1 Laboratory of Neurogenetics, Department of Human Genetics, Flanders
Interuniversity Institute for Biotechnology (VIB), KU Leuven, 3000 Leuven,
Belgium
2 Developmental Biology unit, Department of Molecular Biomedical Research,
Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University,
9000 Ghent, Belgium
3 Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA
4 Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX
77030, USA
* Author for correspondence (e-mail: bassem.hassan{at}med.kuleuven.ac.be)
Accepted 16 December 2003
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SUMMARY |
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Key words: Neural precursor, Drosophila, Xenopus, bHLH, Proneural gene, Evolution
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Introduction |
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The initiation event in neural lineage development is the selection of
NPCs. The study of the PNS of various model systems, such as Drosophila,
Xenopus and mouse, shows that expression of basic helix-loop-helix (bHLH)
proteins in the neuroectoderm confers the ability to generate NPCs
(Anderson, 1999;
Brunet and Ghysen, 1999
;
Campuzano and Modolell, 1992
;
Chitnis, 1999
;
Guillemot, 1999
;
Jan and Jan, 1994
;
Okano et al., 1997
). bHLH
proteins are the key proteins in nervous system development and evolution.
Their expression determines the position, timing and extent of neural stem
cell selection as well as the identity of the neural cells in each lineage.
These proteins, which are known as proneural proteins, promote NPC formation
by forming heterodimers with a widely expressed bHLH protein, called
Daughterless (DA) in Drosophila
(Cabrera and Alonso, 1991
),
and E12/E47 in vertebrates (Murre et al.,
1989
). The proneural-DA heterodimer regulates transcription of
target genes by binding, via some of the residues in the two basic domains, to
a DNA motif called the E-box. The function of bHLH proteins is thought to
reside mostly within the bHLH domain, a structural motif, encoded by a stretch
of 50-60 highly conserved amino acid residues.
Expression of a proneural gene in a presumptive NPC regulates, and is
regulated by, a cell-cell communication process mediated by the highly
conserved Notch signaling pathway
(Artavanis-Tsakonas et al.,
1999). Expression of Notch receptor ligands, such as Delta (DL),
is under the transcriptional control of proneural genes
(Fode et al., 1998
;
Kunisch et al., 1994
). Ligand
engagement in a signal receiving cell leads to the repression of proneural
genes partly by activation of the Enhancer of split E(spl) complex
genes (Bailey and Posakony,
1995
; Jennings et al.,
1994
; Lecourtois and
Schweisguth, 1995
). Thus, the signaling cell elevates levels of
proneural genes and adopts the NPC fate, while at the same time preventing the
neural specification of its neighbours. The genes required for these steps are
highly conserved both structurally and functionally across species.
What determines which cell becomes the NPC is not clearly established. In
some cases, the future NPC autonomously expresses a higher level of proneural
proteins (Culi and Modolell,
1998). A zinc (Zn)-finger transcription factor, Senseless (SENS),
has been shown to be an essential element in the cascade of events that allows
cells to differentiate as NPCs. SENS appears to interact synergistically with
proneural proteins in a positive genetic feedback loop in Drosophila
(Nolo et al., 2000
).
Similarly, the Zn-finger protein X-MyT1 appears to be involved in the
selection of NPCs in Xenopus: it synergizes with NGN1 rendering cells
apparently less sensitive to Notch inhibition
(Bellefroid et al., 1996
).
However, SENS and X-MyT1 belong to different classes of Zn-finger protein, and
what mediates their synergy with proneural proteins remains unclear.
