
superficial) neurons (Figure S10). Loss of Cas or Svp activity,
or prevention of temporal series progression in several other
ways all lead to a blocked Chinmo / Br-C transition, a failure
to die at 70–75 hr, and the subsequent generation of many
supernumerary progeny (Figures S11, S12, 6A, 6B, and 6D–6F).
These results show that the temporal series performs similar
functions in type I and type II neuroblast lineages, regulating
both the Chinmo / Br-C neuronal switch and the cessation of
Figure 6. The Temporal Series and Its Targets Grh and D Regulate
Type II Neuroblast Apoptosis
(A and B) Neuroblasts in abdominal cas24MARCM clones (arrows and dotted
areas), induced during early embryogenesis and marked with tub > nGFP, fail
to undergo apoptosis and to express Grh (A) or repress D (B) at 96 hr. Insets
show single confocal sections, enlarged from one abdominal clone (dotted
area) containing a Grh?neuroblast (Mira+) or a D+neuroblast (large cell adja-
cent to smaller PH3+GMC).
(C) Ventral views of abdominal neuromeres from wt (tub-GAL80ts; nab-GAL4,
UAS-mGFP) and UAS-D (UAS-D/tub-GAL80ts, nab-GAL4, UAS-mGFP) larvae
at 96 hr. Continuous postembryonic D expression leads to abdominal lineages
with supernumerary cells and a persistent Grh+neuroblast. Eighty-nine
percent (n = 25/28) of dl lineages retain a persistent Grh+neuroblast (e.g.,
arrowhead and enlarged inset).
(D and E) The Grh+D?state is retained at 96 hr by persistent dl neuroblasts
misexpressing Cas (elavC155-GAL4; tub-GAL80ts/UAS-cas) or lacking Svp
activity (svpe22MARCM clones induced at 0–24 hr and marked with elav >
genetically manipulated dl neuroblast lineages. Progression (arrows) and
blockade (dotted lines) of the temporal series are indicated. For clarity,
Chinmo/Br-C expression is omitted from the neuroblast (large circle) and
only one last-born neuron (small circle) is shown in the manipulated lineages.
898 Cell 133, 891–902, May 30, 2008 ª2008 Elsevier Inc.
progenitor activity. Next, we investigated the mechanism linking
the temporal series to RHG-dependent death of type II neuro-
blasts. As for type I neuroblasts, dl progenitors in cas24clones,
inducedin the embryo,fail to activate Grhand repress D (Figures
6A and 6B). Moreover, if grh activity is reduced, or if D is contin-
uously misexpressed, dl progenitors persist long after 75 hr (Fig-
ures 6C and S13A) (Cenci and Gould, 2005). Therefore, both of
these early Cas-dependent events are essential for subsequent
type-II neuroblast apoptosis. However, in contrast to members
of the temporal series themselves, persistent misexpression of
their target D does not block the Chinmo / Br-C switch in the
dl lineage (Figures S13B and 6G). Thus, the D?Grh+state of
type I and type II neuroblasts, installed via an embryonic Cas
pulse, appears to be necessary for progenitor termination but
not for Chinmo / Br-C switching. Nevertheless, dl neuroblasts
stalled at a postembryonic stage in svpe22and UAS-cas clones
6D and 6E). Therefore, as with type I cell-cycle exit, timely type II
apoptosis requires both embryonic Cas-dependent and post-
embryonic Svp-dependent inputs from the temporal series.
