First published online 15 March 2006
doi: 10.1242/dev.02321
Development 133, 1445-1455 (2006)
Published by The Company of Biologists 2006
Specification of Drosophila aCC motoneuron identity by a genetic cascade involving even-skipped, grain and zfh1
Alain Garces1 and
Stefan Thor2,*
1 INSERM U 583, INM-Hopital St Eloi, 80 rue Augustin Fliche, 34091 Montpellier
Cedex 5, France.
2 Division of Molecular Genetics, Department of Physics, Chemistry and Biology,
Linkoping University, S-581 83 Linkoping, Sweden.
*
Author for correspondence (e-mail:
steth{at}ifm.liu.se)
Accepted 10 February 2006
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SUMMARY
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During nervous system development, combinatorial codes of regulators act to
specify different neuronal subclasses. However, within any given subclass,
there exists a further refinement, apparent in Drosophila and C.
elegans at single-cell resolution. The mechanisms that act to specify
final and unique neuronal cell fates are still unclear. In the
Drosophila embryo, one well-studied motoneuron subclass, the
intersegmental motor nerve (ISN), consists of seven unique motoneurons.
Specification of the ISN subclass is dependent upon both even-skipped
(eve) and the zfh1 zinc-finger homeobox gene. We find that
ISN motoneurons also express the GATA transcription factor Grain, and
grn mutants display motor axon pathfinding defects. Although these
three regulators are expressed by all ISN motoneurons, these genes act in an
eve
grnzfh1 genetic cascade unique to one of the ISN
motoneurons, the aCC. Our results demonstrate that the specification of a
unique neuron, within a given subclass, can be governed by a unique regulatory
cascade of subclass determinants.
Key words: Axon pathfinding, Even-skipped, Grain, Neuronal fate specification, Combinatorial code, Drosophila
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INTRODUCTION
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During the past decade, motoneuron specification has been intensely studied
and work from both invertebrates and vertebrates has shown that motoneuron
subclass identity is determined by combinatorial transcription factor codes
(Briscoe and Ericson, 2001
;
Shirasaki and Pfaff, 2002;
Thor and Thomas, 2002).
However, how individual identities, within a related pool of motoneurons, are
determined is still not understood. In the abdomen of the developing
Drosophila embryo, reiterated sets of 
80 motoneurons are
generated in each segment of the ventral nerve cord (VNC). These motoneurons
project along distinct nerves to innervate peripheral target muscle fields
and, based upon their peripheral axonal projections, they are typically
grouped into six well-defined classes
(Landgraf et al., 1997). The
motor nerve innervating the dorsal-most muscle field, the intersegmental nerve
(ISN), contains axons from seven well-defined motoneurons; the aCC, RP2 and
the five U motoneurons, each with a well-defined and specific muscle target
(Jacobs and Goodman, 1989;
Johansen et al., 1989;
Landgraf et al., 1997). The
even-skipped (eve) regulatory gene is specifically expressed
in ISN motoneurons and eve is both necessary and sufficient for ISN
motor axon pathfinding (Landgraf et al.,
1999). However, eve is expressed in all ISN motoneurons
and is cell-autonomously crucial for their axonal exit out of the VNC
(Fujioka et al., 2003). Recent
studies reveal that the zinc-finger/homeodomain gene zfh1 is also
expressed by ISN motoneurons (Layden et
al., 2006). However, zfh1 is expressed by most if not all
motoneurons, and important for many motor axons to exit the VNC. Together,
these results suggest that regulators other than eve and
zfh1 are necessary to explain the specification of each individual
ISN motoneuron identity.
To gain further insight into motoneuron specification, we have addressed
the role of the Drosophila GATA transcription factor grain
(grn). We find that grn is specifically expressed within the
ISN motoneuron subclass and plays a crucial role for ISN axon projections.
Genetic analysis reveals that the regulatory interplay between eve,
grn and zfh1 varies between the different ISN motoneurons.
Within the postmitotic aCC motoneuron, these three regulators act in a unique
evegrnzfh1 genetic cascade that is crucial for the
correct specification of aCC identity. Misexpression of zfh1
(Layden et al., 2006) or
co-misexpression of eve with grn, can trigger lateral axonal
exit from the ventral nerve cord. grn and zfh1 are,
furthermore, sensitive to Notch signaling within this ISN motoneuron, whereas
they are insensitive to Notch in other ISN motoneurons. These findings reveal
the existence of a unique genetic program for the aCC motoneuron fate,
consisting of factors expressed by all ISN motoneurons.
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MATERIALS AND METHODS
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Drosophila stocks
The grnlacZ allele l(3)05930 was identified in
a survey of the BDGP lacZ collection
(Spradling et al., 1999) for
lines with restricted expression pattern in the embryonic VNC.
grnGAL4 was generated by P element conversion of
grnlacZ as previously described
(St Pierre et al., 2002). For
grn mutant analysis, grn7l and
grnSPJ9 (Brown and
Castelli-Gair Hombria, 2000) were placed over deficiency
Df(3R)dsx3, and both allelic combinations showed the same pathfinding
phenotype and no detectable Grn expression (not shown). For grn
misexpression and rescue experiments, we used UAS-grn#2
(Brown and Castelli-Gair Hombria,
2000). UAS-mEGFPF is a c-myc epitope-tagged
membrane-targeted EGFP reporter line (Allan
et al., 2003). Other lines used were:
islet-
-myc-EGFP (S.T., unpublished); RN2-GAL4, CQ2-GAL4,
Df(2R)eve,
RP2A/CyO,P[wg-lacZ];RN2-GAL4,UAS-lacZ,
Df(2R)eve/CyO,P[wg-lacZ];RP2B
(Fujioka et al., 2003);
UAS-eve and eveID19
(Landgraf et al., 1999).
zfh12, zfh165.34, zfh175.26 alleles
were obtained from R. Lehmann and UAS-zfh1 from the Bloomington stock
center. Hb9GAL4, Hb9KK30, UAS-vnd,
maml(2)04615, spdoG104 were provided by J. B.