Two families of proneural bHLH proteins have been found and are conserved
across species: the Achaete-Scute proteins (AS) and the Atonal related
proteins (ARPs) (Bertrand et al.,
2002; Hassan and Bellen,
2000
). The ARPs consist of several subgroups, two of which,
Neurogenin (NGN) and Atonal (ATO) groups appear to act at the earliest steps
of NPC selection (Fode et al.,
1998
; Goulding et al.,
2000
; Huang et al.,
2000b
; Jarman et al.,
1993
; Ma et al.,
1996
). In the ATO group, gene substitution and misexpression
studies within and across species suggest that there is a very high degree of
functional similarity, and sometimes, but clearly not always, functional
identity (Ben-Arie et al.,
2000
; Goulding et al.,
2000
; Wang et al.,
2002
). Although this has not been directly tested by gene
replacement, expression and mutant analyses suggest that it may be true for
the NGN group as well (Begbie et al.,
2002
; Ma et al.,
1999
). Both flies and vertebrates have PNS expressed genes
belonging to the NGN and ATO groups. These two groups of proteins show very
high similarity in the bHLH domain. Interestingly, TAP, the fly NGN group
protein is not expressed during NPC selection in the fly PNS and does not
appear to have proneural activity (Bush et
al., 1996
; Gautier et al.,
1997
). Conversely, ATO proteins are generally not expressed during
early NPC selection in vertebrate neural plate
(Ben-Arie et al., 2000
;
Brown et al., 1998
;
Helms et al., 2001
;
Kanekar et al., 1997
;
Kim et al., 1997
). Therefore,
does this reversal in the use of ARP proteins in NPC selection represent (1) a
divergence in the mechanisms by which these genes act to specify NPCs, or (2)
a functionally inert change in expression patterns?
To answer this question, we initiated a comparative study of the proneural capacities of ATO and NGN group proteins using Drosophila and Xenopus as model organisms. First, we find that ATO group proteins, potent neural inducers in the fly, are extremely weak NPCs inducers in Xenopus. By contrast, NGN proteins, which are potent neural inducers in vertebrates, are extremely weak inducers in flies. Second, the functional specificities of ATO proteins and NGN proteins are differentially encoded within the bHLH domain. We identify the specific residues responsible for proneural activity in each protein. Third, this differential activity between ATO proteins and NGN proteins is not mediated by DA or Notch. Fourth, the specific residues encoding the proneural activity mediate the specificity of genetic interactions with the appropriate Znfinger proteins. The correct combination of bHLH protein and Zn finger protein is highly specific, and necessary for NPC formation. In summary, we identify both extrinsic and intrinsic factors responsible for specificity of NPC selection and demonstrate a mechanistic divergence in bHLH protein function.
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Materials and methods |
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Plasmid construction, microinjection and in situ hybridization
The ato and math1 cDNA and coding region of NGN1 were subcloned
into pCS2+ vector. The pCS2+X-MyT1 plasmid was described earlier
(Bellefroid et al., 1996). DNA
coding for ngnbato, ngnH2ato,
atobngn and atoH2ngn were obtained by
site-directed mutagenesis PCR. The ngnbato and
ngnH2ato fragments were cloned into pUAST vector. The
atobngn and atoH2ngn fragments were
cloned into pCS2+. The mRNAs were injected in a volume of 5 nl at a
concentration of 20-200 pg/nl, into a single blastomere of Xenopus
laevis embryos at the two-cell stage. Embryos were collected at stage 15
and 19. Whole-mount in situ hybridisation was performed as described
(Harland, 1991
), using a
digoxigenin labelled antisense N-tubulin probe.
Immunohistochemistry
Third instar larval wing discs were dissected in PBS. Embryos were bleached
for 3 minutes. Discs and embryos were fixed with 4% formaldehyde in PBT for 15
minutes. Blocking and antibody incubation were performed as described
(Mardon et al., 1994). The
antibodies used were: mouse anti-ß-Gal (Promega, 1:2000), rabbit anti-ATO
(1:1000), rabbit anti-NGN1 (1:250), rabbit anti-Math1 (1:100), rabbit anti-ASE
(1:1000), guinea pig anti-SENS (1:1000) and monoclonal antibody 22C-10
(1:100). Secondary antibodies were always used 1 in 500. Samples were mounted
in Vectashield mounting medium (vector) and detected using confocal microscopy
(BioRad 1024). Adult fly wings and scutella were mounted in 70% ethanol and
documented using Leica microscopes and software.
