RESEARCH ARTICLE 3327
Development 137, 3327-3336 (2010) doi:10.1242/dev.052233
© 2010. Published by The Company of Biologists Ltd
A genetic cascade involving klumpfuss, nab and castor
specifies the abdominal leucokinergic neurons in the
Drosophila CNS
Jonathan Benito-Sipos1,*,†, Alicia Estacio-Gómez1,†, Marta Moris-Sanz1, Magnus Baumgardt2, Stefan Thor2
and Fernando J. Díaz-Benjumea1,‡
SUMMARY
Identification of the genetic mechanisms underlying the specification of large numbers of different neuronal cell fates from
limited numbers of progenitor cells is at the forefront of developmental neurobiology. In Drosophila, the identities of the
different neuronal progenitor cells, the neuroblasts, are specified by a combination of spatial cues. These cues are integrated
with temporal competence transitions within each neuroblast to give rise to a specific repertoire of cell types within each lineage.
However, the nature of this integration is poorly understood. To begin addressing this issue, we analyze the specification of a
small set of peptidergic cells: the abdominal leucokinergic neurons. We identify the progenitors of these neurons, the temporal
window in which they are specified and the influence of the Notch signaling pathway on their specification. We also show that
the products of the genes klumpfuss, nab and castor play important roles in their specification via a genetic cascade.
INTRODUCTION
During development of the central nervous systems (CNS) of
vertebrates and invertebrates, limited numbers of progenitor cells
give rise to vast numbers of differentiated neurons. Each neuron
acquires a specific identity, manifested in particular by the position
of its soma, its axonal projection and neurotransmitter type. Many
of these characteristic traits are acquired during development, but
how multiple transcription regulators cooperate to specify a given
cell fate is poorly understood (reviewed by Jessell, 2000; Shirasaki
and Pfaff, 2002). A central goal of neuroscience is to explain the
development of the nervous system in terms of gene functions and
genetic mechanisms. Because of the relative simplicity of its CNS,
Drosophila is an excellent model system for the study of CNS
development.
The Drosophila CNS can be subdivided into the brain and
ventral nerve cord (VNC). The VNC arises from the
neuroectoderm, a sheet of cells located ventrolaterally on both sides
of the embryo (for a review, see Skeath and Thor, 2003). Two sets
of patterning genes expressed along the anterior-posterior and
dorsal-ventral axes divide the neuroectoderm into a Cartesian grid
of neural equivalence groups. From these cell groups, individual
cells delaminate to become the progenitor cells of the CNS, the
neuroblasts (NBs). The delamination process occurs in five
sequential waves and results in the formation of an invariant pattern
of 30 NBs per hemisegment (Campos-Ortega and Hartenstein,
1
Centro de Biología Molecular-Severo Ochoa, Universidad Autónoma-C.S.I.C.,
Madrid 28049, Spain. 2Department of Clinical and Experimental Medicine,
Linköping University, Linköping SE-581 85, Sweden.
*Present address: Dpto. Biología, Universidad Autónoma, Madrid 28049, Spain
These authors contributed equally to this work
‡
Author for correspondence ([email protected])
†
Accepted 15 July 2010
1985), with each NB acquiring a unique fate based on its position
in the grid (Bossing et al., 1996; Schmid et al., 1999; Schmidt et
al., 1997). After their formation, NBs undergo a stereotyped and
invariant pattern of asymmetric cell divisions that maintains the NB
and produces a secondary cell, the ganglion mother cell (GMC).
Typically, each GMC divides once more to produce two
postmitotic cells that differentiate as neurons or glia. The number
of GMCs generated by each NB and the fate of the postmitotic
cells are NB specific; thus, each NB creates a unique and
reproducible lineage (Udolph et al., 1993).
Ablation experiments in the grasshopper have demonstrated that
the fates of each GMC and its daughter cells are determined by
their birth order from the parental NB (for a review, see Pearson
and Doe, 2004). Recently, part of the underlying genetic
mechanism controlling such temporal transitions in progenitor
competence has been revealed (Brody and Odenwald, 2000; Cleary
and Doe, 2006; Grosskortenhaus et al., 2005; Grosskortenhaus et
al., 2006; Isshiki et al., 2001; Kambadur et al., 1998; Novotny et
al., 2002; Pearson and Doe, 2003; Tran and Doe, 2008; Tsuji et al.,
2008). These studies have identified the temporal identity genes,
which are sequentially expressed in the NB and act to specify
temporal windows in which the different GMCs are produced.
Although NB and GMC determinants, such as spatial and temporal
regulators, are important for establishing neuronal identity, it is
unlikely that they directly control the expression of the unique
characteristics of postmitotic neurons. The functions of many other
genes that are specifically expressed in subsets of NBs, GMCs and
neurons are unknown, and there are very few cases in which the
progenitor NB of a neuron, and the genetic mechanisms underlying
its specification, are known. In addition, the lack of availability of
terminal molecular markers with restricted expression makes it
difficult to unequivocally identify specific subsets of neurons.
The Drosophila embryonic/larval VNC contains ~10,000 cells
(Schmid et al., 1999). Of these, ~150 are peptidergic, as defined by
the expression of one of ~30 identified neuropeptides (Park et al.,
DEVELOPMENT
KEY WORDS: Drosophila, CNS development, Neuronal fate specification, Leucokinin, ABLK
3328 RESEARCH ARTICLE
MATERIALS AND METHODS
Fly stocks
We used the following alleles to analyze wild-type and mutant phenotypes:
Df(2L)ED773 (pdm–), Df(3L)H99, casD1, casD4, Chipe5.5, Chip9.6, crol04418,
da1, dimmrev4, dimmrev8, dve01D01W-L186, el3.3.1 nocD64, esg35Ce-1, grh3776,
grn72, hbP1 hbFB (this genetic combination removes Hb CNS expression,
but, as the larvae do not survive to stage 18, we use UAS-hbRNAi in
combination with UAS-dicer), htlAB42, jumu11.683, klu212IR51C, kr1 krCD (to
remove Kr CNS expression), lz815, mamGA345, mld92, mld47, nabSH143,
nabR52, numb1, pnrv1, pnrvx6, rn20, spdoG104, Df(3R)Dl-KX23 (referred to as
sqzDf), sqzie, sqzGal4, stc05441, tup1, vg83b27r, vn10567, wor1, zfh100865.
lacZ lines: eve-lacZ, gsb01155-lacZ, hkb5953-lacZ, klu09036-lacZ, lbeK-lacZ
[this transgenic line contains a 2 kb genomic fragment of the regulatory
region of lbe driving lacZ, and reproduces the pattern of expression of lbe
(De Graeve et al., 2004)], mirr-lacZ, unpgr37-lacZ, wg-lacZ.