Skeath and H. T. Broihier. UAS-NotchICD was obtained from
S. Artavanis-Tsakonas.
Quantification of pathfinding phenotypes
ISN motor axonal projections were scored at embryonic stage 16/17 in A2-A6
abdominal hemisegments using anti-Fasciclin 2,
RN2-GAL4/UAS-mEGFPF or
CQ2-GAL4/UAS-mEGFPF. Phalloidin-Texas Red (Molecular
Probes) was used to visualize the musculature.
Antibody production and staining of embryos
grn cDNA encoding amino acids 1-166 was cloned into pGEX-2T
(Amersham) for protein expression and purification (J. Castelli-Gair Hombria,
unpublished). Fusion protein was used to immunize rabbits and rats (Covance).
Grn antibodies were used at 1:200 and their specificity was verified by the
absence of staining in grn mutants. Immunolabeling was carried out as
previously described (Thor et al.,
1999). The following antibodies were used: 
-c-Myc 9E10
(1:50), -Fas2 1D4 (1:50), -Even skipped 2B8 (1:5) and
-ß-gal 40-1a (1:10) (all from Developmental Studies Hybridoma
Bank). Rabbit -ß-gal (Cappel; 1:5,000), rabbit -pMad
(Tanimoto et al., 2000)
(1:2,000), rabbit -Zfh1 (Van Doren
et al., 2003) (1:5,000), rabbit -Hb9
(Broihier and Skeath, 2002)
(1:500) and rabbit -Vnd (Shao et
al., 2002) (1:1,000). Double-labeled images were false colored for
the benefit of color-blind readers. Prior to use, the polyclonal
-ß-gal, -pMad, -HB9, -Vnd and -Grn antibodies were pre-absorbed
against early-stage wild-type embryos.
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RESULTS
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grain is expressed in subsets of developing motoneurons and interneurons
To identify genes controlling motoneuron specification, we analyzed the
expression patterns of a number of lacZ enhancer trap lines,
surveying for lines with expression in the embryonic VNC (see Materials and
methods). One line that showed restricted expression in subsets of cells in
the VNC is an insertion in the grain (grn) gene.
grn encodes a GATA transcription factor previously shown to control
cell rearrangements in the developing leg imaginal disc and in the posterior
spiracle (Brown and Castelli-Gair Hombria,
2000). Previous studies revealed that grn expression
commences at the cellular blastoderm stage, and rapidly becomes localized to
the dorsal part of the embryo, being most prominent in the procephalic region.
From stage 11, expression is evident in the posterior spiracles, in the midgut
and in a patch of cells in the lateral ectoderm
(Brown and Castelli-Gair Hombria,
2000; Lin et al.,
1995b). We generated Grn-specific antibodies and found that the
expression of Grn closely matches the grnlacZ and
grnGAL4 reporter expression in these structures (not
shown), as well as in the VNC (Fig. 1A1-3,
1E1-3; not shown).
In the VNC, grn expression commences at early stage 12. The
position and morphology of grnlacZ- and
grnGAL4-expressing cells suggested a postmitotic and
neuronal identity. Using grnGAL4/UAS-lacZ, we
observed that grn is expressed in a diverse set of interneurons and
motoneurons that extend axons along the major axon tracts
(Fig. 1B,F). Double labeling
with the glial-specific marker Repo showed that, with the exception of one
dorsal glial cell per hemisegment (Fig.
1I), Grn (and grnlacZ or
grnGAL4) expression is restricted to neurons. To resolve
the identity of grn-expressing neurons further, we assayed for
overlap with regulators known to be expressed in restricted sets of neurons,
such as isl, lim3, Hb9, zfh1, apterous and even-skipped
(eve) (Fig. 1B-D,F-H;
not shown). Of these genes, only eve and zfh1 showed
apparent overlap with grn, specifically in the intersegmental nerve
(ISN) motoneurons: aCC, RP2 and the five Us (U1-5 or CQ)
(Fig. 1D,H). The ISN
motoneurons are born during early embryogenesis with aCC and RP2 born at stage
9, and the U motoneurons born sequentially during stage 9-11
(Broadus et al., 1995;
Doe et al., 1988a;
Weigmann and Lehner, 1995).
Expression of grn and Grn in ISN motoneurons commences at stage
11-12, subsequent to Eve expression, and expression of grn and Grn is
maintained in ISN motoneurons into larval stages (not shown). Thus,
grn is expressed in subsets of interneurons, and in a distinct
subclass of motoneurons that innervate the dorsal-most muscles in the
Drosophila embryo (Fig.