Evolutionary trace analysis
A multiple sequence alignment and a sequence identity tree were generated
using the pairwise sequence comparisons algorithm PILEUP
(Feng and Doolittle, 1987),
from the GCG sequence analysis package
(Devereux et al., 1984
). The
Evolutionary Trace was performed as described previously
(Lichtarge et al., 1996b
).
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Results |
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One explanation for the very small number of bristles obtained after strong
expression of NGN1 may be that the protein is able to induce NPCs, but most of
these NPCs fail to differentiate properly and do not give rise to sensory
organs. To test this possibility, we examined NPC formation directly upon
expressing NGN1, ATO and MATH1 with dppGal4 in
A101-lacZ flies. A101-lacZ is an NPC
specific enhancer trap (Huang et al.,
1991). The normal pattern of NPCs is revealed by anti-ß-GAL
staining in third instar larval (L3) wing discs
(Fig. 1I). Misexpression of ATO
along the AP axis of the wing disc results in the induction of ectopic NPCs
within the domain of ATO expression (Fig.
1J). By contrast, despite high levels of NGN1 expression, no
detectable increase in NPCs is observed upon expression of NGN1
(Fig. 1K). Similarly, ATO, but
not NGN1, induced asense expression, another marker of NPC
specification (Fig. 1L-N).
Is the weak activity of NGN1 specific to ectopic expression in the wing
disc? We find that wide expression of NGN1 in embryos using da-Gal4
does not result in ectopic neurons (Fig.
1O,P). Finally, we attempted to rescue the loss of ato in
the eye imaginal disc using Gal4-7 and uasngn1. Gal4-7
induces expression anterior to the morphogenetic furrow and has been used to
restore photoreceptors to ato mutant eye discs using scute
(Sun et al., 2000) and
Math1 (Wang et al.,
2002
). Expression of NGN1 in ato mutant discs did not
result in any rescue (Fig. 1Q)
nor did it induce ectopic R8 cells when expressed in control discs
(Fig. 1Q, inset). For
simplicity, we used the number of bristles as a quantitative assay for NPC
formation for the remainder of the study.
Differential encoding of proneural activity in the bHLH domains of NGN proteins and ATO proteins
Three non DNA-binding basic domain residues determine the differential proneural activities of NGN proteins and ATO proteins
To explore whether the differential activities of NGN proteins and ATO
proteins can be understood at the level of the proteins themselves, we turned
to the comparative analysis of the amino acid sequence of the basic domain.
Several studies have shown that important information is encoded by the basic
domain, or specific residues therein
(Chien et al., 1996;
Davis and Weintraub, 1992
;
Talikka et al., 2002
). In
addition, the 12 amino acids in the basic domain are sufficient to
phylogenetically delineate ATO proteins and NGN proteins, arguing that
sequence differences within the basic domain are of functional significance
(Hassan and Bellen, 2000
).
However, these studies did not investigate the genetic basis or address the
evolutionary implications of the variation in basic domain sequence. ATO
proteins and NGN proteins share eight residues out of 12 in the basic domain.
One is variable, and the other three residues (4, 7 and 11) show almost
absolute group specificity: they are highly conserved within each group but
are essentially never the same between the two groups
(Fig. 2A, green). To
investigate whether this sequence specificity can explain the species-specific
activities of ATO proteins and NGN proteins, we created a chimeric protein
exchanging the three group-specific amino acids in the basic domain of NGN1 to
those present in ATO, named NGNbATO
(Fig. 2B). Expression of
NGNbATO induces the appearance of bristles along the AP axis of the
wing in all transgenic lines examined (Fig.