Gal4 lines: elav-Gal4, en-Gal4, wgMD758-Gal4 (gift from M. Calleja, ,
Centro de Biología Molecular, Madrid, Spain), ins-Gal4, wor-Gal4.
UAS lines: UAS-dicer on chromosomes II and III, UAS-cas, UASgrh15M, UAS-hbF4A, UAS-klu, UAS-Nintra, UAS-nab, UAS-p35, UAS-sggCA,
UAS-hbRNAi (Vienna Drosophila RNAi Center #44892), UAS-osaRNAi,
and UAS-zfh2RNAi (http://flybase.org/).
Immunohistochemistry
We used the following antibodies at the dilutions indicated: rabbit anti-Lk
(1:50; gift from D. Nässel, Stockholm University, Stockholm, Sweden);
mouse anti--galactosidase (1:2000; Promega); rat anti-Gsb-d (1:3; gift
from R. Holmgren, Northwestern University, Evanston, IL, USA); guinea
pig anti-Cas (1:500) and guinea pig anti-Hb (1:200) (gifts from T. Isshiki,
National Institute of Genetics, Mishima, Japan); rabbit anti-Dpn (1:500;
Bier et al., 1992); mouse anti-En (1:50; Developmental Studies Hybridoma
Bank #4D9); rabbit anti-Nab (1:1000) and rabbit anti-Pdm1 (1:1000)
(Terriente et al., 2007); and rabbit anti-Runt (1:500; gift from A. Brand,
University of Cambridge, Cambridge, UK).
Embryos were staged according to Campos-Ortega and Hartenstein
(Campos-Ortega and Hartenstein, 1985). Lk expression is first detected in
ventral ganglia at embryonic stage 18, which corresponds to late stage 17
(from 18 hours after egg laying until hatching); at this stage the mouth
hooks are prominent and the trachea are air filled. For antibody staining,
the CNS of stage 18 embryos or early first instar larvae were dissected in
PBT (PBS with 0.3% Tween20), fixed for 20 minutes in 4% formaldehyde
(Polysciences #04018) and processed with antibodies in BBT-250 (PBT
with 0.1% BSA and 250 mM NaCl). Slides were mounted in Vectashield.
Embryos from earlier stages were stained as whole-mount preparations
using the same protocol.
Lineage tracing
Embryos of genotype y w UAS-FLP122; Act5C >y+>lacZ/wg-Gal4; tubGal80ts/+ were collected for 2 hours at 25°C and shifted to 17°C. At 7 hours
of embryonic development they were moved to 30°C for 2 hours, then
shifted back to 17°C, and, as third instar larvae, dissected and stained with
anti-Lk and anti--galactosidase. Cells expressing wg-Gal4 between 7 and
9 hours of embryonic development activate flp-mediated recombination of
the Act5C>y+>lacZ cassette and they and all their progeny permanently
express -galactosidase (Act5C>lacZ) (Struhl and Basler, 1993).
RESULTS
Identification of the ABLK progenitor neuroblast
We first wanted to identify the progenitor NB that gives rise to the
ABLK neurons. So far, none of the lineages that generate the
leucokinergic neurons has been identified, and their locations in the
CNS suggest that they arise from different progenitors. Work over
the last two decades has yielded a detailed lineage map of most of
the 30 NBs generated in each hemisegment, providing a set of
genetic markers that permit unambiguous identification of the
different NBs in the embryonic ventral ganglia from stage eight to
11 (Bossing et al., 1996; Doe, 1992; Prokop and Technau, 1991;
Schmid et al., 1999; Schmidt et al., 1997).
Based on morphological data, it has been suggested that ABLKs
arise from NB2-4 (Schmid et al., 1999). Nevertheless, we consider
this conclusion mistaken because we found that ABLKs did not
express genetic markers such as mirror (mirr) and Even-skipped
(Eve) found in NB2-4 (Fig. 2A-B). Instead, we found that ABLKs
expressed gooseberry (gsb) (a neuroblast row five and six marker)
but did not express engrailed (en) (a row six and seven marker),
suggesting a row five origin. wingless (wg) is expressed in row five
NBs, so to confirm that the ABLK progenitor NB belongs to row
five, we traced the lineage of the cells expressing wg at late stage
DEVELOPMENT
2008). Because of their highly restricted expression, neuropeptides
have emerged as particularly useful markers for addressing the
molecular genetic mechanisms controlling neuronal subtype
specification in both vertebrates and invertebrates (Brohl et al.,
2008; Thor, 2008). In particular, studies of one subgroup of
peptidergic neurons, the thoracic Apterous neurons, have resulted
in a detailed understanding of how spatial and temporal cues are
translated into combinatorial regulatory codes of cell fate
determinants (Allan et al., 2005; Allan et al., 2003; Baumgardt et
al., 2009; Baumgardt et al., 2007; Miguel-Aliaga et al., 2004). To
understand whether or not such genetic cascades are commonly
used during neuronal subtype specification, we have initiated
studies of another peptidergic cell type: the Drosophila abdominal
leucokinergic neurons.