Finally, we dissected the regulatory relationship between the
temporal series and AbdA, a Hox protein transiently expressed
in postembryonic type II (but not type I) neuroblasts and required
for their apoptosis (Bello et al., 2003). dl neuroblasts lacking
postembryonic Svp activity or persistently expressing Cas still
retain AbdA expression yet do not die (Figures S14A and
S14B). This suggests that AbdA is unable to kill neuroblasts un-
less they progress, in a Svp-dependent manner, to a late Cas?
temporal state (Figures S14A and S14B). To test this prediction
directly, we made use of the previous finding that ectopic
AbdA is sufficient to induce neuroblast apoptosis, albeit only
within a late time window (Bello et al., 2003). We find that consti-
tutive AbdA-induced apoptosis is efficiently suppressed by
persistent Cas, not only in type II but also in type I neuroblasts
(Figures 7A–7C). This result demonstrates that, in order to termi-
nate, type II neuroblasts must progress to a late Cas?state, thus
acquiring a D?Grh+Cas?AbdA+code. It also suggests that
AbdA is sufficient to intercept progression of the temporal series
in type I neuroblasts, inducing an early type II-like termination.
We found that the Drosophila CNS contains two distinct types of
self-renewing progenitors: type I neuroblasts terminate divisions
Figure 7. The Temporal Series Installs
(A–C) Distribution ofMira+neuroblasts inwild-type
(A), elavC155-GAL4; tub-GAL80ts/UAS-abdA (B),
UAS-cas (C) larvae at 96 hr. AbdA-induced neuro-
blast apoptosis in both the thorax and abdomen
tent postembryonic Cas.
(D) An indirect feedforward model for neural pro-
genitor aging. Transient bursts of members of
the temporal series (such as Hb, Kr, Pdm, Cas,
Svp) in type I and type II neuroblasts are trans-
duced into feed-forward activation/repression of
targets controlling the mitotic activity, apoptosis,
and cell-cycle exit of neuroblasts (such as D and
Grh) and targets regulating neuronal identity
(such as Chinmo and Br-C). In both type I and
type II neuroblasts, embryonic Cas switches D
(an inhibitor of RHG-activity) off and Grh (an inhib-
itor of nuclear Pros) on. For type II neuroblasts,
a subsequent larval input from the temporal series
‘‘default’’ type I program, triggering ?72 hr RHG
activity and thus early neuroblast apoptosis.
Type I neuroblasts do not express AbdA and so
can progress to an even later temporal series in-
put, scheduling ?120 hr nuclear Pros and cell-cy-
cle exit. Indirect feedforward allows early temporal
factors to install the competence to respond ap-
propriately to later factors and could, in principle,
regulate transitions in many different progenitor
and neuronal/glial properties. Note that it is not
yet clear whether Cas expression in postembry-
onic neuroblasts results from maintained embry-
onic Cas during quiescence (Q) or from a distinct
Cell 133, 891–902, May 30, 2008 ª2008 Elsevier Inc. 899
by cell-cycle withdrawal and type II neuroblasts via apoptosis.
Despite these different exit strategies, both progenitor types
use a similar molecular timer, the temporal series, to shut
down proliferation and thus prevent CNS overgrowth. These
findings demonstrate that the temporal series does considerably
more than just modifying neurons; it also has multiple inputs into
neural proliferation. The identification and analysis of several
pan-lineage targets of the temporal series also begins to shed
light on the mechanism by which developmental age modifies
the properties of neuroblasts and neurons. Two targets, Chinmo
and Br-C, are part of a downstream pathway temporally regulat-
ing the size and identity of neurons. Two other temporal series
targets, Grh and D, function in neuroblasts to regulate Pros-
pero/RHG activity, thereby setting the time at which proliferation
ends. We now discuss how the temporal series regulates both
cell proliferation and cell identity and propose a feedforward
mechanism for generating combinatorial transcription factor
codes during progenitor aging.
The Temporal Series Regulates Pan-Lineage Aspects
of Neural Proliferation and Cell Identity
We found that the temporal series regulates a widespread post-
embryonic switch in neuronal identity. Most, if not all, type I and
type II neuroblasts first generate a deep layer of large Chinmo+
neurons and then switch to producing a superficial layer of small
Br-C+neurons. Two lines of evidence argue that this postembry-
onic neuronal switch is likely to be regulated by a continuation of
the same temporal series controlling early/late neuronal identi-
ties in the embryo. First, the postembryonic Chinmo / Br-C
neuronal switch is promoted by the transient redeployment of
two known components of the embryonic temporal series, Cas
and Svp. Second, this switch remains inhibitable by misexpres-
Cas and Svp are expressed somewhat earlier than the neuronal-
size transition, it is likely that they promote bursts of later, as yet
ulate Chinmo and Br-C. Although neuronal functions for both
BTB zinc-finger targets have yet to be characterized, a progres-
to regulate the temporal identities of mushroom-body neurons
(Zhu et al., 2006). Our results now suggest that this postmitotic
gradient mechanism may be linked to, rather than independent
from, the temporal series.