1J).
grain is required for ISN motor axon pathfinding
To determine if grn plays a role in ISN motoneuron specification,
we analyzed motor axon projections in grn mutants. In
Drosophila embryos, motor axonal projections are stereotyped and can
be revealed using an antibody directed against the surface molecule Fasciclin
2 (Fas2) (Vactor et al.,
1993). The aCC and U1 motor axons are known to innervate the
dorsalmost muscles 1 and 9, respectively, while the RP2 and U2 motor axons
innervate the dorsal muscles 2 and 10 respectively
(Fig. 1J)
(Jacobs and Goodman, 1989;
Johansen et al., 1989;
Landgraf et al., 1997). Fas2
reveals the high reproducibility of these projections in the wild-type embryo
(Vactor et al., 1993)
(Fig. 2A; 100% innervation,
n=96; throughout the text, n refers to the numbers of
hemisegments counted). In grn mutants, we find that the ISN motor
axons are stalled at muscles 2/10, leading to a near complete loss of
innervation of the dorsal-most muscles 1/9 (12% innervation; n=136)
(Fig. 2B). To better resolve
the grn pathfinding phenotype we used both an aCC/RP2-specific and a
U-specific GAL4 driver line (RN2-GAL4 and CQ2-GAL4,
respectively) (Fujioka et al.,
2003; Landgraf et al.,
2003) and expressed a membrane targeted EGFP reporter
(UAS-mEGFPF) (Allan et
al., 2003). In the wild type,
RN2-GAL4/UAS-mEGFPF clearly visualizes the peripheral
projections of aCC and RP2 onto muscles 1 and 2 (arrow and arrowhead,
respectively, in Fig. 2D), as
well as their terminal processes (Fig.
2G). In grn mutants, muscle 2 is innervated with near
wild-type frequency, but, by contrast, muscle 1 is innervated in only 15% of
hemisegments (n=146) (Fig.
2E,K). Using CQ2-GAL4/UAS-EGFPF in
grn mutants, we observed a similar phenotype - apparently normal
innervation of muscles 2/10 but only 18% muscles 1/9 innervation
(n=88) (Fig. 2I,J,K).
In addition, using Fas2, RN2-GAL4 or CQ2-GAL4 as markers, we
noticed aberrant projections onto muscle 8
(Fig. 2B,E,H,J). We quantified
this phenotype using RN2-GAL4 or CQ2-GAL4, and found that
whereas control embryos (RN2-GAL4/UAS-EGFPF or
CQ2-GAL4/UAS-EGFPF) displayed no innervation of muscle 8
(0%; n=87 and n=72, respectively), grn mutants
displayed frequent innervation of muscle 8. This phenotype was observed more
often with RN2-GAL4 than with CQ2-GAL4 as marker (35%;
n=140 versus 21%: n=146). In affected hemisegments, we
observed a grossly normal pattern of axonal projections to the dorsal muscles
2/10 (Fig. 2B,E,J). This
indicates that aCC and/or RP2, and at least one of the U motoneurons project
aberrantly to muscle 8. These results show that grn is crucial for
proper motor axon pathfinding of ISN motoneurons.
grain acts cell-autonomously in ISN motoneurons
Although grn is expressed in ISN motoneurons, it is also expressed
in a patch of ectodermal cells in the lateral body wall that underlie the SNa
muscle field, muscles 21-24 (Brown and
Castelli-Gair Hombria, 2000) (not shown). In grn mutants,
we observe a partially penetrant muscle patterning phenotype, evident as an
imprecise insertion of muscles 21-24 into the body wall
(Fig. 2A-F,I,J). Although the
ISN motoneurons do not normally innervate this muscle field, it still raised
the concern that the motor axon pathfinding defect observed in grn
mutants may not result from a cell-autonomous role for grn in ISN
motoneurons. To address this issue, we used the RN2-GAL4 and
CQ2-GAL4 drivers to provide grn activity in aCC/RP2 and U
motoneurons, respectively. We find that RN2-GAL4 efficiently rescues
grn mutant axon pathfinding (100% muscle 1/9 innervation;
n=88) (Fig. 2C,K). By
contrast, the CQ2-GAL4 driver only partially rescued the grn
phenotype; 54% of muscles 1/9 (n=132)
(Fig. 2K). Together, these
results show that grn acts cell-autonomously in ISN motoneurons to
ensure proper axon pathfinding to the dorsal-most muscles
(Fig. 2L,M).
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Fig. 1. Grain is expressed in ISN motoneurons and in subsets of
interneurons. Stage 14 (A-H) and stage 12 (I) embryos
stained for Grn (A,E,I), Repo (I), ß-gal (A,C,E,G) and Myc (B,C,D,F,G,H).
Dorsal (A-D,I), mid-dorsal (C') and intermediate (E-H) focal planes of
the VNC. Anterior is upwards in all panels. Grn expression within one
(C,C',I), two (A,E) or three (B,C,F-H) segments.
grnlacZ is expressed in all Grn-positive neurons (A,E). We
noticed consistently weaker expression of Grn and grnlacZ
(or grnGAL4) in the RP2 motoneuron (arrowhead) compared
with other grn-expressing neurons (A,D). Mutually exclusive
expression patterns of grnGAL4/UAS-lacZ with
islet-mycEGFP (B,F), and with Hb9-GAL4 (C,C',G)
in subsets of motoneurons and interneurons. Overlap of Grn with Eve and Zfh1
in aCC, RP2 (D) and the U motoneurons (asterisks, H). The pCC interneuron,
which is located posterior to aCC does not express grnlacZ
(A) or Grn (D). In stage 12 embryos, we find overlap of Grn and Repo (I) in
one glia cell (*). This glia cell rapidly becomes Grn negative at
later stages (compare with A1) but maintains ß-gal expression when probed
with grnlacZ (A2) probably owing to the stability of the
ß-gal protein. (J) Schematic showing grn-expressing cells
in the VNC and the grn-expressing ISN motor axon projections in the
periphery. The five U motoneurons are depicted in dark brown.
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An evegrnzfh1 regulatory cascade in the aCC motoneuron
eve is expressed in a small subset of transiently identified GMC
(ganglion mother cell) and derived aCC, RP2 and U motoneurons. Studies show
that eve is both necessary and, at least in part, sufficient for
dorsal motor axon projections (Landgraf et
al., 1999). Given that eve and grn show similar
mutant phenotypes in dorsally projecting motoneurons, we wanted to address
whether these two genes regulate each other or act at the same genetic level.