The abdominal leucokinergic neurons (ABLKs) belong to the set
of neurons that synthesize the neuropeptide Leucokinin (Lk). Lk is
widespread among invertebrates and is known to stimulate fluid
secretion by Malpighian tubules, a type of excretory and
osmoregulatory system found in arthropods (Hewes and Taghert,
2001; Nassel, 2002; Terhzaz et al., 1999). More recently, it has
been shown that the Lk pathway is also involved in meal size
regulation (Al-Anzi et al., 2010). The larval leucokinergic system
is formed by four different sets of neurons, which are easily
identified in the CNS of first instar larvae by immunostaining with
an anti-Lk antibody: anterior LK (ALK), lateral-horn LK (LHLK),
subesophageal LK (SELK) or abdominal LK (ABLK) (Fig. 1A-B)
(Cantera and Nassel, 1992; Herrero et al., 2003). We have focused
on the ABLKs, which form a group of 14 cells, one per
hemineuromere, in the abdominal ganglia (A1-7). These neurons
are detected by immunostaining with specific anti-Lk antibodies
during late embryonic development, from stage 18 and onward
during larval and pupal development, as well as in adult flies. In
this report, we describe our initial approach aimed at identifying
the genetic mechanisms involved in specification of the ABLKs.
First, we identify the NB progenitor of ABLKs. Second, we find
that the ABLKs are generated within a castor (cas)/grainyhead
(grh) temporal window, and show that cas is crucial for the
specification of ABLKs. Third, we find that the genes nab and
klumpfuss (klu) act both upstream and downstream of cas,
suggesting that cas has two temporal windows and that they both
play important roles in the ABLK specification process. We also
find that Nab functions to prevent the repression of the ABLK fate
by the Sqz transcription factor. Finally, we find that the ABLK
sibling cell probably dies by apoptosis and that the Notch pathway
is required to prevent the ABLK from similarly undergoing
apoptosis. These findings help set the stage for in-depth
comparative analyses of cell fate specification within the
Drosophila CNS.
Development 137 (19)
Neuropeptidergic cell fate specification
RESEARCH ARTICLE 3329
Fig. 1. The leucokinergic system. (A)Expression of Leucokinin (Lk) in
the Drosophila CNS detected by antibody staining. (B)Schematic of the
leucokinergic system. ALK, anterior Lk; LHLK, lateral horn Lk; SELK,
subesophageal Lk; ABLK, abdominal Lk.
Fig. 2. Identification of the ABLK progenitor neuroblast.
(A-H)Expression of Lk (red) and mirr-lacZ (A), eve-lacZ (B), gsbn-lacZ (C),
wg-lacZ (D), Engrailed (E), lbe-lacZ (F), hkb-lacZ (G) and unpg-lacZ (H)
(green) in the ventral ganglia of Drosophila first instar larvae. Dashed
outline, indicates an ABLK (I-M)Expression of unpg-lacZ in embryos of
stage 11(I), 13 (J), 14 (K), 16 (L), and Lk in first instar larvae (M). Arrows
point to NB5-5 (I) and their putative progeny (J-M). (N)Expression of Lk
(red) and -galactosidase (Act5C>lacZ; green) in lineage-tracing
experiment (see main text and Materials and methods for further
explanation). (O)Schematic summarizing these results. Circles represent
the pattern of NBs in late stage 11 (Doe, 1992). The different colors
represent the patterns of expression of the various NBs. In A-H and N
the separate channels are shown at the bottom of each figure.
et al., 2008). The Lk cells extend along the SNa, project across
muscle eight, then follow the intersegmental nerve (ISN) out and
terminate on the alary muscles (Cantera and Nassel, 1992;
Landgraf and Thor, 2005).
Identification of the sequence of expression of
temporal factors in NB5-5
The fates of the different GMCs and neurons generated by each NB
are in part controlled by the sequential expression of temporal
identity genes (Brody and Odenwald, 2000; Cleary and Doe, 2006;
Grosskortenhaus et al., 2005; Grosskortenhaus et al., 2006; Isshiki
et al., 2001; Kambadur et al., 1998; Novotny et al., 2002; Pearson
DEVELOPMENT
11 (see Materials and methods). To this end we induced early
permanent expression of -galactosidase in all row five cells,
including NB5-5, and thereby labeled all of their progeny. These
experiments confirmed that the ABLK was born from NB row five
(Fig. 2C-E,N).
Given the limited migration of neurons within the VNC and the
lateral position of ABLKs within row five, this pointed to NB5-4,
5-5 or 5-6 as the likely progenitor NB. To distinguish between
these possibilities, we used markers specific for different row five
NBs. We found that ABLKs did not express ladybird early (lbe), a
specific marker for NB5-6 and its progeny (Fig. 2F) (De Graeve et
al., 2004), but did express huckebein (hkb), a marker for NB5-4 and
5-5 (Fig. 2G) (Bossing et al., 1996; Chu-LaGraff et al., 1995), as
well as unplugged (unpg), which was expressed in NB5-5 but not
in NB5-4 (Fig. 2H) (Doe, 1992).
Since Lk expression is first detected in ABLKs during late
embryonic development (from stage 18 onward), the present
coexpression analysis was carried out in the ventral ganglia of first
instar larvae (L1). Although it has been reported that expression of
gsb is lineage specific (Buenzow and Holmgren, 1995), it is not
known if expression of the other genetic markers used to identify
NBs at stage 11 changes late in embryogenesis. In the absence of
markers that allow us to detect ABLKs at early times, we used
unpg expression to trace the development of the ABLK progenitor
NB. We followed the expression of unpg-lacZ in NB5-5 from its
initial activation at stage 11 to stage 18, when we were first able to
detect Lk expression in the ABLKs (Fig. 2I-M). Using this marker,
NB5-5 was first detected at stage 11, and the expanding cluster of
cells expressing unpg-lacZ could be followed into L1, when one of
the cells coexpresses Lk. From these results we conclude that the
ABLKs are generated by NB5-5 (Fig. 2O).