Type I neuroblasts in clones lacking postembryonic Cas/Svp
activity or retaining an early temporal factor, fail to express nu-
clear Pros during pupal stages and thus continue dividing long
only a single neuroblast, sharply contrasting with adult clones
lacking Brat or Pros, in which there are multiple neuroblast-like
progenitors (Bello et al., 2006 and this study). Hence, manipula-
tions of the temporal series and its progenitor targets offer the
prospect of immortalizing neural precursors in a controlled man-
ner, without disrupting their self-renewing asymmetric divisions.
Step-wise Acquisition of Combinatorial Codes
for Progenitor Cell-Cycle Exit and Apoptosis
This study demonstrates that type I and type II neuroblasts must
progress through at least two critical phases of the temporal
series in order to acquire the D?Grh+Cas?combinatorial tran-
scription factor code that precedes Pros/RHG activation. The
early phase corresponds to embryonic Cas activity switching
neuroblasts from D+Grh?to D?Grh+status. The equally essen-
tial, but less well-defined, late postembryonic phase of the tem-
poral series requires transition to a Cas?state and a late Svp
burst. For type I neuroblasts, Grh and a late Cas?temporal iden-
tity are both required for timely expression of nuclear Pros and
subsequent cell-cycle withdrawal. For type II neuroblasts, these
two inputs are also necessary for RHG-dependent apoptosis,
with the additional requirement that D must remain repressed.
Although the temporal series and its targets are similarly ex-
pressed in type I and type II neuroblasts, only the latter progen-
itors undergo a larval burst of AbdA. This AbdA expression is
likely to be the final event required to convert the D?Grh+
Cas?state, installed by the temporal series, into the D?Grh+
Cas?AbdA+combinatorial code for RHG-dependent apoptosis.
This code prevents type II neuroblasts in the abdomen from
reaching the end of the temporal series and accounts for why
they generate fewer progeny and terminate earlier than their
type I counterparts in the central brain and thorax.
The data in this study support an indirect feedforward model
for neuroblast aging (Figure 7D). Key to this model is the finding
that, although members of the temporal series are only ex-
pressed very transiently, some of their targets can be activated
or repressed in a sustained manner, as observed for Chinmo/
Br-C in neurons and also for Grh/D in neuroblasts. In principle,
this indirect feedforward allows aging progenitors to acquire
step-wise the combinatorial transcription factor codes modulat-
ing cell-cycle speed, growth-factor dependence, competence
states, and neural potential. Like Drosophila neuroblasts, iso-
lated mammalian cortical progenitors can sequentially generate
neuronal fates in the correct in vivo order (Shen et al., 2006). Our
studies suggest that it will be important to investigate whether
the transcription factors controlling this process also regulate
cortical proliferation and whether their targets include BTB-
zinc finger, Grh, SoxB, Prox, or proapoptotic proteins. Some in-
sect/mammalian parallels seem likely as it is known that Sox2
downregulation and Prox1 upregulation can both promote the
cell-cycle exit of certain types of vertebrate neural progenitors
(Dyer et al., 2003; Graham et al., 2003). Thus, although insect
and mammalian neural progenitors do not appear to use the
same sequence of temporal transcription factors, at least
some of the more downstream components identified in this
study might be functionally conserved.