As eve-null mutants display severe segmental defects, a
temperature-sensitive (ts) allele (eveID19), was
previously used to study the role of eve in motoneuron specification
(Landgraf et al., 1999).
However, recent studies have shown that the eve ts allele does not
completely remove eve function in ISN motoneurons. Using a
sophisticated strategy, Fujioka et al. have succeeded in restoring
eve function in all eve-expressing cells, except in the aCC
and RP2 neurons, in an otherwise eve-null background
(Fujioka et al., 2003). Using
this `composite' eve allele,
eveRP2 (denoted eve
mosaic herein), we reproduced the recently described aCC/RP2 eve-null
phenotype; a failure of these two motoneurons to project out of the VNC
(Fig. 3A,B,F,G). This is
coupled both with ectopic expression of the Hb9 homeobox gene and
loss of Grn expression within these cells. In aCC, these effects are highly
penetrant and observed at several stages, whereas in RP2 the effects are
partly penetrant at stage 12 and almost absent at stage 15
(Fig. 3C-E,H-J). However, in
grn mutants, we did not observe any evidence of Eve downregulation in
aCC, RP2 or U motoneurons (Fig.
5A,B,D,E; not shown). We also addressed whether grn is
important for repressing Hb9 in these motoneurons, but found no
evidence for ectopic expression of Hb9 in aCC (or in RP2) in grn
mutants (Fig. 5G,H).
Zfh1, a Zn-finger-homeodomain protein, has been reported to be expressed in
aCC and RP2, as well as in many other motoneurons
(Lai et al., 1991). Recent
analysis of zfh1 reveals that is indeed expressed in all identifiable
motoneurons, and genetic analysis reveals that it is necessary for proper
motor axon pathfinding (Layden et al.,
2006). In stage 15 embryos, we find that Zfh1 expression is
dependent both upon eve and grn, but only in aCC and not in
RP2 (Fig. 4A-E,
Fig. 5D,E). As expected, when
grn function is rescued (RN2-GAL4/UAS-grn;grn), Zfh1
expression is restored in aCC (Fig.
5I). In line with the notion that eve and grn
act upstream of zfh1, Eve or Grn expression is unaffected in
zfh1 mutants (Fig.
5C,F).
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Fig. 2. grain is required for ISN motor axon projections. Stage 16
embryos stained with -Fas2 (green in A-C,G), RN2-GAL4 driving
UAS-mEGFPF (green in D-H), CQ2-GAL4 driving
UAS-mEGFPF (green in I,J) and Phalloidin-TX (magenta in
A-F,H-J). Arrows and arrowheads indicate axons terminals contacting dorsal (2
and 10) and dorsal-most (1 and 9) muscles, respectively. (A) In wild
type, the ISN nerve innervates muscles 2/10 and 1/9. (B) In
grn mutants, ISN fails to innervate muscles 1/9, but axonal
projections are seen contacting muscles 2/10. Bracket denotes a partially
penetrant muscle patterning phenotype, evident as an imprecise insertion of
muscles 21-24 into the body wall. (C) In grn rescue
(RN2-GAL4/UAS-grn; grn-/-) ISN innervates muscles 2/10 and
1/9 as in wild type. (D) In control,
RN2-GAL4/UAS-EGFPF reveals muscle 1 innervation by aCC and
muscle 2 innervation by RP2. (E) In a grn mutant background,
RN2-GAL4/UAS-EGFPF reveals that although muscle 1/9 is not
innervated by aCC, axon terminals from aCC and/or RP2 contact muscles 2/10.
(F) zfh1 can partially rescue grn mutants
(RN2-GAL4/UAS-zfh1; grn-/-) and the lack of muscle 1
innervation (arrowheads) is less severe than in grn mutant.
(G) Overlap between RN2-GAL4/UAS-EGFPF (green) and
-Fas2 (magenta) revealing axons terminals for aCC and RP2. This
reporter allows for a precise analysis of aCC and RP2 terminals in the
periphery. (H) In grn mutants, 36% of hemisegments
(n=69) show ectopic innervation of muscle 8 together with
defasciculation of aCC and RP2 motor axons (see also oblique arrow in E).
(I) In control, CQ2-GAL4/UAS-EGFPF reveals muscle 9
innervation by U1 and muscle 10 innervation by other U motoneurons. (J)
In a grn mutants, CQ2-GAL4/UAS-EGFPF reveals that
muscle 9 is not innervated (by U1), while U axon terminals contact muscles
field 2/10. (K) Quantification of muscles 1/9 innervation in different
genetic backgrounds. (L,M) Schematic showing the grn
mutant phenotypes (M) compared to wild type (L).
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Drosophila motoneurons depend upon a target-derived BMP signal for
proper maturation (Aberle et al.,
2002; Marques et al.,
2002). Consistent with the failure of aCC and RP2 axons to exit
the VNC in eve mosaic mutants, we observe a complete loss of pMad
staining in both aCC and RP2 (0% pMad in aCC and RP2; n=32)
(Fig. 4F,G), indicating that
these neurons are unable to receive the peripheral BMP retrograde signal. By
contrast, in grn and zfh1 mutants, where aCC and RP2 still
project into the periphery, we detect wild-type staining for pMad (100% pMad
in aCC and RP2; n=46 and n=48, respectively)
(Fig. 5A-C). These observations
indicate that in grn and zfh1, ISN motoneurons maintain a
`generic' motoneuronal identity and further indicate that embryonic activation
of the BMP pathway does not rely on the establishment of functional contacts
between motoneurons and their proper muscle targets.