NB5-5 is one of the NBs that delaminates later in
embryogenesis, at late stage 11 (Doe, 1992). A previous analysis
of NB lineages by in vivo DiI (a lipophilic fluorescent tracer)
labeling reported a single abdominal NB5-5 clone, of eight to 11
cells (Schmid et al., 1999). This clone included local interneurons
and neurosecretory cells extending along a branch of the segmental
nerve (SNa). This previous study described the axonal projections
of these neurons at stages 15-17, but as Lk expression commences
in embryonic stage 18, it is not surprising that none of the axonal
projections described coincided with the axonal projection of the
ABLKs observed in larval stages. The ABLKs further belong to a
lateral cluster of four dimm-expressing cells referred to as the
peptidergic lateral cluster (PLC) (Miguel-Aliaga et al., 2004; Park
3330 RESEARCH ARTICLE
Development 137 (19)
Fig. 3. Sequence of expression of
temporal factors in NB5-5.
(A-F)Expression of gsb-lacZ (red),
Deadpan (A,B,D; blue) and Hb (A), Kr (B),
Pdm (C), Cas (D-F) (green) and Grh (E,F;
blue) in NB5-5 at late stage 11 (A-D),
stage 12 (E) and stage 13 (F). The
separate channels are shown at the
bottom of each figure. For clarity the
blue channel is shown in white.
Identification of the temporal factor that
specifies the ABLKs
To identify the temporal window in which the ABLKs are born, we
followed the expression of Lk in the CNS of first instar larvae in
which expression of Hb (wor-Gal4>UAS-hbRNAi UAS-dicer), Kr
(kr1 krCD), Pdm [Df(2L)ED773], Cas (casD1) or Grh (grh376) was
compromised. In agreement with our observation that NB5-5 does
not express early temporal genes, we observed little or no change
in the number of ABLKs in larvae in which the expression of hb
was knocked down, or in Kr mutant larvae (Fig. 4A-C; Table 1).
Moreover, in spite of the fact that NB5-5 expresses Pdm, we found
no effect upon Lk in pdm mutants (Fig. 4D). By contrast, we
observed a complete absence of ABLKs in cas mutants and a
pronounced reduction in grh mutants (Fig. 4E,F; Table 1). These
results suggest that ABLKs are not generated in the Pdm window
but rather in the Cas/Grh temporal window (Fig. 4L). In agreement
with this, we found that Cas is expressed in the ABLKs (Fig. 4K),
although we did not observe expression of Grh in the ABLKs in
first instar larvae (data not shown).
To test whether the cas and grh temporal genes might be sufficient
to trigger ectopic Lk expression, we analyzed the effect of
misexpressing cas and grh with a pan-neural driver elav-Gal4. We
found that cas misexpression significantly increased the number of
ABLKs (Fig. 4G; Table 1). These extra ABLKs appeared in
proximity to the normal ABLK cells. By contrast, grh misexpression
did not affect the number of ABLKs (Fig. 4H). The basic helix-loop-
helix (bHLH) protein Dimmed (Dimm) controls neuroendocrine cell
differentiation (Allan et al., 2005; Hamanaka et al., 2010; Hewes et
al., 2003), is expressed in the ABLKs (Park et al., 2008), and in
dimm mutants the Lk expression in the ABLKs is downregulated
(Table 1) (Hewes et al., 2003). Similar to previous studies
(Baumgardt et al., 2007), misexpression of dimm alone had no effect
upon the number of ABLKs (Table 1). However, recent findings
indicate that dimm can act potently with grh to trigger ectopic
neuropeptide expression (Baumgardt et al., 2009), and, similarly, we
find here that co-misexpression of grh and dimm significantly
increased the number of ABLKs (elav-Gal4>UAS-grh UAS-dimm;
Fig. 4I; Table 1). By contrast, co-misexpression of cas and dimm did
Fig. 4. ABLKs are specified in a Cas/Grh temporal window.
(A-F)Expression of Lk in wild-type (A), wor-Gal4 UAS-hbRNAi UAS-dicer
(B), kr1 krCD (C), Df(2L)ED773 (pdm–) (D), cas1 (E) and grh376 (F) in
ventral ganglia of Drosophila first instar larvae. (G-J)Expression of Lk in
elav-Gal4>UAS-cas (G), elav-Gal4>UAS-grh (H), elav-Gal4 UAS-grh
UAS-dimm (I; a cluster of three ABLKs is shown in the inset) and elavGal4>UAS-cas UAS-grh (J) in the ventral ganglia of first instar larvae.
(K)Coexpression of Cas (green) and Lk (red) in a wild-type first instar
ganglion (a magnification of an ABLK is shown in the inset).
(L)Schematic summarizing these results.
DEVELOPMENT
and Doe, 2003; Tran and Doe, 2008; Tsuji et al., 2008). The
transcription factors Hunchback (Hb), Krüppel (Kr), Nubbin (Nub)
and Pdm2 (henceforth Pdm), Castor (Cas) and Grainyhead (Grh)
are transiently expressed in a temporal sequence in NBs, and their
expression is correlated with the production of distinct cell types.
GMCs are born intermittently as the NBs progress through the
sequence of temporal genes, and the GMCs and their neuronal
progeny initially maintain the gene expression profile of the NB
when the GMC was born (Isshiki et al., 2001).
Although all NBs are believed to progress through the same
sequence of temporal factors, it is unclear whether late-born NBs,
such as NB5-5, actually express early temporal genes. To
determine the sequence of expression of temporal factors in NB55, we used Deadpan (Dpn), an NB-specific marker, and gsb-lacZ,
which labels NBs in rows five and six. In line with NB5-5 being
late-born, we did not observe expression of the early temporal
genes Hb or Kr in the newborn NB5-5 at late stage 11. It initially
expressed Pdm, but this expression was rapidly lost in early stage
12, when Cas expression started (Fig. 3A-E); at this stage we did
not observe Grh expression. Finally, we observed coexpression of
Cas and Grh at stage 13 (Fig. 3F). Thus, as might be anticipated
from its late birth, NB5-5 expresses a truncated sequence of
temporal genes, initiating at Pdm, i.e. downstream of Hb and Kr.