2C, inset). Strong UAS-NGNbATO lines mimic strong
UAS-ATO lines and result in significant lethality and more than 60 bristles
per wing in the few surviving flies (data not shown). Moderate
UAS-NGNbATO lines behave like moderate UAS-ATO lines and induce an
average of 33 bristles per fly along the AP axis (n=30) when compared
with an average of seven for strongest UAS-NGN1 lines (n=45,
Fig. 2C). Conversely, we
generated a chimeric protein exchanging the three group-specific amino acids
in the basic domain of ATO to NGN1, named ATObNGN
(Fig. 2D). Whereas the
injection of Ato mRNA in Xenopus embryos has no significant
effect on the N-tubulin expression pattern
(Fig. 2E), the injection of
AtobNGN mRNA induces N-tubulin expression
(Fig. 2F), indistinguishable
from that caused by the injection of NGN1. Therefore, the NGNbATO
mutant recovers the NPC inducing activity of ATO in Drosophila, and
the ATObNGN mutant recovers the NPC inducing activity of NGN1 in
Xenopus.
|
Five Helix2 residues are required for proneural activity of NGN proteins but not for ATO proteins
To investigate whether other functionally specific motifs exist in the bHLH
domain of ARP proteins, we turned to the evolutionary trace (ET) analysis
method. ET tracks residues whose mutations are associated with functional
changes during evolution. This approach has been used to identify novel
functional surfaces (Lichtarge et al.,
1996a), and has recently been shown to be widely applicable to
proteins (Madabushi et al.,
2002
). In practice, ET relies on the phylogenetic tree of a
protein family and identifies residues of the alignment that are invariant
within branches but variable between them. These positions are called `class
specific'. The smallest number of branches at which a position first becomes
class specific defines its rank. The top ranked positions (1) do not vary.
Very highly ranked positions (2-8) are such that they vary little and,
whenever they do, there is also a major evolutionary divergence. By contrast,
poorly ranked positions vary more often, and their variation does not seem to
correlate with divergence. Thus, highly ranked positions tend to be
functionally important, while poorly ranked ones tend not to be. When
examining ARP bHLH domains, ET identified a number of positions that are
jointly important in different bHLH domains, yet that undergo significant
variation between them (Table
1). These residues varied in rank from 2 to 7, suggesting that
they can undergo non-conservative mutations that are likely to correspond to
functional divergence events. These positions tend to be most conserved
between NeuroDs and NGN proteins and then undergo variations in ATO proteins,
suggesting that they are important for an activity shared by NGN proteins and
NeuroDs, but absent in ATO proteins. The data above show that the ability to
induce NPCs in vertebrates is precisely such an activity. To investigate the
role of these group-specific residues on functional specificity further, a
chimeric protein, named NGNH2ATO (exchanging amino acids 37, 39,
43, 44 and 46 in Helix2 of NGN1 to those present in ATO), was created and
tested in Drosophila (Fig.
2G). Expression of the strongest NGNH2ATO transgenic
line induces a maximum of two bristles along the AP axis of the wing per fly
in 50% of the flies. Quantitative analysis shows that, unlike ATO,
NGNH2ATO induces an average of 0.8 bristles along AP axis per fly
(n=30, Fig. 2H). These
data indicate that the group-specific motif in Helix2 of ATO does not encode
proneural activity in Drosophila. Conversely, we generated a chimeric
protein, named ATOH2NGN, exchanging the same five amino acids in
Helix2 of ATO to those found in NGN1 (Fig.
2G). Injection of ATOH2NGN mRNA causes ectopic
N-tubulin expression, indistinguishable from the injection of NGN1
(Fig. 2I). Therefore,
ATOH2NGN recovers the activity of NGN1 in Xenopus. Taken
together, the mutational analysis results agree with the predictions of the ET
analysis indicating that the identified residues in the Helix2 mediate the
activity of NGN proteins but not of ATO proteins.