Within the aCC motoneuron, we are thus able to place these three genes in
an evegrnzfh1 regulatory cascade, with the added
complexity that eve also acts to suppress Hb9. By contrast,
there is only partial crossregulation between eve, grn, zfh1 and
Hb9 in the RP2 motoneuron.
eve and grain play additional roles outside of the evegrnzfh1 regulatory cascade
Do eve and grn act solely in the
evegrnzfh1 regulatory cascade to specify aCC motoneuron
identity, or do these regulators play additional roles during aCC
specification? To address this question, we attempted to rescue the motoneuron
pathfinding phenotype of eve mutants with UAS-grn, and,
similarly, to rescue grn mutants with UAS-zfh1 (using in
both cases RN2-GAL4). First, we find that grn does not
rescue the eve phenotype in aCC; a failure of aCC to project its axon
out of the VNC and activate Zfh1 expression
(Fig. 6A-E). Second, we find
that UAS-zfh1 can only partially rescue the grn motoneuron
phenotype; muscle 1/9 innervation is increased to 34% (n=136)
compared with the more severe (12%) grn mutant phenotype
(Fig. 2F,K).
The dMP2 peptidergic neurons project posteriorly in the VNC
(Hidalgo and Brand, 1997) and
exit the VNC to innervate the hindgut
(Miguel-Aliaga and Thor,
2004). dMP2 neurons do not express Eve, Grn or Zfh1
(Fig. 6F; not shown). Recent
studies show that misexpression of zfh1 in dMP2 neurons can potently
trigger lateral axonal exit from the VNC (45% lateral exit)
(Layden et al., 2006). To test
whether misexpression of eve and/or grn can similarly alter
axonal projections of dMP2 neurons, we misexpressed them alone and in
combination. We find that although eve can trigger lateral VNC exit
at low frequency (5.5%; n=36; Fig.
6H), grn has no such effect (0%; n=28). By
contrast, co-misexpression of eve and grn leads to a high
frequency of lateral exit (40.5%; n=84;
Fig. 6G,H). To our surprise,
the combinatorial misexpression of eve and grn alters axon
pathfinding without any obvious sign of ectopic Zfh1 expression
(Fig. 6G). Thus, misexpression
of either zfh1 alone or of eve/grn together, can act equally
well in triggering dMP2 lateral axonal exit. These rescue and misexpression
results indicate that although eve and grn act in an
evegrnzfh1 regulatory cascade within aCC, both genes play
additional roles to ensure proper aCC identity.
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Fig. 3. eve is necessary for grain expression and for
Hb9 repression in both aCC and RP2 motoneurons. Stage 12 (A-D) or
stage 15 (F-I) eveRP2A/+
heterozygote (eve mosaic/+) (A,C,F,H) and
eveRP2A homozygote mutant
(eve mosaic) (B,D,G,I) embryos. Arrows and arrowheads indicate aCC
and RP2, respectively (visualized using RN2-GAL4/UAS-lacZ).
(A,C,F,H) eve mosaic/+
RN2-GAL4/UAS-lacZ showing that Grn is expressed in aCC and RP2
at stage 12 and stage 15, while Hb9 is not. (B,D) In stage 12
eve mosaic mutant, Hb9 is derepressed in aCC and RP2 while Grn
expression is not detectable in aCC but maintained in RP2. (G,I)
In stage 15 eve mosaic mutants, Hb9 remains derepressed in aCC and
partly in RP2, while Grn expression is not detectable in aCC but maintained in
RP2. At this stage, Grn expression in RP2 appears even stronger in
eve mosaic compared with wild type. (E,J)
Quantification of these phenotypes.
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The evegrnzfh1 regulatory cascade and integration of the Notch pathway
In the aCC neuron, grn and zfh1 are positively regulated
by eve. aCC and its sibling, the pCC interneuron, is a well-studied
sibling pair. The pCC neuron also expresses Eve, as well as the Nkx-family
member vnd (ventral nervous system defective)
(McDonald et al., 1998). Using
eve mosaic mutants, we find that Vnd expression in pCC is completely
dependent upon eve (Fig.
4H,I). Thus, eve acts in both sibling cells to regulate
different downstream genes in each neuron; grn and zfh1 in
aCC, and vnd in pCC. Studies have shown that the aCC versus pCC cell
fate decision is dependent upon Notch signaling, with pCC being dependent upon
Notch activation (Skeath and Doe,
1998). Although Eve expression in aCC and pCC does not respond to
alterations in the Notch pathway, expression of both Zfh1 and Vnd in these
siblings has been shown to be sensitive to Notch signaling
(Lear et al., 1999). To
address whether grn also responds to Notch activity in the aCC/pCC
cell pair, we analyzed grnlacZ, grnGAL4 and Grn
expression in two mutants affecting the Notch pathway, sanpodo
(spdoG104) and mastermind
(maml(2)04615). spdo facilitates N signaling
specifically during asymmetric cell divisions, and mutants permit normal N
signaling during early neurogenesis
(O'Connor-Giles and Skeath,
2003). Likewise, mam is needed for nuclear events
downstream of N signaling, but has a maternal contribution
(Skeath and Doe, 1998). This
allows, in both cases, for the examination of N function at later stages of
neuronal development. In spdo and mam mutants, we find
activation of both Grn (and grnGAL4) expression in pCC
(Fig. 7E,F,H). As previously
reported, we find that Vnd expression is lost in pCC
(Fig. 7A,B,B').
Conversely, ectopic Notch activation in aCC, using the RN2-GAL4
driver to express the intracellular (activated)
UAS-NotchICD transgene
(Doherty et al., 1996),
produces the reverse phenotype: de-repression of Vnd in aCC (but not in RP2)
and repression of grn in aCC and RP2
(Fig. 7C,G,I). Thus, in the
evegrnzfh1 regulatory cascade, only grn and
zfh1 respond to Notch signaling.