Neuropeptidergic cell fate specification
RESEARCH ARTICLE 3331
Table 1. Expression of Lk in different genetic combinations
Genotype

n
4A
4B
4C
4D
4E
4F
4G
4H
Wild type
elav-Gal4>UAS-hbRNAi UAS-dicer
kr1 krCD
Df(2L)ED773 (pdm–)
casD1
grh3776
elav-Gal4>UAS-cas
elav-Gal4>UAS-grh
elav-Gal4>UAS-dimm
elav-Gal4>UAS-grh UAS-dimm
elav-Gal4>UAS-cas UAS-grh
elav-Gal4>UAS-cas grhIM
elav-Gal4>UAS-p35
Df(3L)H99
spdoG104
mamGA345
elav-Gal4>UAS-Nintra
numb1
mamGA345 elav-Gal4>UAS-p35
elav-Gal4>UAS-cas Df(3L)H99
chipe5.5
da1
dimmrev8
el3.3.1 nocD64
esg35Ce-1
grn72
htl
jumu11.683
mld97
pnrv1e
stc05441
vn10567
wor1
zfh100865
zfh2LP30-Gal4>UAS-zfh2RNAi
nabSH143/nabR52
klu212IR51C
elav-Gal4>UAS-nab
elav-Gal4>UAS-nab UAS-cas
elav-Gal4>UAS-nab casD1/casD3
elav-Gal4>UAS-cas nabR52
sqzie
elav-Gal4>UAS-klu
elav-Gal4>UAS-klu UAS-p35
elav-Gal4>UAS-cas klu212IR51C
elav-Gal4>UAS-nab klu212IR51C
nabR52 sqzGal4/nabR52 sqzie
7
7
4.8
6.6
0
2.6
14.6
6.8
7.8
11.5
12.4
21
11.5
13.2
1
0
8.8
11.5
13.5
19
6.8
6.5
4.9
6.3
6.3
6.8
6.5
4.4
6.1
6.6
6.5
6.7
6.6
6.6
6.6
0
0
8.1
13.7
0
25
7
0
3.5
21
8.4
7.8
45
11
9
31
14
20
4
18
18
6
10
15
10
6
2
6
8
21
6
4
13
18
10
16
10
8
12
20
18
16
14
13
8
6
5
10
18
8
16
30
20
15
20
6
8
19
32
4I
4J
5A
5B
5C
5D
5E
5F
5G
5H
6A
6B
6C
6D
6E
6F
6G
6H
5I
6J
7A
7B
7E
7G
7H
7I
7J
7M
7N
7Q
Average number of ABLKs found per hemiganglion () and number of hemiganglia
analyzed (n) in each experiment. The column on the left indicates the figure showing
each result.
not increase the phenotype observed, misexpressing only cas (not
shown). We also co-misexpressed cas and grh (elav-Gal4>UAS-cas
UAS-grh) and observed that the number of ABLKs does not increase
more than when misexpressing only cas (Fig. 4J; Table 1);
considering that Cas activates grh, this result is not surprising
(Baumgardt et al., 2009). However, when we misexpressed cas in
grh mutants (elav-Gal4>UAS-cas grhIM), strikingly, we observed
extra ABLKs (Table 1; data not shown) (see Discussion).
The role of the Notch pathway in the specification
of ABLK fate
The GMC is programmed to divide only once and generate two
invariant cells that will differentiate as neurons or glia, or undergo
apoptosis (reviewed by Karcavich, 2005). To determine the fate of
Fig. 5. Requirement for Notch signaling in ABLK specification.
(A-H)Expression of Lk in elav-Gal4 UAS-p35 (A), Df(3L)H99 (B),
spdoG104 (C), mamGA345 (D), elav-Gal4>UAS-Nintra (E), numb1 (F),
mamGA345 elav-Gal4>UAS-p35 (G), and elav-Gal4>UAS-cas Df(3L)H99
(H) in ventral ganglia of Drosophila first instar larvae. Arrows in G point
to two duplicated ABLKs. (I)Schematic summarizing these results.
the ABLK sibling cell, we labeled Lk expression in ganglia in
which programmed cell death (PCD) was repressed by the
overexpression of the baculovirus caspase inhibitor p35 (elavGal4>UAS-p35) (Hay et al., 1994). We observed that the ABLK
neuron was duplicated in many hemisegments (Fig. 5A; Table 1).
A stronger result was obtained in individuals homozygous for the
deficiency Df(3L)H99; deletion of this region completely abolishes
PCD (White et al., 1994) (Fig. 5B; Table 1). These results indicate
that the ABLK sibling cell dies by apoptosis but forms an ABLK
if apoptosis is inhibited.
It has been shown in several lineages that Notch signaling
between the two GMC daughter cells is required for them to
assume different cell fates (Lundell et al., 2003; Schuldt and Brand,
1999; Skeath and Doe, 1998; Spana and Doe, 1996). It has also
been reported that Notch signaling drives PCD in postmitotic cells
in a lineage-specific manner (Karcavich and Doe, 2005; Lundell et
al., 2003; Novotny et al., 2002). To study the effect of removing
Notch on ABLK fate we looked at the expression of Lk in sanpodo
(spdo) mutants. Spdo is required for Notch signaling during
asymmetric cell division but permits Notch signaling during early
neurogenesis (Babaoglan et al., 2009; Dye et al., 1998; O’ConnorGiles and Skeath, 2003; Skeath and Doe, 1998). In spdoG104 mutant
ganglia, the number of ABLKs was strongly reduced (Fig. 5C;
Table 1). The same result was obtained in mastermind (mam)
mutant embryos (Fig. 5D). Mam is a transcriptional co-factor that
interacts with the Notch intracellular domain (Petcherski and
DEVELOPMENT
Fig.
Kimble, 2000a; Petcherski and Kimble, 2000b). We next
overexpressed a constitutively active form of Notch (elavGal4>UAS-Nintra) (Rebay et al., 1993; Struhl et al., 1993) and
observed an increased number of ABLKs (Fig. 5E). The same
result was obtained in numb mutant embryos (Fig. 5F; Table 1).