Drosophila Genetics and Staging
Drosophila were raised at 25?C on cornmeal/yeast/agar medium supple-
mented with live yeast, unless otherwise stated. Stages were calculated chro-
nologically, relative to hatching (at 0 hr) for larvae or pupariation (at 96 hr) for
pupae. The 96 hr larval stage was determined by morphology and wandering
Embryonic MARCM clones were induced using hsFLP122at 37?C for 45 min,
either at 8 hr (for D analysis) or at 16 hr (for Chinmo and Br-C analyses) after
egg laying.Postembryonic MARCMcloneswereinduced at0–24 hrafter larval
hatching for 80 min at 37?C unless otherwise stated. Neural expression of UAS
transgenes was temporally restricted to postembryonic stages by combining
900 Cell 133, 891–902, May 30, 2008 ª2008 Elsevier Inc.
elav-GAL4C155or nab-GAL4 with tubP-GAL80ts(McGuire et al., 2003) and
shifting from 18?C to 30?C from ?4 hr for induction during all larval stages.
elav-GAL4C155; UAS-cas; tub-GAL80tslarvae do not detectably express
UAS-cas when raised at 18?C, but the switch to 30?C induces robust expres-
sion. For transient induction of Pros, UAS-YFP-pros (Choksi et al., 2006), tub-
GAL80ts; nab-GAL4 larvae were raised at 18?C and shifted to 30?C during the
66–96 or 45–75 hr time windows. Five recombinant chromosomes were con-
structed for this study: UAS-cas, FRT2A; UAS-cas, UAS-abdA; FRT82B,
svpe22, and FRT82B, cas24. Details of genetic strains can be found in Supple-
mental Experimental Procedures available online.
BrdU Labeling and Immunohistochemistry
BrdU was incorporated by raising larvae on a diet containing 0.4 mg/ml of
BrdU from 0–96 hr. Then, prior to immunostaining, fixed dissected tissues
were pretreated with 50 Units/ml of RQ1 Rnase-free Dnase (Promega) for
Embryonic and larval tissues were fixed and immunostained essentially as
described (Bello et al., 2003). Details of antibodies used can be found in Sup-
plemental Experimental Procedures available online.
Confocal Microscopy and Image Analysis
All images are projections of several confocal sections, unless stated other-
wise. Clone/lineage sizes were calculated from confocal sections spaced by
?1.5 mm as described (Cenci and Gould, 2005). Diameters of elav > mGFP-
labeled neurons in MARCM clones were calculated from the average of two
orthogonal measurements using Leica SP5 confocal images and LAS AF soft-
ware. For live imaging, CNS explants from His2AvDGFP larvae were cultured
as described (Brown et al., 2006) and time-lapse recordings made using a
Leica SP5 confocal microscope with a 63X oil immersion objective and 30 s
frame intervals. AVI files were assembled from single z sections using ImageJ,
and neuroblasts were identified by their ability to divide and their larger size
compared to nearby GMCs/neurons. For all numerical measurements, P
values were calculated assuming equal sample variance, using two-tailed
Student’s t tests.
Supplemental Data include 14 figures, 2 movies, Supplemental Experimental
Procedures, and Supplemental References and can be found with this article
online at http://www.cell.com/cgi/content/full/133/5/891/DC1/.
Hoch, T. Lee, M. Mlodzic, A. Nose, W. Odenwald, S. Russell,G. Struhl, and the
Bloomington and Kyoto Institute of Technology stock centers for flies. We
acknowledge B. Bello, M. Buescher, W. Chia, Y. Hiromi, T. Kaufman, D.
Kosman, T. Lee, J.-A. Lepesant, F. Matsuzaki, J. Nambu, W. Odenwald, J.
Reinitz, and S. Russell for sharing antibodies and thank J. Briscoe, R. Lovell-
Badge, I. Miguel-Aliaga, I. Salecker, R. Sousa-Nunes, J.-P. Vincent, and D.
Wilkinson for advice and critical reading of the manuscript. C. M., L. C., and
A. G. were supported by the MRC, and C.M. also by EMBO Long-Term and
Marie Curie EIF fellowships.
Received: July 3, 2007
Revised: January 29, 2008
Accepted: March 25, 2008
Published: May 29, 2008
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