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Fig. 4. Zfh1, pMad and Vnd expression is affected in eve mutants.
(A-D,F-I) Stage 15 eve mosaic/+ (A,C,F,H) and eve mosaic
(B,D,G,I). Arrows and arrowheads indicate aCC and RP2, respectively
(visualized using RN2-GAL4/UAS-lacZ). (A,C) Zfh1
expression is robust in control aCC and RP2 motoneurons. (B,D)
In eve mosaic mutants, Zfh1 is lost from aCC, but unaffected in RP2
motoneurons. (E) Quantification of these phenotypes. (F) In
control, pMad staining is evident in both aCC and RP2, but lost from these
neurons in eve mosaic mutants (G). (H) In control, Vnd
is specifically expressed by the pCC interneuron (double arrowhead) but
expression is lost in eve mosaic mutants (I).
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We next asked whether grn was sufficient to activate aCC-specific
or to suppress pCC-specific genes, respectively? Although grn is
necessary for Zfh1 expression in aCC, we find that misexpression of
grn in pCC neither suppresses Vnd nor activates Zfh1
(Fig. 8A-C; not shown). This is
in agreement with the fact that we never observed Vnd expression in aCC in
grn mutants (data not shown). Likewise, using
RN2-GAL4/UAS-vnd, we asked whether vnd was sufficient to
suppress aCC-specific markers but find that vnd cannot suppress Grn
expression in aCC (Fig.
8D-F).
In summary, we have shown that Notch signaling acts downstream of, or in
parallel to, eve to restrict grn and zfh1 to aCC,
and vnd to pCC. However, these determinants are not involved in
cross-repressive interactions within these post-mitotic sibling cells
(Fig. 9). We furthermore find
that although both aCC and RP2 express eve, grn and zfh1,
their regulatory interactions differ between aCC and RP2.
|
DISCUSSION
|
|---|
Specification of unique motoneuron identities
During motoneuron generation, combinatorial codes of regulators act to
specify important aspects of subclass identity
(Briscoe and Ericson, 2001;
Shirasaki and Pfaff, 2002;
Thor and Thomas, 2002).
However, within any given subclass, there exists a further refinement,
apparent in Drosophila and C. elegans at single-cell
resolution. Our findings suggest that unique motoneuron identities may be
defined by the unique interplay between subclass determinants (i.e.
eve/grn/zfh1 in the ISN subclass). Our findings,
combined with previous studies of the aCC/pCC and RP2/RP2sib pairs
(Doe et al., 1988b), reveal a
remarkable difference in the genetics of aCC and RP2 specification. A summary
of the specification of these cells is presented in
Fig. 9 and highlights how a
unique genetic cascade allows for the specification of the aCC motoneuron. But
why do these three genes act in a unique fashion in aCC, and why is
grn and zfh1 sensitive to Notch specifically in this ISN
motoneuron? One explanation may be that the differential input from upstream
regulators, such as Ftz, Pdm1, Hkb and Pros
(McDonald et al., 2003), acts
to modify the genetic interactions between eve, grn and
zfh1. Another possibility is that the relative level of each factor
plays an important role in dictating different cellular fates. Studies of the
related Isl1 and Isl2 LIM-homeobox genes suggest that their
involvement in motoneuron subclass specification is not primarily the result
of the unique activity of each gene, but rather by the combined `generic',
tightly temporally controlled, Isl1 and Isl2 levels
(Thaler et al., 2004).
Similarly, the different expression levels of the transcription factor Cut
have been shown to play instructive roles during the specification of neuronal
cell identities within the PNS (Grueber et
al., 2003). We have as well noticed different levels of expression
of Grn and Zfh1; while Grn is strongly expressed in aCC and weakly in RP2,
Zfh1 expression shows an opposite distribution. It is tempting to speculate
that these levels may be instructive for ISN motoneuron specification.
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Fig. 5. In grain mutants, loss of Zfh1 expression is restricted to the
aCC motoneuron. Stage 15 wild-type (A,D,G), grn mutant (B,E,H),
zfh1 mutant (C,F) and grn rescue (I) (using
RN2-GAL4/UAS-grn; grn-/-) embryos stained for Eve and pMad (A-C), Eve
and Zfh1 (D-F) or Grn and Zfh1 (I). (G,H)
RN2-GAL4/UAS-mEGFPF embryo stained with -Hb9.
(A-C) pMad staining in grn and zfh1 mutants appears
unaffected within aCC and RP2. (D-F) In grn mutants, Zfh1
expression is not detectable in the aCC motoneuron, but RP2 maintains Zfh1
expression. Grn expression is not affected in aCC or RP2 in zfh1
mutants. (G,H) Hb9 expression is unaffected in grn
mutants. (I) In grn rescue experiments, Zfh1 expression is
restored in aCC showing the cell autonomous effect of grn on Zfh1
expression in this motoneuron. Arrowheads and asterisks indicate aCC and RP2,
respectively.
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Fig. 6. eve and grain play additional roles outside of the
evegrnzfh1 cascade. (A-E) Stage 15 embryo
stained for Grn (A,B) ß-Gal (A-D) and Zfh1 (A,C,E). B-E are identical to
A but with different combinations of color channels to facilitate the
observation of Grn and Zfh1 expression in aCC (arrows) and RP2 (arrowheads).
grn is unable to rescue eve mosaic mutants (UAS-grn,
eve mosaic; RN2-GAL4, UAS-lacZ), evident as a failure of
aCC and RP2 to project axons out of the VNC, and of aCC to express Zfh1.