With respect to these results, we consider two alternative
scenarios: one is that Notch signaling is required to activate
ABLK neuronal fate which then represses apoptosis; the other,
that Notch signaling does not play an instructive role in ABLK
specification, but directly represses PCD in one of the two cells
that have the potential to acquire the ABLK fate. Our results
support the second hypothesis as p35-overexpressing embryos
contained extra ABLK. To test this notion, we analyzed the
expression of Lk in embryos in which Notch signaling was
compromised and PCD was simultaneously repressed (mamGA345
elav-Gal4>UAS-p35), and observed extra ABLKs (Fig. 5G; Table
1). This result suggests that (1) once PCD is suppressed, Notch
signaling has no function and (2) that the specification of the
ABLK fate does not require Notch signaling. Thus, we conclude
that both of the postmitotic sibling cells have the potential to be
ABLKs, but are fated to die unless Notch signaling is activated
and rescues one of them (Fig. 5I).
We have observed that cas misexpression duplicates the number
of ABLKs (Table 1; 14.6), indicating that an additional cell per
hemisegment takes on the ABLK fate. The same result was
observed in Df(3L)H99 embryos (13.2), indicating in this case
that the lethality of the ABLK sibling cell is rescued. When we
misexpressed cas in Df(3L)H99 embryos, we expected to obtain
four times the number of ABLKs, and, although in some
hemineuromers we found this result, as an average their number
only tripled (Fig. 5H; 19). We consider that these three cells
probably correspond to the normal ABLK, its sibling that is
rescued by the lack of cell death, and an extra ABLK originated by
the cas misexpression that has no sibling cell.
The genes nab and klumpfuss are required to
specify ABLK neuronal fate
There is increasing evidence that neuronal fate specification is a
multistep process involving combinatorial gene expression codes
that specify neuronal properties (Allan et al., 2005; Baumgardt et
al., 2007; Certel and Thor, 2004; Garces and Thor, 2006). In order
to identify genes involved in specification of the ABLK fate, we
analyzed the expression of Lk in embryos mutant for genes known
to be required for CNS development. We identified a number of
mutants in which the pattern of ABLKs was not altered: crooked
legs (crol), defective proventriculus (dve), lozenge (lz), osa, rotund
(rn), seven up (svp), tailup (tup) and vestigial (vg) (data not
shown). There were other mutants in which the number of ABLKs
was reduced: chip, daughterless (da), elbow/no oceli (el/noc),
escargot (esg), grain (grn), heartless (htl), jumeau (jumu), molting
defective (mld), pannier (pnr), shuttle craft (stc), vein (vn), worniu
(wor), zinc finger homeodomain 1 (zfh1) and zinc finger
homeodomain 2 (zfh2) (Fig. 6A-H; Table 1); it is possible that the
maternal effect was the cause of the weak phenotype observed in
these mutations. In some mutants ABLKs were completely absent:
nab and klumpfuss (klu) (Fig. 7A-B; Table 1). We focused our
analysis on these last two genes.
nab encodes a nuclear co-factor without a DNA-binding domain
(Svaren et al., 1996), whereas klu encodes a zinc-finger protein
(Klein and Campos-Ortega, 1997). Both nab and klu are expressed
in ABLKs at stage 18 (Fig. 7C-D); klu is expressed in the newborn
NB5-5 in late stage 11 (Yang et al., 1997), but we did not observe
Development 137 (19)
Fig. 6. Mutant background with altered pattern of ABLK.
(A-J) Expression of Lk in the ventral ganglia of Drosophila first instar
larvae mutant for chipe5.5 (A), da1 (B), el3.3.1 noc64 (C), esg35Ce-1 (D),
grn72 (E), htl (F), jumu11.683 (G), mld97 (H), pnrv1e (I) and vn10567 (J).
Percentage of ABLKs present compared with wild type (100%) is
indicated.
expression of Nab in NB5-5 at this stage (data not shown).
Overexpression of nab with a pan-neural driver (elav-Gal4>UASnab) increased the number of ABLKs (Fig. 7E; Table 1) and these
extra ABLKs expressed Cas (Fig. 7F). In cas mutants, there is a
complete loss of nab RNA and protein expression, with the
exception of Nab midline expression (Baumgardt et al., 2009;
Clements et al., 2003). We expressed nab in a cas mutant (elavG4>UAS-nab casD1/casD3), but found no evidence of rescue (Fig.
7G). We next overexpressed Cas in a nab mutant (elav-G4>UAScas nabR52) and found ectopic ABLKs (Fig. 7H). To determine
whether or not these ectopic ABLKs belong to the NB5-5 lineage,
i.e. if Cas overexpression could rescue nab, we analyzed the
expression of gsb, lbe and Runt. We observed that, similar to the
wild-type ABLKs, all the extra ABLKs expressed gsb and Runt,
but did not express lbe (Fig. 7O-P). These results indicate that they
are generated by a row five or six NB that is not NB5-6. As all
these neurons appear clustered, we conclude that they are most
probably generated by NB5-5 and, therefore, that Cas acts either in
parallel to, or downstream of, Nab in ABLK specification (see
Discussion).
Next, we wondered whether Nab and Klu could crossregulate
one another at the level of transcription, but observed that
expression of neither gene was altered in embryos mutant for the
other (data not shown).
We analyzed the phenotype resulting from klu misexpression
(elav-Gal4>UAS-klu) and, surprisingly, found that all the ABLKs
were missing (Fig. 7I). Since Klu is involved in PCD during retinal
development (Rusconi et al., 2004; Wildonger et al., 2005), we
attempted to rescue the phenotype of klu mutants with elavGal4>UAS-p35 or Df(3L)H99 and found that the pattern of ABLKs
was partially restored (Fig. 7J), indicating that klu overexpression
has a deleterious effect.