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ATO proteins and NGN proteins interact genetically with different co-factors during NPC selection
NPC formation in Drosophila requires the Zn-finger protein
Senseless (SENS). Fly proneural proteins first induce sens expression
and then synergize with it in a positive feedback loop
(Nolo et al., 2000). This
appears to enhance the ability of proneural genes to downregulate Notch
signaling in the presumptive NPC. In vertebrates, Senseless-like proteins
appear not to act in NPC formation, although they are expressed in the PNS
(Wallis et al., 2003
). To test
the possibility that SENS shows group specific interactions with bHLH proteins
during NPC selection, we compared the abilities of ATO and NGN1 to induce
SENS. SENS expression in wild-type L3 wing discs marks NPC formation
(Fig. 3B). Ectopic SENS
induction is detected along the AP axis of wing discs when ATO is misexpressed
(Fig. 3C). However, SENS
expression is not induced by NGN1 (Fig.
3D). These data suggest that unlike ATO, NGN1 does not efficiently
induce SENS expression. We further tested whether lowering endogenous levels
of Notch would allow NGN1 to induce SENS. Expression of NGN1 in Notch
heterozygous animals, although significantly increasing the number of induced
bristles (Fig. 3A), fails to
induce SENS expression (Fig.
3E) when compared with N+/ controls, arguing
that NPCs induced by NGN proteins are specified via a different mechanism not
normally used in Drosophila. The data above are quantified in
Fig. 3F. Although NGN1 does not
induce SENS, it is possible that synergy might occur if the requirement for
SENS induction is bypassed. We therefore compared the ability of NGN1 and
MATH1 to synergize with SENS in vivo by co-expressing either NGN1 or MATH1
with SENS using a moderate scutellar Gal4 driver (C5Gal4). Neural
induction was examined by counting the ectopic bristles induced on the
scutellum. Wild-type flies have four large bristles, or macrochaete, on their
scutella. Expression of SENS (Fig.
4A) or MATH1 (Fig.
4B) alone with C5-Gal4 induces a number of ectopic
microchaete, or small bristles, on the scutellum. No ectopic sensory bristles
were found when NGN1 was expressed alone (data not shown). Co-expression of
NGN1 and SENS has the same effect on the scutellum as the misexpressing SENS
alone (Fig. 4C). Co-expression
of MATH1 and SENS, however, causes the appearance of a large number of both
micro- and macrochaete (Fig.
4D). Finally, we misexpressed NGN1 or MATH1 in the absence of one
copy of sens (Fig.
4E). No effect on NGN1 activity in a
sens+/ background was observed. By contrast, the
average number of sensory bristles produced by MATH1 along the AP axis was
reduced by 42% (n=50, P<0.001) if a single copy of
sens was removed suggesting dose-sensitive interactions. Thus,
neither by loss nor gain of function criteria does NGN1 appear to interact
with SENS, thus explaining its weak proneural activity and inability to
efficiently antagonize Notch signaling in Drosophila. Therefore, SENS
is a key extrinsic difference in how ATO proteins and NGN proteins regulate
NPC selection.
In Xenopus, the C2HC-type Zn-finger protein X-MyT1 is expressed in
primary neurons and can be induced by NGN proteins. In addition X-MyT1 has
been suggested to play a role in NPC formation and to synergize with NGN
proteins (Bellefroid et al.,
1996). In order to test if X-MyT1, like SENS, shows specificity in
its interaction with ARP proteins, we compared its ability to interact with
NGN1 and ATO in Xenopus. X-MyT1 mRNA was injected alone or
co-injected with either Ngn1 or Ato mRNA. As expected, the
injection of X-MyT1 increases the number of
N-tubulin-expressing cells in the neural plate domains where neurons
normally form (Fig. 4F), while
the injection of Ngn1 mRNA alone leads to induction of
N-tubulin expression (Fig.
4G). Co-injection of Ngn1 and X-MyT1 mRNAs
results in very strong N-tubulin induction, pointing to a synergistic
interaction between the two proteins (Fig.