(F-H) Stage 15 embryo stained for Myc and Zfh1, expressing only
UAS-EGFPF (F), UAS-eve (G) or co-misexpressing
both eve and grn (H). (F) In the control, dMP2 axons project
posteriorly in the longitudinal connective and never exit the VNC laterally
(n=62). (G) Ectopic eve triggers lateral VNC exit, but only
in 5% of hemisegments. (H) Ectopic eve and grn (UAS-eve,
UAS-grn, dMP2-GAL4; UAS-EGFPF) triggers lateral VNC exit in
40% of hemisegment (n=84). There is no evidence of Zfh1 expression in
dMP2 neurons (yellow circles), in the control (F) or in the misexpression
backgrounds (G). Arrowheads indicate dMP2 axons exiting the VNC.
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Fig. 7. grain expression is under the control of Notch signaling.
(A) In wild type, Vnd is expressed in the pCC interneuron, but this
expression is lost in spdoG104 mutant (B,B').
(C,G) Vnd is derepressed in the aCC motoneuron when Notch signaling is
activated using RN2-GAL4/UAS-NotchICD (intracellular
domain of a constitutive activated form of Notch). The arrow indicates
aberrant axonal projection (probably from aCC and/or RP2). (D) In
grn mutants, derepression of Vnd is not observed in aCC (or in RP2)
suggesting that grn does not repress Vnd in this sibling neuron.
(E) In wild type, Grn is not expressed in pCC. (F,H) In
spdoG104 mutants, Grn (and grnGAL4) is
derepressed in the pCC neuron. Grn is also derepressed in the RP2sib; 4
Eve-positive neurons (observed in B) are indicated by a vertical bar and an
asterisk. (G,I) Activation of Notch (RN2-
GAL4/UAS-NotchICD) led to a loss of Grn (and
grnlacZ) expression in aCC and RP2. Arrowheads and
asterisks indicate aCC and RP2, respectively.
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Cross-repressive interactions and Notch signaling specify neural fates
In the VNC, we observe mutually exclusive expression between Grn and Hb9
(and Islet) in different subsets of interneurons and motoneurons.
Cross-inhibitory interactions between eve and Hb9 has been
shown to contribute to their mutually exclusive expression patterns, and
functional studies demonstrate that eve and Hb9 regulate
axonal trajectories of dorsally and ventrally projecting axons, respectively
(Broihier and Skeath, 2002;
Doe et al., 1988b;
Fujioka et al., 2003;
Landgraf et al., 1999). These
observations are reminiscent of the cross-repressive interactions between
classes of regulators that act to determine, refine and maintain distinct
progenitor domains along the dorsoventral axis of the vertebrate neural tube
(Briscoe et al., 2000). We have
shown that eve is important for proper grn and zfh1
expression in aCC, but not in RP2. These results are consistent with
previously reported observations that the requirement for eve in
axonal guidance is somewhat more stringent in aCC than in RP2, leading the
authors to propose that there may be different target genes for Eve in these
two motoneurons (Fujioka et al.,
2003).
Zfh1 expression was previously shown to depend upon Notch signaling
activity in the aCC/pCC sibling pair as mutations in spdo or
mam, members of the Notch signaling pathway, lead to de-repression of
Zfh1 in pCC (Skeath and Doe,
1998). Using the same allelic combinations, we also observed
de-repression of grn in pCC. Whether or not grn is directly
suppressed by the Notch pathway remains to be seen, but it is interesting to
note that in vertebrates, gata2/3 have been identified as
targets of Notch during the differentiation of specific hematopoietic lineages
(Amsen et al., 2004;
Kumano et al., 2001).
aCC, RP2 and U motoneurons - several pioneers for ISN?
Within the ISN subclass, the aCC motoneuron pioneers the ISN to innervate
the dorsal-most muscle, muscle 1 (Jacobs
and Goodman, 1989;
Sanchez-Soriano and Prokop,
2005; Thomas et al.,
1984). A number of genetic and cell-ablation studies have
convincingly shown that aCC plays an instructive pioneer role and guides the
follower U motoneurons along the ISN nerve
(Fujioka et al., 2003;
Lin et al., 1995a;
Sanchez-Soriano and Prokop,
2005). Our results lend support for the proposed instructive role
of aCC in ISN formation. However, our studies indicate that aCC may not be
essential for ISN formation. First, using RN2-GAL4 to visualize aCC
and RP2, we frequently find (35% of hemisegments) aberrant innervation of
muscle 8 in grn mutants. However, we simultaneously observe an axonal
projection at the vicinity of the dorsal muscles 2/10. In grn
mutants, zfh1 expression is specifically lost in aCC but maintained
in RP2. Given the role for zfh1 in motor axon pathfinding, we propose
that aberrant innervation of muscle 8 in grn mutants, is caused by
aCC and not by RP2, and that RP2 pathfinds normally to the muscles 2/10. If
so, RP2 may function as a pioneer motoneuron for muscle 2 and project there
without the aCC axon. Second, although the rescue of grn mutants
using RN2-GAL4 is complete, we do find that using CQ2-GAL4
to specifically rescue U motoneurons does lead to a partial rescue (54%
muscles 1/9 innervated compared with 15% in grn mutants). Thus, even
in the absence of aCC pioneer function, the Us (presumably U1) can still
project to the dorsal-most muscles. This is in line with previous studies
showing that in eve aCC/RP2 mosaic mutants and in aCC/RP2 cell ablation
experiments, there is still partial innervation of muscle 1/9
(Fujioka et al., 2003;
Lin et al., 1995a;
Sanchez-Soriano and Prokop,
2005).
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Fig. 8. grain and vnd do not act in a cross-repressive manner
in aCC, pCC and RP2. (A-F) Stage 15 embryos stained for Grn and Vnd.