To determine whether the expression of klu depends on cas, we
analyzed the expression of klu in the cas mutant (klu-lacZ
casD1/casD3); klu expression appeared normal in midline cells but
failed to spread to NBs (Fig. 7K-L). We next assessed whether cas
misexpression rescues the klu phenotype of a lack of ABLKs (elav-
DEVELOPMENT
3332 RESEARCH ARTICLE
Neuropeptidergic cell fate specification
RESEARCH ARTICLE 3333
Fig. 7. Nab and Klu are required for ABLK specification.
(A,B)Expression of Lk in nabSH143/nabR52 (A) and klu212IR51C (B) in ventral
ganglia of Drosophila first instar larvae. (C,D)Coexpression of Lk (red)
and nab-lacZ (C; green) and klu-lacZ (D; green) in first instar ganglia.
(E,F)Expression of Lk (E), and Cas (green) and Lk (red) (F) in elavGal4>UAS-nab. (G-J)Expression of Lk in elav-Gal4>UAS-nab cas1/cas4
(G), elav-Gal4>UAS-cas nabR52 (H), elav-Gal4>UAS-klu (I), and elavGal4>UAS-klu UAS-p35 (J). (K,L)Anti--galactosidase staining (green)
in klu-lacZ/+ (K) and klu-lacZ cas1/cas4 (L) embryos. (M,N)Lk
expression in elav-Gal4>UAS-cas klu212IR51C (M) and elav-Gal4>UAS-nab
klu212IR51C (N). (O,P)High magnification of single hemineuromers
(outlined) in elav-Gal4>UAS-cas showing the expression of Lk (red) and
gsb-lacZ (O) and Runt (P) (green). (Q)Expression of Lk in nabR52
sqzGal4/nabR52 sqzie. In C,D,O-P the separate channels are shown.
Gal4>UAS-cas klu212IR51C) and indeed we observed up to 24
ABLKs per hemisegment (Fig. 7M; 21), which suggests that Cas
acts either in parallel to, or downstream of, Klu. We also observed
that nab overexpression in klu mutant embryos (elavG4>UAS-nab
klu212IR51C) rescued expression of Lk (Fig. 7N; Table 1).
Nab is required to block the repressive activity of
Squeeze on the ABLK fate
nab plays a role in specification of the FMRFamide-expressing Tv
neuron of the dorsal apterous cluster and physically interacts with
the zinc-finger transcription factor Squeeze (Sqz) (Baumgardt et al.,
2009; Terriente et al., 2007). Since sqz is expressed in the ABLKs
(Herrero et al., 2007), we tested whether the ABLK pattern was
affected in sqz mutants and in nab sqz double mutants. No phenotype
was observed in sqz embryos but, surprisingly, nab sqz showed an
almost wild-type pattern of ABLKs (Fig. 7Q) (see below).
ABLKs are specified in the Cas/Grh temporal
window
Recent findings on NB5-6 demonstrate that Cas and Grh act as
crucial temporal genes to specify several cell fates at the end of this
lineage (Baumgardt et al., 2009). Our findings with NB5-5 reveal
similar roles for Cas and Grh, and indicate that the ABLKs are
specified in a Cas/Grh temporal window. We have observed that
cas mutants generate no ABLKs, that cas misexpression leads to
clusters of two to four ABLKs per hemisegment, and that Cas is
expressed in all the ABLKs. Thus, our data confirm that cas plays
a role as a temporal identity gene, which remains compatible with
its proposed role as a switching temporal factor (Tran and Doe,
2008).
The proposed role of grh as a temporal identity gene remains
open to question. It has also been reported that it is required to
regulate mitotic activity and apoptosis of post-embryonic NBs
(Almeida and Bray, 2005; Cenci and Gould, 2005; Maurange et al.,
2008). However, recent evidence has emerged indicating that Grh
also temporally regulates FMRFamide neuropeptide cell fate and
can act in a combinatorial manner with dimm and apterous to
trigger ectopic FMRFamide expression (Baumgardt et al., 2009).
Similarly, we find here that Grh is required for correct specification
of the ABLKs. Together, these results suggest that Grh also
plays an instructive role in ABLK specification. Thus, many
neuropeptidergic neurons are generated late in several lineages, and
depend upon the late temporal genes cas and grh for their
specification.
We have shown that NB5-5 does not express the temporal genes
hb and Kr, and genetic analysis confirms that these two genes are
not required for specification of ABLK fate. We have observed that
NB5-5 initially expresses Pdm at the time of delamination in late
stage 11. Pdm is downregulated at early stage 12, when Cas is
activated, and there is a brief period in which both proteins can be
detected. The lack of molecular markers does not permit us to
determine whether the Pdm/Cas coexpression stage generates a
GMC.
It is of interest to note that the phenotype observed
misexpressing cas with NB-specific drivers was very mild
(inscuteable-Gal4: 8.1, n18; worniu-Gal4: 7.3, n28)
compared with that obtained using a pan-neuronal driver (elavGal4: 14.6). NB5-5 expresses cas soon after delamination and
generates six to nine neurons (Schmid et al., 1999; Schmidt et
al., 1997). This suggests that NB5-5 probably has a broad Cas
temporal window. Thus, the phenotype obtained upon
misexpressing cas with elav-Gal4 indicates that Cas might have
a later requirement in postmitotic cells that generates
subtemporal windows. Consistent with this interpretation, cas
misexpression rescues the grh phenotype of loss of ABLKs,
which also suggests that Grh, in addition to being required as a
temporal factor, would be indirectly required to activate cas
expression in postmitotic cells.
DEVELOPMENT
DISCUSSION
To better understand the mechanisms involved in the generation of
cellular diversity in the CNS, we performed an analysis of the
specification of ABLK neuronal fate. The small number of ABLKs
present in the ventral ganglia of first instar larvae is easily
identifiable. Although the lack of molecular markers does not
permit identification of the complete repertoire of fates generated
by the ABLK progenitor NB, we have been able to draw
significant conclusions concerning the specification of ABLK
neuronal fate.