4I). By contrast, co-injection of Ato and X-MyT1
mRNAs does not cause a detectable increase in N-tubulin expression
compared with the injection of X-MyT1 mRNA alone
(Fig. 4J). Similarly, the few
ectopic N-tubulin-expressing cells observed when Math1 mRNA
is injected (Fig. 4H) are not
increased by co-injection of Math1 and X-MyT1
(Fig. 4K). Thus, X-MyT1
interacts specifically with NGN1 and not with ATO or MATH1. The data above
demonstrate that the correct combination of ARP protein and Zn-finger protein
is necessary for NPC induction.
NGNbATO and ATObNGN have reversed interactions with Zn finger proteins
Does the coding sequence difference mediate the divergence in the genetic
interactions of ARPs? To test this, we investigated whether the chimeric
proteins recover the ability to interact with the respective Zn-finger
proteins. Indeed, expression of NGNbATO in Drosophila
results in the induction of SENS (Fig.
5A-C), and the number of bristles induced by NGNbATO in
absence of one copy of sens (sens+/) is
reduced by 44% (n=30, P<0.001)
(Fig. 5D). In addition, strong
synergy was observed by co-expression of NGNbATO and SENS using the
dppGal4 driver (data not shown). Therefore, NGNbATO is
able to induce and interact with SENS in Drosophila. In
Xenopus, just like Ngn1, co-injection of
AtobNGN and X-MyT1 mRNAs
(Fig. 5F) results in synergy
and very strong ectopic N-tubulin expression when compared with the
injection of X-MyT1 (see Fig.
4F) or AtobNGN
(Fig. 5E) alone. Similarly,
co-injection of ATOH2NGN and X-MyT1 mRNAs results in synergy and
very strong induction of N-tubulin expression
(Fig. 5G,H) suggesting that
ATOH2NGN and ATObNGN use the same mechanism of action as
NGN1.
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Discussion |
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ATO proteins and NGN proteins act by divergent mechanisms to regulate neural lineage development
At the developmental level, the data presented here can be explained by two
possibilities (Fig. 6A,B). The
first is that Drosophila and vertebrates use different bHLH proteins
with divergent mechanisms for selecting similar cell types: the earliest born
neural progenitors (Fig. 6A).
Alternatively, NGN proteins may be involved in selecting neuronal (versus
glial) rather than earliest born neural progenitors in vertebrates
(Fig. 6B)
(Nieto et al., 2001;
Sun et al., 2001
;
Tomita et al., 2000
). This is
certainly the case in the mammalian inner ear
(Bermingham et al., 1999
;
Ma et al., 2000
) and it should
be determined whether it is a more generally applicable rule, at least in the
PNS. Given that there is no direct evidence in the literature to support a
role for NGN proteins in selecting multipotent progenitors, we propose that
the situation in Fig. 6B is
likely to be more representative of the events in vivo. The latter scenario
raises the question of whether Drosophila-like proneural proteins are
needed in the vertebrate neural plate. If they are not, then the strict
definition of a proneural gene as derived from work in Drosophila may
need to be re-examined (Ledent and
Vervoort, 2001
). However, these two models for NGN function are
not mutually exclusive. It is possible that in different lineages, NGN
proteins select first neural, and then neuronal, precursors. This would be
compatible with data from both flies and vertebrates showing that Notch
signaling, in addition to having anti-neural effects, has also anti-neuronal
and pro-glial effects during neural lineage development
(Morrison et al., 2000
;
Udolph et al., 2001
;
Umesono et al., 2002
;
Van De Bor and Giangrande,
2001
). Analysis of the fly NGN protein, TAP, may shed some light
on this issue. At any rate, a comparative approach should provide a powerful
tool for the systematic analysis of the pathways which program neural stem
cells.
|
Finally, genes common to protostomes and deuterostomes including atos,
ngn genes, Notch signaling genes, sens and
X-MyT1 most probably derive from the last common bilaterian ancestor
(Erwin and Davidson, 2002).
This implies that such an ancestor already possessed all the tools to specify
a large diversity of neural cell types and lineages, suggesting a
structurally, and consequently behaviorally, complex animal.
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ACKNOWLEDGMENTS |
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