(A-C) Ectopic grn expression in pCC (double arrowhead;
RN2-GAL4/UAS-grn) does not suppress Vnd expression in this cell.
(D-F) Ectopic Vnd expression in aCC (arrow) and RP2 (arrowhead;
RN2-GAL4/UAS-vnd) does not suppress Grn expression in these
cells.
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Fig. 9. An evegrnzfh1 genetic cascade specifies aCC motor
axon identity. Within the VNC, GMC1-1a is one of the first GMCs to divide
and produces two postmitotic neurons: the aCC pioneer motoneuron and its
sibling the pCC interneuron. In aCC and pCC, eve expression is
independent of the activity of Notch signaling, whereas grn and
zfh1 are suppressed by Notch signaling, and vnd is activated
by Notch. The GMC4-2a divides later and produces the RP2 motoneuron and the
RP2sib. In contrast to aCC/pCC, in the RP2 neuron, eve, grn and
zfh1 do not regulate each other and in addition expression of all
three genes is dependant upon Notch signaling. Although pros function
is essential for proper GMC1-1a fate, GMC4-2a specification is under control
of concerted activities of pros, hkb, ftz and pdm1. The
orange boxes indicate genes regulated and/or sensitive to Notch signaling.
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The evegrnzfh1 genetic cascade contra other roles for eve and grain
We find that grn is part of an evegrnzfh1
transcriptional cascade crucial for specification of aCC motoneuron identity.
However, the failure of grn to rescue eve, and of
zfh1 to completely rescue grn, combined with the
misexpression results, indicate additional roles for both eve and
grn. These roles could be either in the regulation of other aCC
determinants and/or in the regulation of genes directly involved in aCC axon
pathfinding. Although we are unaware of obvious candidates for additional aCC
determinants, recent studies point to a candidate axon pathfinding gene. The
Drosophila unc-5 gene encodes a netrin receptor and is expressed in
subsets of neurons in the VNC (Keleman and
Dickson, 2001). Misexpression of unc-5 is sufficient to
trigger ectopic VNC exit in subsets of interneurons
(Allan et al., 2003;
Keleman and Dickson, 2001).
Recent studies now show that unc-5 is specifically expressed in
eve motoneurons, and that eve is necessary, but only partly
sufficient for unc-5 expression
(Labrador et al., 2005). In
line with these findings, we find that whereas single misexpression of
eve or grn in dMP2 neurons has very minor effects,
co-misexpression of eve and grn can efficiently trigger dMP2
lateral axonal exit. This combinatorial effect of eve/grn
occurs without apparent activation of zfh1. However, misexpression of
zfh1 can also trigger dMP2 lateral exit
(Layden et al., 2006). Thus,
these genes appear to be able to act in an independent manner to trigger VNC
exit, but in a highly context-dependent manner. A speculative explanation for
not only the mutant and rescue results, but also these misexpression results,
would be that all three regulators are needed for robust and
context-independent activation of axon pathfinding genes such as, for example,
unc-5.
Evolutionary conservation of GATA gene function
grn encodes a GATA Zn-finger transcription factor and is the
ortholog of the closely related vertebrate gata2 and gata3
genes. In vertebrates, gata2/3 are expressed in overlapping domains
in the nervous system, but relatively little is known about their function.
Expression data and evidence from gene targeting suggest an involvement in
neurogenesis, neuronal migration and axon projection
(Karis et al., 2001;
Nardelli et al., 1999;
Pandolfi et al., 1995;
Pata et al., 1999). A role in
specifying neuronal subtypes within the context of neural tube patterning is
emerging (Karunaratne et al.,
2002; Zhou et al.,
2000) and recently a role for gata2/3 during
5-HT neuron development has been reported
(Craven et al., 2004;
Tsarovina et al., 2004;
van Doorninck et al., 1999).
The role of gata3 in the development of the inner ear has been of
particular interest, and in humans, mutations in this gene have been linked to
HDR syndrome, which is characterized by hypoparathyroidism, deafness and renal
defects (Muroya et al., 2001;
Van Esch et al., 2000). In the
mouse, gata3 is expressed in auditory but not vestibular ganglion
neurons during development (Lawoko-Kerali
et al., 2002; Rivolta and
Holley, 1998). The mouse gata3 mutant shows auditory
ganglion neuron loss and efferent nerve misrouting, revealing that
gata3 regulates molecules associated with neural differentiation and
guidance (Karis et al., 2001).
These vertebrate studies, combined with our results, suggest that
gata2/3 genes, similar to other transcription factors specifying
neuronal identities, such as islet1/2, evx1/2 or Hb9, and
their respective orthologs in Drosophila, have maintained similar
functions throughout evolution (Broihier
and Skeath, 2002; Fujioka et
al., 2003; Thor and Thomas,
2002).
|
ACKNOWLEDGMENTS
|
|---|
This study was initiated while we were at the Department of Neurobiology,
Harvard Medical School, and we are grateful for the support from our former
colleagues there. We thank J. Castelli-Gair Hombria, M. Fujioka, J. B. Skeath,
M. Nirenberg, R. Lehmann, S. Artavanis-Tsakonas, P. ten Dijke, the
Developmental Studies Hybridoma Bank at the University of Iowa and the
Bloomington Stock Center for reagents. We also thank Michele L. Ocana for
assistance with confocal microscopy. We are grateful to J. B. Thomas and C. Q.
Doe for helpful comments on the manuscript. This work was funded by grants
from NIH (RO1 NS39875-01), by the Freudenberger Scholarship Fund at Harvard
Medical School, by the Swedish Research Council, by the Swedish Strategic
Research Foundation and by the Swedish Royal Academy of Sciences to S.T.; and
by the Fondation Recherche Médicale (FRM) to A.G.
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