Notch signaling prevents death of the ABLK-fated
cells
The Notch pathway is involved in many cell fate decisions in neural
development (reviewed by Cau and Blader, 2009). Here, we have
shown that the ABLK and its sibling are equivalent cells committed
to die, and that activation of the Notch pathway in the ABLK
prevents its death. A similar situation has been described for
specification of the anterior and posterior Corner Cells (CaCC/pCC)
neurons in the grasshopper NB1-1 lineage, in which the siblings start
as equivalent cells and interaction between them leads to different
fates (Kuwada and Goodman, 1985). By contrast, activation of
Notch in the NB7-3 lineage drives PCD (Karcavich and Doe, 2005).
Here, activation of the Notch pathway, or misexpression of p35 in
the sibling cell, is sufficient to generate two ABLK neurons. A
systematic analysis of the lineage of apoptotic cells in embryos in
which apoptosis is prevented has shown that the lineage of
abdominal NB5-5 contains twice the normal number of cells, but that
they have wild-type-like axonal projections (Rogulja-Ortmann et al.,
2007). These findings are in agreement with ours. We conclude that
in this lineage, Notch does not play an instructive role in specifying
ABLK neuronal fate, but influences a fate decision by regulating the
competence to respond to a program of cell death.
A genetic cascade involving Cas and Nab specifies
ABLK neuronal fate
We have identified a set of mutants that produce an altered number
of ABLKs. In most cases the effect is very mild. Among the mutants
with the strongest phenotypes we highlight jumu, nab and klu. The
jumu phenotype was expected because it has been shown that Jumu
is required in the NB4-2 lineage for normal segregation of Numb in
the asymmetric cell divisions (Cheah et al., 2000). Consistent with
this interpretation, the fact that the phenotype of jumu in the NB5-5
lineage is similar to that seen in spdo explains its phenotype and
indicates that in jumu embryos Notch is off in both siblings.
nab and klu embryos display a strong reduction in the number of
ABLKs, suggesting that both genes have direct roles in ABLK
specification. Interestingly, Cas activates the expression of both
genes via repression of Pdm. The lack of availability of markers for
identifying ABLKs in earlier stages did not permit us to establish
whether Nab and Klu are required in the NB or in postmitotic cells.
We did observe that misexpression of cas in nab embryos
showed ectopic ABLKs, suggesting that Cas acts either parallel to,
or downstream of, Nab. The lack of molecular markers specific to
the NB5-5 lineage does not allow us to determine whether all
ectopic ABLKs are generated by the NB5-5 or by other lineages.
Nevertheless, several results suggest that, most probably, all of
them are produced by NB5-5. First, it has been observed that
neurons that belong to one lineage form a coherent cluster
(Dumstrei et al., 2003), and we find that ectopic ABLKs appear
clustered (see Fig. 4G and insets in Fig. 7H,M). Second, all of them
express gsb, which labels rows five and six NB, and do not express
lbe, an NB5-6-specific marker. Third, ABLKs are the unique cells
expressing Lk in the ventral ganglion. However, we have not
observed downregulation of cas in nab mutants, and the same has
been reported in the better characterized lineages of NB3-3 and
NB5-6 (Baumgardt et al., 2009; Tsuji et al., 2008); together, these
results indicate that the molecular relationship between Cas and
Nab requires a more complex interpretation than a linear genetic
cascade.
klu encodes a zinc-finger protein but does not appear to interact
directly with Nab (Clements et al., 2003), and we did not find
evidence that nab and klu regulate each other. Surprisingly, we
Development 137 (19)
have found that nab misexpression rescues the phenotype of a lack
of ABLKs observed in klu. By contrast, we have found that cas
misexpression produces more ABLKs in grh, nab or klu (21, 25
and 21, respectively) than in wild-type background (14.6). As
proposed above, these results suggest that Cas plays a role in
postmitotic cells that is crucial for ABLK specification.
We have observed that the sqz phenotype is epistatic over the nab
phenotype. Thus, although nab embryos have no ABLKs, sqz and
nab sqz show a normal pattern of ABLKs. It has been shown by pulldown assay that Nab physically interacts with Sqz (Terriente et al.,
2007), and in vertebrates the Nab homologs act as transcriptional cofactors. Since both genes, sqz and nab, are expressed in the ABLKs,
we propose that the function of Sqz in NB5-5 lineage is to repress
the ABLK fate. In normal development, as both genes are expressed
in the ABLKs, Nab binds to Sqz and blocks its repressor activity; in
nab embryos Sqz represses the ABLK fate, but in nab sqz the pattern
is wild-type because there is no repression by Sqz. This intimate
interplay between Sqz and Nab is also found in the NB 5-6 linage,
in which sqz is first required to activate cell fate determinants, and
then acts with nab to suppress the same determinants (Baumgardt et
al., 2009).
The findings reported here extend our understanding of the
mechanisms of ABLK specification. However, more precise
analysis of the genes and the mechanisms involved in specification
of the different cell fates in the NB5-5 lineage will require
additional molecular markers. This would permit us to identify the
different neurons generated from this NB and the genes required to
specify their various fates.
Acknowledgements
We are grateful to A. Brand, M. Calleja, J. Castelli-Gair, W. Chia, S. Cohen, C.
Doe, R. Hewes, Y. Hiromi, R. Holmgren, T. Isshiki, D. Nässel, W. Odenwall, P.
Taghert and G, Technau for flies and reagents and to P. Herrero for comments
on the manuscript. This work was supported by grants from the Ministerio de
Educación y Ciencia (BFU2005-00116 and BFU2005-00116), the Ministerio de
Ciencia e Innovación (BFU2008-04683-C02-01 and CSD2007-00008) and an
institutional grant from the Fundación Ramón Areces to the CBM-SO to F.J.D.B., and by the Swedish Research Council, the Swedish Strategic Research
Foundation, the Knut and Alice Wallenberg foundation, the Swedish
“Hjärnfonden”, “Cancerfonden” and the Swedish Royal Academy of Sciences,
to S.T.
Competing interests statement
The authors declare no competing financial interests.
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